Various imaging techniques are used to assess the structures of equine limbs and hooves. However, each technique has its limitations. Ultrasonography, for instance, is not currently considered sufficient for fully evaluating the dermoepidermal junction (DEJ) of the hoof. To address this limitation, we hypothesized that immersing the hoof wall in water could produce better results. After a 24 h period of immersion of the digit, the normal appearance of the middle stratum , the internal stratum (lamellae), the sublamellar dermis, and a few structures of the distal phalanx could be described using a transmural technique. Our findings demonstrate that a transmural technique, used after a 24 h immersion of the digit, permits the ultrasonographic identification and characterization of the middle stratum , internal stratum (lamellae), sublamellar dermis, and some structures of the distal phalanx. This transmural technique may have specific utility in evaluating structures involved in hoof conditions, particularly laminitis.Ultrasonography is commonly used to evaluate equine lameness-related structures, but traditional hoof approaches cannot adequately visualize internal structures such as the lamellar layer. To address this limitation, we used 62 healthy hooves without digital radiographic abnormalities, made up of thirty anatomical pieces (phase 1) and thirty-two hooves from living horses (phase 2). In both phases, half of the digits were submerged in water (group 1) and the other half in water and ice (group 2) for 24 h. Ultrasonographic views and temperature and humidity measurements were taken every two hours, and sagittal sections were obtained in phase 1. Good quality images were obtained in both phases. The transmural technique allowed the evaluation of several structures of the DEJ  and of the distal phalanx . Our findings suggest that the transmural technique following hoof submersion can be useful for internal evaluation of hoof conditions, such as laminitis. However, further validation of the technique in natural or experimental cases is required. Ultrasonography is a diagnostic technique widely used to evaluate structures associated with lameness in the distal part of the limbs of horses . SeveralThe parietal DEJ—more commonly known as the internal (lamellar) layer of the wall—is the interdigitation between the dermal and epidermal layers of the hoof, which makes up the suspensory apparatus of the distal phalanx (SADP), which aims to stabilize the digit and protect it from load forces ,14,15. AThe diagnostic value of images obtained of the lamellar layer and the sublamellar dermis requires the recognition and characterization of their normal status, to interpret changes derived from clinical conditions such as laminitis. The parietal dermis is divided into two layers: (1) the lamellar dermis—composed of primary and secondary dermal lamellae that are attached to the basement membrane—and (2) the sublamellar dermis—composed of sublamellar blood vessels and collagen fibers that connect to the foramina on the distal phalanx parietal surface, suspending it inside the hoof ,14,27. TBased on our pilot studies, the objective of this research is to describe a transmural ultrasonographic technique that can evaluate the internal structures of the equine hoof after a submersion period. We also aim to determine the optimal immersion time in the conduction medium and the positioning of the transducer to obtain high-quality diagnostic images.® HFX90V , with 1.2 mAs–66 kv settings. The location and normal anatomy of the distal phalanx were determined by measuring angles and other factors  with relevance to laminitis [This research was approved by the Ethics Committee for Animal Experimentation of the Universidad de Antioquia, Colombia . Sixty-two healthy hooves from thoracic limbs (TL) and pelvic limbs (PL) of Colombian creole horses were divided in two study phases. In phase 1, 30 hooves collected in a slaughterhouse were disarticulated from the fetlock joint, transported fresh for 4 h, and finally frozen at −21 °C for 150 days. In phase 2, 32 hooves from eight healthy mares without lameness and with an average age, weight, and body condition score [aminitis ,34,35,36® v25-19, 220v/60/2f-1hp). The sagittal sections were measured and analyzed with a digital stereomicroscope , as shown in In phase 1, a total of 16 hooves (4 TL and 4 PL by group) were randomized, selected and sectioned in a sagittal plane using a band saw  and a mixture of ice and water in group 2 (G2) . These m® Shenzhen Emperor Electronic Technology Co., Ltd. Nanshan district, Shenzhen, China) and the temperature measured with an infrared thermometer  and the hoof surface humidity measured with a hygrometer  were recorded.Before being immersed for 24 h, each hoof was thoroughly cleaned and sanded. The bags containing the commercial solutions (with a capacity of 3 L) were changed as needed to ensure that the immersion process was adequate. Throughout the immersion period (every 2 h), ultrasonographic evaluations were carried out using a G30 Color Doppler ultrasound machine , the inner layer (lamellar), and the sublamellar dermis of the hoof, along with the transitions between them, at the proximal and distal levels of the third phalanx, to categorize the images. An image was deemed of high quality only when all these structures could be clearly differentiated.2) nonparametric test, the association between temperature, humidity, and immersion time  and the prevalence of high-quality ultrasound images  was estimated, considering a significance level of p ≤ 0.05. Data were analyzed using STATA®, version 16.1 .Descriptive statistics were first calculated as measures of central tendency , mean (average), median) and measures of dispersion , coefficient of variation (CV)). For non-normally distributed data, the analyses were performed based on the median (50th percentile). Using Pearson’s chi-square  with the immersion time of G1 in phase 2, an approximate time of 7 h. Although the immersion time of G2 was similar, no statistical significance was found.Association test results for each variable that could influence the transmural technique’s ability to obtain high-quality images are shown in Regardless of the position of the transducer, the experimental phase, and the conduction medium used, the transition between the middle layer (tubular), the inner layer (lamellar), and the sublamellar dermis could be identified at the proximal and distal levels of the distal phalanx after the preparation procedures. At the proximal level and close to the extensor process, the coronary papillae were observed to be parallel to the parietal surface, indicating the normal anatomical configuration of the hoof-wall growth c. HoweveAt the distal level, it was found that the sole papillae were located distal to the sole border b. Such fTwo transition zones can be seen, in both Once the structures inside the hoof were located, their textures were described . The tubThe ultrasonographic technique through the hoof wall , as described and implemented in this study, allowed the identification and delimitation of tissues inside the hooves of normal limbs, including structures of interest such as the lamellar layer (which contains the DEJ), previously observed in studies using MRI and CT ,37,38. TAlthough high-quality images were obtained using both immersion mediums, an important difference was observed regarding the immersion time, in that phase 2 hooves required at least 7 h, compared to 16 h in phase 1. Phase 2 hooves kept the hydration mechanisms intact and active , allowinDespite the association between different immersion times and obtaining better images with ultrasonography, humidity was the most homogeneous variable while temperature was the most heterogeneous in both phases. When ice was used in phase 2, the variability in temperature tended to be higher, possibly due to the activation of the digit thermoregulation mechanisms ,41. The Although maintaining constant temperature control can be challenging, one of the reasons for using water and ice as a conduction medium was its practicality since digital cryotherapy is recommended and widely used in cases of acute laminitis ,44,45,46The benefit of using anatomical pieces was the ability to confirm and validate the strata/layers obtained by the radiographic and ultrasonographic studies through anatomical sections. However, the use of frozen pieces made it difficult and delayed the obtaining of ultrasonographic images, as they required a thawing time that was delayed when a water and ice mixture was used as a conduction medium. It is possible to obtain better effects with fresh pieces, although the animal study showed better results.Despite some limitations in methodology, such as the researcher performing all the examinations and image evaluations being aware of the sample group, the present study successfully described and characterized the acquisition of ultrasonographic images of the middle stratum , internal stratum (lamellar), and sublamellar dermis in healthy hooves. Its findings encourage the application of this technique in cases of natural or induced laminitis for further validation.In the evaluation of the images obtained using the transmural technique at the proximal level, the coronary papillae were clearly visible. These structures have also been observed in MRI and CT imaging studies ,38 and rThe concave shape of the coronary band made it difficult to complete the contact with the transducer, producing changes in the inclination of the coronary papillae in the image in At the distal level, the images revealed the inclination of the dermal papillae, which plays a crucial role in determining the growth pattern of the sole epidermis. Similar to the coronary papillae, the horn tubules also join the dermal papillae , which eThe heterogeneous appearance of the middle layer   Figure  is due tThe parietal dermis can be divided into two layers. The lamellar dermis is composed of primary and secondary dermal lamellae that are attached to the basement membrane, and the highly irrigated sublamellar dermis, composed of sublamellar blood vessels and collagen fibers ,41,50,51The transmural technique allowed the differentiation of the layers that make up the lamellar and sublamellar dermis , as wellUltrasonographic evaluation of the sublamellar dermis revealed the presence of larger caliber vessels , also reThe technique implemented herein revealed images of structures that are usually affected in cases of laminitis ,19,20,21Discrepancies have been found between clinical and radiographic findings—basic diagnosis to assess severity and prognosis in laminitis, and null information on soft tissues —demonstrMRI is superior to digital radiography for determining normality and changes in the hoof soft tissues ,23,37. IThe transmural technique, used after a 24 h immersion of the digit in water, permitted the ultrasonographic identification and characterization of several structures of the DEJ  and of the distal phalanx . In addition, a statistical association between high-quality images and submerging the hooves of living horses in water for seven (7 ± 1.4) hours was found. Likewise, quality images were obtained using water and ice as the immersion medium.To the authors’ knowledge, this is the first time this technique has been described and used. To increase its reliability, it is necessary to contrast the findings of this study with magnetic resonance imaging (both are able to differentiate the components of the hoof dermis and epidermis). Although preliminary, our findings suggest that the transmural technique following hoof submersion can be useful for internal evaluation of hoof conditions, such as laminitis. However, further validation of the technique in natural or experimental cases is required.


Tether breakage is the most common complication of Vertebral Body Tethering (VBT) occurring in up to 52% of Adolescent Idiopathic Scoliosis (AIS) patients and risks continued progression and revision. Radiographical diagnosis of tether breakage is commonly defined by a 5° increase in inter-screw angle and associates breakage with loss of correction. However, the sensitivity of this method was 56% only, suggesting that tethers can break without an increase in angulation, which was supported by other studies. To our knowledge, current literature lacks a method merely focusing on the diagnosis of tether breakage radiographically that does not associate the breakages with loss of correction.This was a retrospective review of prospectively collected data of AIS patients who underwent VBT. The “inter-screw index” is defined as the percentage increase in inter-screw distance since post-op, with ≥ 13% increase defined as tether breakage as suggested by our mechanical tests. CTs were reviewed to identify the breakages and compared with inter-screw angle and inter-screw index.94 segments from 13 CTs were reviewed, and 15 tether breakages were identified. Use of inter-screw index correctly identified 14 breakages (93%), whereas ≥ 5° increase in inter-screw angle only identified 12 breakages (80%).Use of inter-screw index is proven to be more sensitive than inter-screw angle in identifying tether breakages. Therefore, we propose the use of inter-screw index to diagnose tether breakages radiographically. Tether breakages were not necessarily accompanied by a loss of segmental correction leading to an increase in inter-screw angle, especially after skeletal maturity.Level 3. Vertebral Body Tethering (VBT) has gained popularity in recent years as a non-fusion growth modulation method for treating scoliosis in skeletally immature patients . DespiteTether breakage was reported as the most common complication of VBT and reported to be risking continued progression and subsequent revision if it occurs prior to skeletal maturity . FurtherTether breakage was reported to be occurring in up to 3–52% of patients, where a huge variation between the reported breakage rate data throughout the literature was observed –10. RaitInter-screw angle criterion, which is an index derived from the Cobb angle method, associates the tether breakage with loss of correction. However, recent studies showed that the loss of correction may not always be an indicator of the tether breakages, and explain why a change in inter-screw angle is recognised to underdiagnose tether breakages. Trobish et al. reported the poor accuracy of this method in diagnosing the tether breakages, and showed that tether breakage was not always accompanied by loss of correction . SimilarPreviously, we performed tensile tests on single unit VBT constructs  and investigated the structural mechanics and the failure mechanisms of the VBT. Our results showed that the inter-screw distance can only increase up to 10–13% prior to failure, which corresponds to an elongation on the length of PET tether. We reported that the inter-screw distance has the potential to be the bridge between mechanical investigation and radiographical examination as the measurement of elongation between the post-op and follow-up radiographs is possible, and suggested its use to diagnose the tether breakages radiographically over inter-screw angle method. However, further clinical validation was necessary to establish its reliability .The aim of this study was to derive a tether breakage diagnosis index employing the inter-screw distance, the inter-screw index, assess its sensitivity compared to inter-screw angle, and to validate it based on confirmed breakage on CT scans. To the authors’ knowledge, this is the first study providing an alternative method to diagnose tether breakages radiographically which is developed based on mechanical investigation and failure mechanisms of VBT.On coronal radiographs, inter-screw distance was measured as the distance between the centers of the staples of two adjacent screw heads, where centers of the staples were geometrically located as the intersection of the diagonals of the staples. On sagittal radiographs, the distance between the centers of the circles surrounding the two adjacent screw heads were used to measure the inter-screw distance Figs.  and 2. IEquation Inter-screw angle was measured as the angulation between two adjacent screw axes on coronal radiographs, with convex angles as negative (–) and concave angles as positive (+) Fig. . An incrterature , 9.Toshiba Aquilion ONE and PRIME CT Scanners  were used for the collection of CT scans. The resolution of the images as well as the other CT scanning settings were modulated by the Automatic Exposure Control (AEC) system, which is a system aiming to maximize the image resolution while minimizing the radiation exposure, and is a part of the regular practice in our clinic . On averTether breakage on CT was defined as a clear discontinuity between tether segments on 2D CT scan slices Fig.  16].Fig.Fig16].FThis was a retrospective analysis of prospectively collected radiographic data of patients who underwent VBT at a single center between February 2019 and December 2020 for the treatment of Adolescent Idiopathic Scoliosis (AIS). Ethics approval was obtained from the local institutional review board (UW 19-002). All patients underwent surgery using implants from the same manufacturer . Patients who have taken CT scans post-VBT were included in the study. Indications for scanning included the assessment of implant position and suspected tether breaks based on inter-screw index.Patient demographics , skeletal maturity, curve type, and instrumented levels  were reviewed. Biplanar standing full-length radiographs  taken at the first postoperative visit were compared with radiographs taken at the time of CT scanning (± 2 months). At each of the instrumented segments, Cobb angle of the instrumented curve, inter-screw distance, and angles were measured. To minimize bias, the measurements were conducted by two independent investigators (SW and MY) blinded to clinical and CT information. Inter-observer reliability was quantified using Intraclass Correlation Coefficient (ICC) with 95% confidence interval based on a two-way random-effects model . ReliabiAll CTs were assessed by two investigators (KC and SW) who were blinded to the radiographic findings and the tether breakage locations were identified. The distance between two broken ends of the tether at the diagnosed tether breakage locations was measured on 2D CT scan slices. To identify that the breakages observed in CT scans are not a by-product of the CT image quality and the other imaging artefacts, a known dimensional parameter, the diameter of the tether, was measured by two blinded investigators  at each broken level, and the standard error of measurement (SEM) was calculated by directly comparing it with the known diameter value of the tether . Tether All measurements were conducted on the local Picture Archiving and Communication System (PACS) using high image resolution and standardised calibration . StatistNine patients fit the eligibility criteria within the study period. 94 segments from 13 CT scans were reviewed. The mean age at surgery was 11.1 ± 1 years and the mean number of instrumented levels were 7.9 ± 1. There were 7 main thoracic curves, 2 double major curves, and 1 thoracolumbar curve. Mean duration between index surgery and CT scan was 16.3 months (range 2–36 months). Seven CT scans were performed for assessment of implant position, and six CT scans were performed because of suspected tether breakages.On CT, tether breakages were observed in 15 segments in 7 of the patients that fit the eligibility criteria. SEM was calculated to be ± 0.35 mm Fig. . The disFour of the breakages observed on CT were located at the apex of curves, while 11 of them were found 1–2 levels distal to the apex. No breakages were found above T9–T10. The rate of tether breakage was 15% for thoracic segments (9 out of 59) and 60% for lumbar segments (6 out of 10). Out of 15 tether breakages, 9 of them was located at the thoracic spine and failed at the screw–tether junction, while the remaining 6 were located at the lumbar spine and broke at the mid-substance , while ≥ 5° increase in inter-screw angle was observed in 12 broken segments (sensitivity 80%) Fig. . No falsThere were 5 patients with breakages at consecutive levels. In identifying both breakages in consecutive breakages, inter-screw index showed 80% sensitivity (4 out of 5), while inter-screw angle showed 40% sensitivity (2 out of 5). In three pairs of consecutive breakages, inter-screw angle only identified one breakage from each pair. An example is shown in Figs. Inter-observer reliability ranged from 0.88 to 0.95 for change in inter-screw angle, and 0.89 to 0.96 for change in inter-screw distance Table . Both meDiagnosis of tether breakage through inter-screw angle assumes that angular change will always occur after the tether breaks. However, recent studies showed that angular change may not always be an indicator of the tether breakage. Trobish et al. reported the poor accuracy of this method and showed that tether breakage was not always accompanied by loss of correction . In lineConsecutive level tether breakages were reported previously on 7 out of 10 patients (70%) by Trobish et al. , and intWe observed a strong correlation between our clinical findings and our mechanical tests. Previously, we reported that the failure of the VBT construct occurs at the screw–tether junction and reported that this is the dominant failure mode . SimilarWe observed that the tether breakages were diagnosed relatively early at 10, 14, and 15 months after the surgery, indicating that tether breakage could have occurred even earlier. According to Baroncini et al., tether breakages occurring within the first 12 months is significantly correlated with the loss of correction . MoreoveA limitation of the current study is that only one VBT system is used. Previously, we reported that the failure at the screw–tether junction is a design-related complication . SimilarIn summary, this is the first study to present inter-screw index as a rational indicator of tether breakage, and the first to offer an alternative diagnosis method of tether breakage independent of loss of correction. In our series of tether breakages confirmed on CT scans, a change in inter-screw distance, as represented by an increase in index of 13%, was able to identify more breakages than the inter-screw angle. Moreover, in the case of consecutive breakages, it was shown to be superior to the inter-screw angle. While larger studies would be needed to validate the generalizability of our results to other VBT systems, we propose that the concept of the inter-screw index has the potential to provide insights into the pattern and mechanism of tether breakage, as well as its effect on clinical outcome in the form of longitudinal studies.


More importantly, the coating can also autonomously self-heal at −20 °C, as a result of multiple dynamic bonds in its structure, to inhibit defect-induced icing processes. The healed coating sustains high anti-icing and deicing performance even under various extreme conditions. This work reveals the in-depth mechanism of defect-induced ice formation as well as adhesion, and proposes a self-healing anti-icing coating for outdoor infrastructures.Anti-icing coatings on outdoor infrastructures inevitably suffer from mechanical injuries in numerous icing scenarios such as hailstorms, sandstorms, impacts of foreign objects, and icing–deicing cycles. Herein, the mechanisms of surface-defect-induced icing are clarified. At the defects, water molecules exhibit stronger adsorption and the heat transfer rate increases, accelerating the condensation of water vapor as well as ice nucleation and propagation. Moreover, the ice–defect interlocking structure increases the ice adhesion strength. Thus, a self-healing (at −20 °C) antifreeze-protein (AFP)-inspired anti-icing coating is developed. The coating is based on a design that mimics the ice-binding and non-ice-binding sites in AFPs. It enables the coating to markedly inhibit ice nucleation (nucleation temperature < −29.4 °C), prevent ice propagation (propagation rate < 0.00048 cm Nepenthes pitcher-inspired materials based on textured solids infiltrated with lubricants. SLIPSs were created to address the void problem and expand the potential application of surface fabrication technologies [Throughout the story of human survival, the selection or design of well-suited materials inspired from nature has been important to withstand environmental challenges –5. For enologies –16. The nologies ,18.However, anti-icing coatings for large-scale outdoor infrastructures still face substantial challenges ,20. FirsNatural antifreeze proteins (AFPs) help polar fish avoid freezing . Herein,Outdoor anti-icing coatings are inevitably subjected to mechanical injuries caused by storm, foreign-object impacts, and icing–deicing cycles. Defects on the coating surfaces reduce their anti-icing performance and even promote the icing process. Here, the defect-induced icing promotion mechanism was clarified to reveal the importance of self-healing coatings in practical applications.adE) of a water molecule at a defect was calculated using density functional theory (DFT) simulations. The PDMS coating was represented by a single silicone chain, whereas defects were modeled using a broken silicone chain containing a broken Si–Si or Si–O bond . This indicates that the water adsorption on the surface defect is markedly promoted, leading to preferential ice crystallization [An unhealable pristine polydimethylsiloxane (PDMS) matrix, similar to the developed self-healing anti-icing PDMS-based coating matrix, was used to investigate the defected coating model in the following tests. First, the adsorption energy  of the water droplets on the coatings were investigated. According to previous reports, when the cooling rate is 5 °C/min, the HT of water could be considered as the temperature of the whole water droplet that was frozen within 1 s. As shown in Fig. HT of the water droplet on the intact PDMS coating surface was −22.6 °C, whereas that on the coating with 1, 2, and 3 defects were −19.5 °C, −12.9 °C, and −9.5 °C, respectively. The HT on the coating with 3 defects was even higher than that on an uncoated steel surface (−13.6 °C), confirming the defect-induced icing phenomenon. The ice nucleation process was also observed on the surface with defects using a high-speed camera  and cooled from 20 °C to −15 °C before observed. During the cooling process, water was preferentially condensed at the defect, at which it was susceptible to freezing Fig. . The hetera Fig. . The iniIce propagation was then investigated on the intact surfaces and those with defects. Ice from a frozen droplet propagate to other droplets through ice bridges . As showNext, an in-depth investigation of the defect-induced icing mechanism was conducted by simulating the heat transfer process on the intact coatings and those with defects using finite element simulations. Figure adE of the H2O molecule cause a preferential condensation of the water vapor for ice nucleation and formation. Small ice crystals can propagate via the defect, which acts as an ice bridge, finally leading to a massive ice accretion on the surface with defects. In addition, the ice–defect interlocking structure induces very high ice adhesion strength, which hiders deicing performance. Moreover, the defect-induced icing was further verified on other surfaces, including steel, glass, and epoxy resin, as shown in Fig. Overall, the defect-inducing icing mechanisms were systemically investigated and clarified. At the defects, higher heat transfer rates and absolute value of PaAFP contain specific spatial arrangement of hydroxyl and methyl groups, whereas NIBS consist of charged groups [1H nuclear magnetic resonance (NMR) spectrum characterization of PDnSBm, and Fig. Natural AFPs can effectively control ice formation as a result of their Janus effect ,35. The d groups ,30. HereadE of ice/water molecules on PDMS, PSBMA, and PDSB. In addition, the copolymers were simplified to PDMS2, PSBMA2, and PD1SB1 shown in Fig. adE of ice and water molecules on a PDMS2 chain was 1.25 and 0.72 eV, respectively, indicating the higher energy stability of ice crystals on PDMS2 than that of water on it. An IBS-mimic mechanism, in which the spacing between the O atoms in the Si–O–Si chain (2.659 Å) well matched the crystal lattice spacing of ice (2.698 Å), was proposed  segments can bind to water molecules through their ionic solvation effects and H bonds in molecular dynamics (MD) simulation results, thus reducing the water freezing point and inhibiting ice nucleation  was −29.4 °C, which is much lower than that of a 100-mg/ml PSBMA solution (−23.4 °C) and pure water (−18.2 °C), demonstrating the ability of AFP mimetics to inhibit ice nucleation. Figure Next, the antifreeze properties of PDSB, i.e., its ability to inhibit ice nucleation, crystal growth, and recrystallization, were systemically investigated. As shown in Fig. lution −2.4 °C andlution −2.4 °C andPDSB with an optimal PDMS/PSB ratio was added into a silicone-based supramolecular polymeric (PSIB) matrix to develop a self-healing anti-icing coating. Four ratios of PSIB-PDSB coatings were prepared and the proportions of each coating are shown in Table −2) of the Si-based matrix provided the coating with a low ice adhesion strength  into the PSIB matrix, HT decreased to −29.4 °C, which is twice that on the uncoated steel, indicating that ice nucleation on the AFP-inspired coating was markedly inhibited (Movie 2/s)  Movie . The resNext, the deicing performance of the coatings was evaluated. The ice shear strength was measured to be 903.6 kPa Fig. F and 155The self-healing performance of the PPSB15 anti-icing coating was investigated at room temperature and −20 °C. As shown in Fig. HT of the damaged PPSB15 coating surface increased from −29.4 °C to −15.9 °C. After healing for 12 and 24 h at freezing temperature, HT decreased to −19.5 °C and −25.0 °C, respectively. The surface scratch was shallow and then disappeared, indicating the restored ability of the coating to inhibit ice nucleation. Figure HT (71.2%), icing delay time (63.8%), and ice adhesion strength (74.6%), after self-healing at −20 °C, indicating that the healed coating retained an anti-icing/deicing performance. The antifreeze self-healing of this coating contributed to the synergistic interaction of multiple dynamic bonds and lower glass transition temperature (gT). Thereinto, the lower S–S bond dissociation energy  of disulfide metathesis could efficiently improve the healing process and shorten the healing time at low temperature [In addition, the anti-icing and deicing properties of the healed PPSB15 coating were investigated. Figure perature .Outdoor anti-icing coatings would inevitably suffer from various complex environmental conditions such as ultraviolet irradiation, temperature variation, sandstorms, and acid rain erosion. Although natural AFPs exhibit superior antifreeze performance, they are more unstable than the AFP-mimic anti-icing coating when subjected to open air. These AFP-mimic coatings were subjected to various extreme conditions, including high temperature (100 °C), ultralow temperature (−196 °C), strong acid (pH = 0)/alkali (pH = 14), sand scouring, and ultraviolet exposure for 7 days. The surface morphology Fig. A and antAs shown in Fig. adE of water molecules and accelerate heat transfer rate from the surface to air/water, resulting in preferential ice nucleation and propagation at the defects. Based on the understanding of key antifreeze sites in natural AFPs, a self-healing (at −20 °C) AFP-mimic-based anti-icing coating was developed. The coating exhibited outstanding anti-icing and deicing performance through the inhibition of ice nucleation, prevention of ice propagation, and reduction in ice adhesion, attributed to the intrinsic antifreeze properties of substrate materials instead of the commonly used artificial surface microstructure. Moreover, the coating exhibited a high resistance to various extreme conditions and an ability to operate outdoors. This work provides a high-efficiency, large-scale, and self-healing anti-icing coating for infrastructures in outdoor complex environments.In summary, a bioinspired self-healing anti-icing coating was developed to inhibit the surface-defect-induced icing processes. Defects can increase the absolute value of


We describe the various stimuli that can be used to switch screw sense, and explore the way that propagation of the resulting conformational preference in a well-defined helical molecule allows screw sense to control chemical events remote from a source of information. We describe the operation of these conformational switches in the membrane phase, and outline the progress that has been made towards using conformational switching to communicate between the exterior and interior of a phospholipid vesicle.Biological systems have evolved a number of different strategies to communicate information on the molecular scale. Among these, the propagation of conformational change is among the most important, being the means by which G-protein coupled receptors (GPCRs) use extracellular signals to modulate intracellular processes, and the way that opsin proteins translate light signals into nerve impulses. The developing field of foldamer chemistry has allowed chemists to employ conformationally well-defined synthetic structures likewise to mediate information transfer, making use of mechanisms that are not found in biological contexts. In this review, we discuss the use of switchable screw-sense preference as a communication mechanism. We discuss the requirements for functional communication devices, and show how dynamic helical foldamers derived from the achiral monomers such as α-aminoisobutyric acid (Aib) and  Helical oligomers devoid of configurational chirality can communicate information when an externally induced screw-sense preference propagates through the chain. Numerous chemical subtleties likewise make G protein-coupled receptors (GPCRs) highly effective communication devices.20 The identification of these subtleties21–23 has allowed chemists to use concepts from evolved natural systems to help inform the design of artificial communication devices.Each of the principal components in Shannon's theory can take molecular manifestations. Biology has evolved molecular mechanisms that allow complex instructions to be relayed on a variety of scales with negligible signal loss. For example, the membrane-bound receptor rhodopsin enables transmembrane communication and phototransduction.The most pertinent requirement for the design of a molecular communication device is that the medium through which the information is communicated (the channel) must be able to adopt more than one state, but not significantly more than two. A single-state device cannot respond to the input of information; conversely having too many states requires an unfeasible degree of complexity in the receiver if it is to distinguish them reliably.24–26): the states of the channel must be local energy minima. These states must be sufficiently similar in energy that their relative population can be biased in response to a stimulus . The channel must also be dynamic: the minima must be separated by a barrier sufficiently low for communication to occur on practically useful timescales. If these criteria are not satisfied, then the stimulus will not be able to induce communication through the channel, either because the induced state is too high in energy, or because the barrier for interconversion of states is too high.Molecular communication devices typically operate under thermodynamic control  in biaryls or heterobiaryls.30,31 However, to communicate information over long distances, molecules must contain more than just a single, localised stereogenic centre or axis, and must adopt a single globally defined chiral conformation.Dynamic molecules that racemise or epimerise on an appropriate timescale are numerous: these processes include pyramidal inversion in chiral aminesM) or clockwise  as they propagate along an axis. They also have translational symmetry – in other words they possess a form of ‘linear chirality’, in which the same dissymmetry is expressed uniformly along the length of the helix. A helix which is left-handed at one location is left-handed everywhere, and this makes helices ideal as communication devices for chiral messages.One class of chiral structure fits this requirement uniquely: the helix. Helices are chiral by nature – they twist anticlockwise  α-helical structures. But this type of helix is not useful as a communication channel of the type outlined above: due to the central chirality in the amino acid residues, P and M α-helices of this type are diastereoisomeric conformers. These conformers differ substantially in energy  and so M α-helices are not observed at equilibrium.33 Any attempt to bias this equilibrium would need to overcome this substantial penalty .Removing the chiral centres from a peptide solves this problem: a helix of achiral monomers can feasibly adopt either a left- or a right-handed structure .34 Conce34 It is well-established that Aib oligomers are helical – in fact they adopt not an α-helical, but a 310-helical structure,37 with exactly 3 residues per turn, built up from ten-membered hydrogen-bonded rings between amino acid i and amino acid i + 3. This remote interaction between non-adjacent monomers in the oligomer chain is important for a rather subtle reason, and one that makes many alternative and apparently helical oligomers of achiral monomers unsuitable as conformational communication devices. Before exploring poly-Aib in more detail, it is worth exploring first why it functions so well as a conformationally responsive structure, in the way that some urea-linked oligomers of meta-phenylenediamine, which we have also explored in this context, did not.38Poly-Aib suits perfectly as an achiral polymer that adopts a chiral ground-state conformation.P and M monomer junctions in equal measure, making the oligomer a random coil s,41 the attractive π-stacking interactions in Huc's helical oligomeric aromatic amides,42–45 the attractive i to i + 3 hydrogen-bonding interactions in Aib oligomers,46 as well as in polymers organised by dipole–dipole or π–π-stacking interactions such as polyisocyanates and polyacetylenes.††I to i + 1 interactions).123 This translated throughout the whole structure, with each pair of axes exhibiting exclusively oppositely oriented amides. By introducing a symmetry-breaking chiral influence on one end, it was possible to induce a global conformational preference in favour of one of the pseudoenantiomers. A Grignard addition was performed at different points along the oligomeric structure giving up to 95% diastereoselectivity as a result of the remote chiral information.We also reported some oligo structures in which dipolar repulsion between the amides induced a single global conformation in extended structures where adjacent axes were forced to adopt the opposite handedness to each other .49 Moreover, Aib oligomers interconvert between these two enantiomeric conformations on the millisecond timescale . As the two conformations populated by an oligo-Aib chain are enantiomers, they will each interact differently with a chiral stimulus differently (13C NMR (or other nuclei) spectroscopy can be used to calculate the degree of screw-sense preference or ‘helical excess’ at any point in the helix by examining the anisochronicity  between pairs of diastereotopic methyl groups in any residue of an Aib helix. A local chiral stimulus can thus be used as a local informational input with a global conformational consequence, allowing the helix to provide a mechanism for molecular communication.Importantly, the situation is different again if a monomer experiences an interaction – either attractive or repulsive – with more remote components of the oligomer. In such a case, a dramatically reduced degree of conformational promiscuity emerges. at arise  are illuigoureas 38,48 ands of Aib .46 The fferently . The potl-α-methylvaline residues s (PQXs).stimuli,  especialR)-2-octyloxymethyl substituents revealed a dramatic dependence of screw-sense preference on solvent.60 Polymers in d8-THF (P helix) and in 80 : 20 d3-1,1,2-TCE : d8-THF mixture (M helix) exhibited equal and opposite Cotton effects in their circular dichroism (CD) spectra, indicating a quantitative inversion of screw-sense preference.Small-angle neutron scattering (SANS) experiments on 100 mer PQXs bearing (R)-2-octyloxymethyl groups well, allowing them to adopt an approximately linear conformation extending out into the solvent, and orienting the quinoxaline-2,3-diyl units in the main chain so they adopted a P-helical conformation.With the aid of computational simulation, the SANS data recorded in these mixtures indicated that THF solvates the (R)-2-octyloxymethyl groups in order to mitigate their interactions with solvent and maximise inter-chain van der Waals interactions. This compacted conformation reoriented the quinoxaline-2,3-diyl units in the main chain into an M-helical conformation, and solvation-dependent screw-sense preference was used to modulate the conformation of PQXs bearing a variety of chiral groups by exploiting very subtle solvent effects. For example, substituted lactate side chains resulted in a switch in screw-sense preference even between similar ethereal solvents 1,2-dimethoxyethane and tert-butyl methyl ether.61 Judicious selection of chiral substituents led to PQXs that could discriminate between a single methylene unit in the solvent, giving rise to a screw-sense preference switch between n-heptane and n-octane.62 Screw-sense preference was also induced by using configurationally achiral PQXs in the chiral solvent limonene.63In contrast, an 80 : 20 1,1,2-TCE : THF mixture induced a more compact conformation in the s adorned with axially chiral 2,2′-bis(methoxymethoxy)biphenyls display helical chirality. Maeda and co-workers discovered that the screw-sense can be quantitatively induced by treatment with chiral secondary alcohols.69–71 Monomers bearing no chiral centres form racemic helices, lending themselves to screw-sense induction by external stimuli. These racemic helices interconvert through a barrier that varies with oligomer length and with solvent.72,73 This feature was exploited by using a chiral group to promote one screw sense in a hydrophilic foldamer in organic solvent, where helical interconversion was fast, then kinetically trapping that screw sense by dissolving the foldamer in water, where helical interconversion was slow -Phenyl ethers induced no screw-sense preference, but an (R)-benzylic morpholine substituent gave rise to an almost exclusive preference for M helicity in 3 : 1 CD3CN : CDCl3, d3-MeOH and d6-DMSO. This was increased to complete M-selectivity by protonation of the quinolines. Quantitative screw-sense induction also arose in hydrophilic pentamers in water. These examples make it clear that environment can alter the dynamics of screw-sense reversal, and may become key to the functioning of molecular communication devices.Hydrophobic pentamers with enantioenriched benzylic morpholines and phenyl ethers at the C terminus were synthesised. -hexylisocyanate, the motor induces a screw-sense preference (trans olefin), its influence on the polyisocyanate is greatly reduced, giving rise to a helically racemic polyisocyanate, as inferred by CD spectroscopy.Conformational switching induced from a single localised controller has been reported by Feringa and co-workers,eference . When thtrans form (λ = 365 nm) at −20 °C gave a cis alkene with a metastable local P configuration  complexes formed depending on the helical chirality in the motor. In the trans forms, intramolecular complexes are impossible, and strands from different molecules became interwoven to form aggregates. This example again constitutes a three-state switch where screw-sense preference could be selected, but additionally, one of the states triggers a reversible supramolecular polymerisation event.This work was extended to the control of metal-coordinating double helices by appendage of an oligo onto each half of a 80–82 An otherwise configurationally achiral 310-helical oligomer with an embedded l-phenylalanine residue in provided stimulus-responsive control of screw-sense preference.83 The embedded l-phenylalanine was sufficient to impart a screw-sense preference to the whole helix, which augmented with decreasing temperature. The screw-sense preference of the helix could however be inverted by binding a Boc-d-proline ligand non-covalently at the N terminus of the helix, with the influence of the ligand on the helix conformation modulated by altering the concentration and temperature of the system -β--α,β-didehydroalanine as a diagnostic spectroscopic reporter with a distinctive chromophore. As expected, no Cotton effect was observed from the achiral oligopeptide alone, but addition of Boc-d-proline effected a CD response at 320 nm, indicating that the biphenyl groups near the C terminus find themselves in a locally chiral environment, despite being over 3 nm away from the binding event providing the chiral input. Chiral information can thus be read at the N terminus of a 310-helical oligopeptide, propagate down the length of the helix, and be reported at the C terminus.The first example of informational communication by spatial translation of screw-sense preference was published by Inai and co-workers, who extended their work on the use of Boc-13C NMR spectroscopy using isotopically enriched residues located remote from the informational input. Screw-sense switching results from diverse signals including binding chiral 1,2-diols,92 enantiospecific Mitsunobu reactions,93 and association of chiral acids with basic N-terminal residues.94 These examples show that a local, switchable conformational signal can induce a remote conformational response, and in a sense are conceptual models of biological communication devices such as GPCRs. However, two key differences remain: (a) biological communication devices pass information between compartments, through membranes; and (b) the ‘output’ of the communication is a change in (bio)chemical function. In order to advance the field of molecular communication devices, artificial systems must likewise be able to perform remote function as a result of informational input. However, to perform function as a result of screw-sense induction, it is crucial to understand the processes underlying the communication event and therefore how the information can be amplified.Our own group have developed a variety of methods for switching helicity in chemically robust Aib oligomers, with the induced screw-sense preference quantified by 47,95 and provide valuable devices for the control of shape, function and communication of information. Important examples of molecules that can change shape and therefore function in response to a stimulus include the modulation or induction of helicity in oligo(azobenzene)s,96,97 2,6-bis(N-imidazolidin-2-onyl)pyridines,98 and aromatic oligoamide foldamers.99Helical foldamers occupy a vast space of chemical architectures and can be used to arrange specific chemical functionality in a precise manner,10-helical Aib foldamers, in which the decay of screw-sense preference with distance from a chiral influence turns out to be solvent-dependent.100 We investigated the origin of the signal decay and proposed it was caused by the stochastic intrusion of ‘tendril perversions’, where breaks in hydrogen bonding occur randomly along the length of the helix and result in a helical reversal at that point.101Effective communication over long distances requires a conformational signal to persist as it propagates through the oligomer. This effect been explored in detail in 3In order to study this phenomenon in Aib helices, a set of Aib foldamers were terminated at each end with two α-methylvaline residues. These residues induced at one end a right-handed helix, and at the other a left-handed helix, enforcing a tendril perversion somewhere in the chain . For comP screw sense along the whole length of the ‘matched’ oligomer. However, the ‘mismatched’ oligomer contained discrete P-helical and M-helical segments. A P helix propagated from the N terminus to the third Aib residue (residue 5), with a ‘tendril perversion’ at the fourth Aib residue, where the amide proton hydrogen bonded to a P helix, and carbonyl oxygen hydrogen bonded to an M helix  closest to the N terminus, decaying through zero to 30% M between the fourth and fifth Aib residues (residues 6 and 7). These findings were attributed to the presence of a single tendril perversion located between residues 5 and 7 . We have described an informational communication system incorporating an Aib oligomer that switches between conformational states by changes in pH.94 A key feature of the oligomer was a basic binding site which could relay information about different Brønsted acids with different pKa values, depending on the pH of the medium. We also extended this work to an Aib foldamer that uses an N,N′-disubstituted urea as a hydrogen-bonding binding site for the recognition of geometrically complementary chiral hydrogen-bond acceptors  on the screw-sense preference of the Aib foldamer could be turned on and off by using the different geometries of the alkene chromophore to modulate their proximity , bringing the l-α-methylvaline closer to the Aib oligomer, meaning it was able to influence the screw-sense preference of the helix. Forming a reactive oxazolone on the opposite end of the helix allowed a light-promoted diastereoselective chain extension: with the (E) fumaramide, chain extension by the methyl ester of racemic valine resulted in a 50 : 50 mixture of diastereomers, but with the (Z) maleamide a diastereoselective reaction resulted in the preferential incorporation of l-valine (74 : 26 dr). This photoswitch serves as an example of screw-sense induction where a chiral influence inherent to the foldamer is modulated by light – an achiral stimulus. This development in intramolecular communication in Aib foldamers represents a significant advance as it greatly expands the range of stimuli that can be used for informational communication. It is also worth noting that the output in this example is a kinetically controlled reaction, in which selectivity may depend rather subtly on the populations and relative rate of reaction of the two screw senses, along with the rate of their interconversion in accordance with the Curtin-Hammett principle.104The need to use a chiral stimulus represents a significant limitation on the types of signal inputs that can be used to those that can exhibit some form of chirality. Many useful stimuli such as light (except circularly polarised light), heat and oxidation state are inherently achiral. Although Aib helices communicate information in the form of screw-sense preference, informational input does not necessarily need to be of a chiral nature – for example, a chiral input can be inherent to the foldamer and its influence on screw-sense preference can be modulated by an achiral stimulus as discussed previously.roximity . In the The approach discussed here is general among communication devices, in which an appropriate receiver adopts a stimulus-dependent state and communicates that state to the channel. In this example, light serves as the stimulus and the chiral fumaramide/maleamide receiver converts the light signal into screw-sense induction.10 helix, Aib foldamers can be characterised as a hydrophobic tube surrounding the twisting polar chains of hydrogen bonds within.105 As a consequence, both Aib foldamers and their parent peptaibols dissolve readily into phospholipid membranes. This has allowed them to function as communication devices within the membrane phase, allowing chiral information to be communicated from the exterior to the interior of a membrane-bound vesicle or cell.With repeating geminal dimethyl substituents directed towards the exterior of the 3106 The polarity of chloroform is similar to that experienced in a phospholipid bilayer, so these studies reveal the influence of the membrane environment on the conformational preference of the foldamers. VCD spectra indicated that the relevant 310 helix VCD signals in the amide I region became more intense in the vesicles for a Cbz-protected l-phenylalanine-capped Aib tetramer, suggesting that the bilayer environment either increases foldamer rigidity or increases the proportion of 310-helical conformers. The enantiomeric d-phenylalanine-capped Aib tetramer gave a spectrum of the opposite sign, but with some significant differences in the amide I region, indicating that the enantioenriched environment of the phospholipid bilayer has a weak but measurable effect on the screw-sense preference of the 310 helix. Additionally, in both enantiomers, several new bands appeared in the amide I region, corresponding to some α-helical character and different forms of bends and turns. Comparison of the signs of the spectra indicate that the screw-sense preference induced in the solution phase is the same as that induced in bilayers. Crucially, this study shows that 310-helical content is persistent in phospholipid bilayers, meaning that Aib-derived foldamers could serve as transmembrane communication devices that can respond to external stimuli.106Raman Optical Activity (ROA) and Vibrational Circular Dichroism (VCD) analysis was carried out in both chloroform and in dioleoylphosphatidylcholine (DOPC)-derived phospholipid bilayer vesicles.8 foldamer  group was suitable reporter of screw-sense preference: both solution and solid-state 19F NMR spectroscopy gave well resolved spectra that could be used to analyse screw-sense preference without the overlapping signals that would necessarily arise from other nuclei. A fluorine-substituted azobenzene group allowed the same 19F spectrum to report on azobenzene geometry.Screw-sense modulation as a result of a configurational change induced by light was translated into the membrane phase by ligation of a chiral photosensitive moiety to the N terminus of an Aibfoldamer .107 An eE configuration, the lone pair of electrons on the proximal diazo nitrogen forms a hydrogen bond with the amide proton of the l-valine. This hydrogen bond was weak, and therefore the l-valine's influence on the screw sense of the foldamer was largely unaffected, giving rise to a left-handed preference in the bilayer. Irradiation with light at 365 nm converted the azobenzene mostly to the corresponding Z configuration (18 : 82 E : Z). As a result, to mitigate the steric encumbrance associated with coplanarity of the arenes, the diazo group is distorted, making the diazo nitrogen more basic, strengthening the hydrogen bond to the adjacent l-valine amide proton, and weakening the l-valine's influence on the Aib foldamer. A racemic mixture of helical conformations results , leading to a detectable remote effect .108 Conceptually, the design constitutes a mimic of the GPCR communication mechanism: binding of a ligand resulted in a conformational change which propagated within a lipid bilayer.Other biomimetic functions in a membrane requires intermolecular interactions that are sufficiently strong to persist even in an aqueous environment. In order to strengthen these interactions, we made use of a metal cation bound within a bis(2-quinolylmethyl)(2-pyridylmethyl)amine (BQPA) pocket, that was also capable of binding anionic ligands such as chiral carboxylates. We used this anion-responsive binding site to transmit chiral information in a DOPC-derived vesicle in the form of an induced screw-sense preference.8 foldamer and bound to a Cu(ii) ‘cofactor’, which itself interacts with enantioenriched amino acid-derived carboxylate ligands. With NMR spectroscopy methods being unsuitable for use in vesicles, a chiral bis(pyrene) fluorophore was developed and used to terminate the Aib8 foldamer. Being chiral, the fluorophore interacted differently with the right- and left-handed screw senses of the Aib helix, meaning that fluorescent responses were dependent on the screw-sense preference of the helix. This bis(pyrene) reporter was carefully selected to ensure that the interaction between the chiral bis(pyrene) and the foldamer was too weak to bias screw-sense preference, but strong enough to respond to a conformational change induced from the N terminus by reorientating the two pyrene rings.109 In the absence of ligand for the Cu(ii), left- and right-handed helices were populated approximately equally.The detailed design of the receptor mimic is shown in ii)-bound foldamer embedded in the DOPC phospholipid bilayer of unilamellar vesicles, the ratio of excimer (E) to monomeric (M) contributions to the fluorescence spectrum could be perturbed by binding of carboxylate ligands. Boc-l-proline was bound to the Cu(ii)-BQPA in the bilayer, inducing a right-handed helix, causing the two pyrenes to move away from each other and decreasing the E/M ratio -BQPA, inducing reversible and opposite fluorescent responses depending on the relative concentrations of leu-enkephalin and Boc-l-proline.With the Cu(/M ratio . With Boii) cofactor, and relays the chiral information through the bilayer to deliver a remote fluorescence response over 2.6 nm away, as a result of conformational communication through the Aib foldamer.This functional GPCR mimic thus binds chiral ligands at a binding site incorporating a Cu, so this high barrier was attributed to rate-limiting hydrogen bond reorganisation. Interestingly, helical inversion is accompanied by a hydrogen-bond directionality reversal and therefore a switch of the N (i.e. hydrogen-bond donating) and C (hydrogen-bond accepting) termini.Guichard and co-workers pioneered the development of urea-derived helical foldamers, which have found use as asymmetric catalysts, peptidomimetics and as bactericides.ad of it . As thes118 For example, in the pentaurea, functionalisation of one terminus with tert-butoxycarbonyl and the other with allyloxycarbonyl gives rise to a 2.1 : 1 ratio of P : M helices in CD3CN at 25 °C, as shown by 1H NMR and CD spectroscopy  tert-butyl carbamate preferring the N terminus and the allyl carbamate the C terminus in the major conformer. Modifications of the termini allowed modulation of the helical excess in these oligourea foldamers, with competitive hydrogen-bond donation and acceptance of the termini making the helices torsion balances for hydrogen-bond strength. Thioureas are much stronger hydrogen-bond donors than their homologous ureas.119 With an N-benzyl thiourea at one terminus and an N-benzyl urea at the other, a 10 : 90 ratio of P : M helices was formed. Replacing the urea with a carbamate, a poorer hydrogen-bond acceptor, inverted the ratio to >95 : 5 P : M.This bias arises from the differing hydrogen-bonding properties of N-Boc-prolinate is a multiple hydrogen-bond acceptor through the carbamate and carboxylate, and is of a suitable geometry to form four hydrogen bonds with the first two ureas of a meso oligourea helix SANSAmall-angle neutron scatteringCDCircular dichroismTCE1,1,2,2-TetrachloroethaneHPLCHigh-performance liquid chromatographyDMSODimethylsulfoxideNMRNuclear magnetic resonanceBoctert-ButyloxycarbonyldrDiastereoisomeric ratioROARaman optical activityVCDVibrational circular dichroismDOPCDioleoylphosphatidylcholineBQPABis(2-quinolylmethyl)(2-pyridylmethyl)amineTBAPTetrabutylammonium phosphateThe authors declare no conflict of interest.


Handwashing with soap is a widely advocated public health measure, but seldom practiced, partly because it is often difficult  to make both soap and water readily available in relevant situations. This study used both Behaviour Centred Design and Human Centred Design to guide development of a novel hand cleansing technology appropriate for the context of post-toilet hand cleansing in resource-poor societies. Extensive prototyping and field testing resulted in the pilot production of ‘tab’ soap, a small but durable single-use, decomposable substrate embedded with soap. It can be produced in dispenser roll or tear-off formats. With this affordable solution, one may use soap without worrying about contamination pretty much anytime and anywhere. A small-scale field test showed that all poor households in rural and peri-urban areas in Tanzania included in the proof-of-concept study (N = 12 households) would use the product reliably over the medium term. Tab soap awaits full-scale production and marketing but could make hand cleansing a more popular practice around the world. However, few studies suggest that these innovations have created lasting effects on consistent handwashing behaviour. Recently developed handwashing facilities have highlighted the importance of working with design professionals and employing several rounds of iterative prototyping and testing to develop functional and desirable options for handwashing. Examples include the ‘Povu Poa’ (‘Cool Foam’) handwashing station in Kenya (http://demandasme.org/cool-foam/), and the ‘Happy Tap’ project in Vietnam: (http://www.wsp.org/sites/wsp.org/files/publications/WSP_Designing_Handwashing_Station_HWWS.pdf). However, they have not proven themselves sustainable at scale . Handwashing with soap has long been known as a highly effective means of preventing diarrhoea, especially among the young  will use it for drinking water’. Other households made similar comments around the bucket being attractive for drinking, laundry, washing dishes or other activities .The Spatap was one of the least popular solutions as the unit was perceived to have too small a capacity for hand washing (MG3). A sense that the rubber would be easy to break (MG3), despite being made from the most durable material was also mentioned; perceptions of durability were linked to the potential to purchase. A positive comment was that the water was enclosed so that it would not be contaminated by the air in the toilet (DSR4).The Supertowel was met with comments such as ‘If it is true, it simplifies things’ (MWS14) and ‘Its fake’ (MWR12) when respondents were prompted for appraisal, indicating a tricky mental model. The fact that the Supertowel’s immediate use was not apparent was an issue. As there was no comparable product, the participants suggested different benefits: MG1 would share it with the entire family whereas the son at MWS14 said he would take it when travelling on long journeys and use it to wipe sweat from his head, not for handwashing. It was seen as desirable for being a modern technology.The Ease of use for handwashing with soap meant having a reliable system that facilitated the current mental model that handwashing is a two-handed job. This means solutions that are better allow for ‘proper’ hand washing which ‘requires two hands’ (MG4). Presence of soap  acted as a reminder. Refilling often is not convenient, the Happy Tap’s large storage capacity in this respect was seen as a benefit.One of the key benefits of the ranking exercises and using existing products for interviews was that it required participants to verbally relay their initial interpretations of the objects presented to them. The aforementioned Supertowel, while an excellent and well-received product was hard to understand. The Sanitap unit had a similar issue: being a novel product made it hard for participants to know or imagine what it could be used for. However, the Sanitap solved this issue by having a clear set of pictographic instructions on the back. The Happy Tap was particularly successful in this regard as it includes a large bay that is clearly used for a bar of soap.Another issue, common to the Sanitap, Oxfam bucket, and Spatap, were that respondents said that they would use the units for storing drinking water or food, not handwashing:“[I] can use Sanitap for drinking as long as it doesn’t go in the toilet” (MG1)Oxfam bucket would be used for storing drinking water (MG1)Spatap would use for drinking (MG2) and showering (DSR4)This is linked to a key observation that handwashing is the lowest priority usage of water and soap, meaning that products will simply be used for other activities. Most households had either no soap or dry slivers present in the toilet indicating that there were no soap products for handwashing. The buckets for cleansing were usually old storage buckets that were close to breaking point.From the daily thematic analysis sessions, the team highlighted 75 different topics through the lens of handwashing and the products, services and systems that facilitated the behaviour. Themes evolved over time. For instance, as some topics emerged previously, but became clarified, the team would shift focus to areas that were less well understood. Each theme highlighted a potential design priority, a topic to address when generating ideas and solutions. The most pertinent themes and observations are addressed below.All participants expressed knowledge or understanding that one should wash their hands with soap after defecation. Throughout the household visits, the team enquired as to how families sourced water for various activities  and the different soaps used for each. Clean water would be kept for drinking, cooking and potentially clothes washing. Water for handwashing was commonly the same water used for flushing and wiping in the toilet. Soap usage had a similar linear downward trend, with fresh bars of soap being reserved for body washing, then clothes and soap slivers sometimes being used for handwashing. While these were often kept in the toilet, it was clear that many were hardly used once they became so small with participants sometimes not knowing the slivers were in the toilet. Liquid soap was generally used for dishwashing or cleaning a floor but was less commonly found in the homes. Powder soap was used for washing clothes.A sense of contamination was an evident barrier to handwashing with soap and took on many different dimensions throughout all visits. The target of contamination included just about anything in the toilet area . Interestingly, in the case of soap, even an object meant to be clean and to clean dirty things it comes in contact with, was perceived to be contaminated. The sense of contamination around sharing soap and the usage of water presented two very clear design challenges in being able to keep products near the toilet while assuring individuals that no cross contamination occurs.The London-based ‘design sprint’ workshop generated more than 150 ideas from ‘How might we’ statements and other provocations, captured using post-it notes see .A variety of clustering and expert ranking exercises during the Design Sprint Workshop reduced the plethora of initial ideas down to 11. Clustering activities included consideration of the type of motive(s) embodied in the intervention and the type of intervention made . Ranking considered opinion around technical feasibility, a viable business model and some intuition around how desirable the idea would be or how likely it would be to work (i.e. influence behaviour).A focus group in Tanzania run immediately following the idea generation helped to make it clear which of the 11 ideas should be further developed. Subsequent concept development and selection stages are described below in The second field visit utilised prototypes  of the fHouseholds were also asked to rank the five concepts based on various desirability indicators  and likeThe choice between both design approaches is non-trivial as selling specialised products is less feasible (the target demographic observed in our research showed that they primarily owned multi-function products). The idea of selling a multipurpose product that has nudges to encourage hand washing with soap seems like an easier product to sell and was reflected in the HH rankings see  where thThe most pertinent points of comparison for each prototype are listed below see .sine qua non of a behaviour change project.An ideal process would afford further exploration of all concepts to address shortcomings and test more broadly. However, resources and time constraints required that we make a selection based on expert intuition. The Tab Soap was selected on this basis, as well as the fact that it had been the only prototype that had been regularly used by the testing household, since this criterion was the Sabom (Swahili for soap) is soap for handwashing that gives you a fresh piece of soap every time you use it, even in a shared context. This contrasts with the common bar of soap found in the toilet which is seen as disgusting.The final design of tab soap was articulated according to unique selling propositions which would later be further tested in a proof-of-concept study. This was articulated as follows: For low-income residents who want to leave cholera behind and become modern, Key unique selling propositions:A soap dispensing solution that delivers individual soap tabsEach serving can be easily transported from dispenser unit to location of useEach serving can be temporarily stored and carried and it remains durableThe servings have just enough soap for handwashing, making it hard to use them for other activities, a core insight was that if soap can be used for other activities then it will always sideline handwashing with soapThe dispenser unit holds enough to serve a family for 1 week (preferable for habit formation)Units can be sold on an individual basis, allowing for flexible price points and quantities to help sellers find the most suitable price point to encourage customers with reduced means to start using the productThe soap can’t be used easily for other purposes, so when you give them to your family you know what they’re doingThe form factor allows for significant behaviour change oriented messagingSeveral form factors were considered and two were developed for testing. The Tab Soap prototypes needed to be turned into working models for use in a field trial for evaluation of the product’s real utility and value. The main criteria feeding the finalization of the product design are listed in The final design was refined based on the above and formalised with a product design specification–a document used by designers that specifies different user requirements and links them to specific features to be created. In line with the development of a hybrid design and behavioural approach, the behavioural determinants from the Assess stage were used to prioritise different features. A Theory of Change developed for the products at the prototype phase was also used to compare and highlight important design features for the team.During the detail design phase, material selection was a key step as it has a direct impact on the manufacturing process with direct consequences onto the feasibility trial size. The process began with the sourcing of numerous alternative candidate materials for the final design. They were then tested and selected based on a balance of user experience and manufacturing speed. The manufacturing process was designed taking into account the potential tools and workforce available in Tanzania. When running small production runs for feasibility trials, access to realistic commercial materials is not always feasible; this means that compromise materials are sometimes selected.Given that the final product involves tabs of soap with a substrate of material, use of the product will produce small pieces of rubbish. A biodegradable material was chosen which decomposes in the pit latrines common to this type of environment. The material chosen was a bamboo-based textile which could be impregnated with various types of soap. Production of test samples of Tab Soap involved ad hoc manual methods due to the small scale of the trial see .The two final designs see  for the In the proof-of-concept test, households were given the roll units. This decision reflects a core objective of seeing if the product would create behaviour change. The main interest was then around understanding if handwashing would increase with access to the product. While a detailed exploration of the business model is beyond the scope of this work, this was tested slightly through two avenues. First, the households were asked if they would buy the roll after they had used the product. This attempted to simulate a free sample model after which one could continue with purchases. Second, the tear-and-share tabs , right wSeveral kinds of tests were initiated to evaluate the reception and use of the final design, tab soap, in Tanzanian households from Morogoro (MG) and Dar es Salaam (DS). For instance, a household from Morogo will be marked as MGHH and the number of the participating household.Primary among these was an in-home trial. Feedback from the trial was generally very good. Everyone liked the soap: it was pleasant on the skin but did not smell too much, it made a lot of foam, and washed off easily with little water. ‘It cleans the hands well and it does not leave it with strong odour that will keep disturbing you later.’ [MGHH9]The tabs were liked as well, and the novelty was generally seen in a positive way: ‘All [my visitors were] amazed and liked it. Paper turning into soap is not something we are used to.’ [MGHH4] They liked the portability too: ‘I would put them in my handbag and take a few and put [them] on the table’ [MGHH4]. This act of collecting tabs and putting around the house  was repeated in other households to keep out of reach of children or just to be more convenient.The dispenser was seen as good-looking [MGHH2], and as protecting the soap inside from weather and insects. This led to a consistent and important report of no contamination. ‘Yes, I like it very much. […] The tabs are good and prevent transmission of diseases to others. The tabs are covered and insects can’t lick them so it remains clean and uncontaminated.’ [MGHH2]However, the mechanism did not work perfectly as sometimes households pulled off tabs without the next one coming out, requiring them to open the container up to fetch the next tab. In one case, the internal wheel did not spin well after refilling. [MGHH2] These experiences imply the container may be difficult to manufacture reliably at the necessary price-point. Even some of those with the container thought it might be better to just have tear-and-share.The dispenser was kept in a wide variety of places, both inside and outside the toilet. One informant noted that the soap itself can serve as a stimulus to handwashing: ‘Yes, I like the soap. It reminds me to wash my hands whenever I use the toilet, unlike the bar soap.’ [MGHH3] This provides evidence for the Theory of Change successfully working.Many of the households appear to have used the soap, often quite regularly. Some of the behavioural demonstrations suggested practiced use, others less so (or perhaps nervousness at having to be filmed while doing a behaviour in their toilet). A number of households reported running out of tabs before refills were made available, suggesting regular use. About half of the households claimed that they finished the roll of tabs before soap sellers came back with weekly refills. Expectedly, many of these were from households with a larger number of members which also led to the related smaller number of soap usage per day.No one had difficulty disposing of the substrate, either in the toilet or throwing onto a trash heap after use. Several households had the idea to throw away the substrate before it lathered up too much, controlling the amount of soap they used from each tab (and thus the amount of water needed to wash it off) [Handwash demonstrations].The system was definitely something newsworthy in the neighbourhood. Every household mentioned showing the container to neighbours or visitors. ‘When a visitor goes to the toilet, I wait for them outside because I know they will ask me about the container. Then I teach them how to use it.’ [MGHH2]Users prefer to have relatively large quantities of tabs on hand, as they fear the uncertainty of visitors coming and needing soap, and don’t want to repeatedly have to go somewhere to purchase tabs, which they find annoying. The price is always a worry, but they seem to fear not having soap more. Even though the dispenser was seen as large, some wanted it to be even bigger to hold more tabs. [MGHH2] ‘I always want them [my guests] to use the tabs after using the toilet. I give them a tab use and tell them I can’t put them [the tabs] in the toilet because there is no roof.’ [MGHH4]The fact that the tab soap was designed to be used only for handwashing purposes frustrated some users. ‘No…I will go back to normal soaps because the tabs are not for multi-usage. Other than handwashing, I cannot use them for anything else.’ [MGHH1] Several households mention trying to use multiple tabs to do other jobs, like bathing , washing hair [MGHH7] or clothes [MGHH5], but mostly as a consequence of not having other soap available. The experiences were also not satisfactory.While novel, most indicated that it was easy to learn how to use, once you explained that it was paper that turned into soap. ‘[My elderly] mother taught us. It was okay, it’s pretty simple to use.’[MGHH3]When asked for pricing, sellers mentioned prices considerably above the break-even costs though it is not clear if this is within the household’s budget.Unsurprisingly, the baseline and endline questionnaires did not show a significant change in reported automaticity of behaviour. This is likely due to the novelty of the device and associated behaviour, and potentially changing circumstances of use, as households grew more used to having the product around the house.The soap seller refill was documented through images gathered each week see  and throTab numbers are measured as per person per day for each household (HH). Household numbers across the top correspond to two locations: Dar es Salaam (DS) and Morogoro (MG). The missing numbers reflects the absence of those households who were given regular soap as a means of comparing.The electronic sensor embedded in each device recorded the time of use and the rotation of the device (an indication of quantity used). However, the electronics were subject to dropping, water damage and faulty wires. The sensor also required a battery being changed halfway through the trial. The result was that data collected was limited due to various failures. Specifically, only nine of the twelve devices provided enough data to indicate some form of interaction. Still, the data  corroborThe response from the sellers was broadly positive. Beyond all having tried the tabs themselves, both groups of sellers in Morogoro and Dar Es Salaam agreed that they would be able to sell units and tabs following the trial. All the soap sellers managed to sell soap initially, however only the kiosk in Morogoro kept a repeat customer. The unit price settled to TZS 1500–2000, but these units included quite a few tabs. In Dar Es Salaam tabs were sold instead in smaller units, with one seller selling 4 tabs for TZS100 and the other selling 1 tab for TZS100. The Morogoro soap sellers all agreed that “there was no challenge on the price”.All the sellers mentioned that without marketing materials they could not sell the soap as customers were not used to a product specifically used for handwashing, let alone paying for one. They stated that this would also be the soap company’s (project facilitator’s) responsibility.Through the formative research, it was clear that awareness and know-how was not limiting handwashing behaviour and that different motivational levers would need to be triggered to encourage people to undertake the behaviour. The team also realised that although promoting the use of bar soap for handwash would seem a possible solution as soap was always present in a home, the general-purpose nature of the product meant that it was always being used for something with a perceived higher priority. There was a clear hierarchy of usage in which use soap for body wash would be the top priority and handwashing was the lowest. This indicated that substantial effort would need to be made to create a product that would not be used for other activities as handwashing would always be de-prioritised.Consequently, the team developed several possible technologies based on the findings from the Build phase. The development consisted of theoretical concepts and tools being applied to physical prototypes, together with feedback from use by the target demographic. Through this process, a single intervention with multiple form factors for delivery emerged. The design has both a compelling Theory of Change and a reasonable route for manufacture that could be developed in future work.The Theory of Change argues that tab soap, made available through business processes of production and distribution, would cause access to this new product, which would in turn cause people in target households to purchase and then use the innovation after the toilet both because it was seen as being part of a ‘modern’ lifestyle, and because it made the practice easier to perform, either in terms of cognitive discounting (don’t have to remember as much) and/or physical discounts  .The solution is thus thought to be effective because it is a product-service system. That is, the product itself has important features of cleanliness and novelty that influence perceptions and create a reminder but the refill of the soap supply, e.g. through soap sellers in local communities, can create a reinforcing environment for the behaviour and stronger assurance that the product is available. This combination addresses shortcomings in past interventions which have relied on one or the other only, and therefore have not seen desired results.The largest challenge was that there was no standardisation of context in households, either from the perspective of infrastructure and props or from the standpoint of behavioural routine. The only constant was the diversity in settings. Looking beyond the households at the markets, a similar situation exists. Products are sold at different price points depending on how far they are being sold from the initial distribution point, and the packaging or quantities are continuously modified by sellers according to local factors. These factors made us restrict the brief to in-toilet use, so that some level of standardization of context could be achieved.Prototyping in this case can be seen as two iterations of a divergent-then-convergent process. First, a conceptual prototype of the intervention was documented with a Theory of Change and a sketch model which addresses broad topics. From this, risks (ways in which the intended Theory of Change could fail) were identified and the conceptual model was updated to mitigate these risks. Next, a divergent process in which specific prototypes for testing each part of the Theory of Change and how these link together was undertaken. These prototypes sought to both embody the final form and mitigate risks associated with practical matters  as was as theoretical ones .sine qua non test of behavioural innovation, we are optimistic about the prospects for use in larger populations with potential for behaviour change.The different kinds of evidence from the field trial—interviews with participants, seller documentation, data logs and field worker interviews—together paint a persuasive picture of use (with an average 1.5 handwashes per household per day). The interviews support the case for these tabs being almost always used for handwashing, which was an important dimension of our objective. The sensor data, although not complete due to technical difficulties, does not appear to be biased by household, and indicates regular use throughout the trial period. As this is the The Theory of Change was also generally supported and shows some areas for improvement. The product was used in the intended context, successfully compensating for the effort required to remember to wash one’s hands. Environmental factors such as rain and the potential of theft meant that many households kept it in their home . More might be done to understand how to get the innovation into a household’s toilet. The use of the device within households or in the toilet will also be important to explore in contexts where one owns, rents or shares a toilets, e.g. with other renters or tenants in a compound. The single-use nature of the tabs also meant that there was no cross-contamination and the product was seen as clean. The product was also seen as novel and desirable as evidenced through people showing it off to neighbours.The simplicity of the design means that it can be manufactured at scale with relatively low costs which appear to be in the price band relevant to the target demographic. However, much of the business model testing needs to be explored further in future work. Specifically, an exploration of modes of delivery, the amount people will be willing to pay to purchase the product and how to motivate this purchase is needed. The product, behaviour and data analysis were aided by innovative use of soap sellers, which can be explored further as the business model continues to develop.There were several limitations to the study. The creative process was constricted due to a limitation of resources, including time. First, we developed prototypes in London and made minor modifications in the field. A better approach would be to design and test more prototypes in the relevant situation–in Tanzania. In this work, we also prioritised behaviour change and desirability with an eye to technical feasibility and business model viability. An ideal process would allow us to test all aspects further as several questions remain with respect to feasibility and viability. Surely these should be prioritised in subsequent work.Another limitation was the practical ability to produce test units and measure outcomes. The electronics also worked only for part of the study and offer a useful, if incomplete view of usage. These sensors could be improved to measure use in a better way. There is also an important question about supply and an ability to continue to distribute products in a way that would be compelling to the user to the point that it would generate repeat purchases.We note that all involved in the study are motivated to see a solution brought to market and serve those in need. Several of the authors remain active in exploring options to develop the concept further, which can include charitable or market-driven solutions to create sustainable supply. These development tracks have differing merits and a goal in much of WASH has long been to motivate people enough to elevate the importance of hygiene that they make the purchase where possible such that any solution does not rely on ongoing charitable contributions which can be subject to disruptions. While this motivation to bring a solution to those who will benefit is right in the eyes of the authors, it also has the potential to generate biased results. To address this, we have made an effort to be transparent and thorough in our communication so the merits of the intervention can be evaluated by others in terms of both output and development process. As is the case with so many interventions, there may be much learning along the way and in the process from which we hope others will benefit.Finally, the current study did provide compelling early evidence for behavioural change but this iteration of tab soap would not be a solution without a viable and scalable model to bring the soap to many. Indeed, providing free soap which carries novelty to end users is bound to be well received. We did endeavour to create a solution which could be sold at a reasonable price point for the target demographic. However, this does not answer the question of market feasibility. Additional work should explore demand for such a solution particularly around repeat purchasing behaviour and associated use in the home. Within that space an important question is around scarcity and whether the product is abundant enough that it is used regularly as desired or if it is only used periodically.The most significant outcome of this study is that a novel hand cleaning product was developed that people in the target demographic are actually willing to use regularly after the toilet. Tab soap appears to have a number of significant advantages over competitor technologies for hand cleansing in this kind of use context:Bar and liquid soaps are perceived as cross-contaminating, which makes these solutions disgusting, whereas tab soap is single-use, so one person cannot be put at risk by another’s use of the product.Unlike nearly all other forms of soap, it is designed to only be used for washing hands; it is difficult to misuse or use for other purposes.Its price-point per use is comparable to alternatives such as a liquid soap bottle push or bar soap rub.You can carry a single tab with you like sanitizer, for use anywhere there is water.Its use probably requires less water than other forms of soap as the substrate holds water throughout use, lubricating the washing process.Unlike soap leaves, ‘singles’ are easier to manipulate and carry, more robust to water and physical damage or degradation, and have post-use value in other contexts.Families in our trial found a number of innovative ways to store and use the product, pointing to its adaptability as a solution to this difficult behavioural problem. For example, one mother removed a number of tabs at once from her dispenser and gave them to her children to take to school in their backpacks, for use during the day. The containers which were not affixed to a toilet wall were kept in many different locations around the toilet or in the house, suggesting use in more contexts than just post-defecation.Given that researchers and marketers have long sought to remove the barriers to hand washing after potential contact with faeces, often with little success, this is a welcome achievement. Admittedly, the pilot was small-scale and not of truly long duration, and users were not required to purchase the soap, so further work remains to be done to establish a working business model for this new product. Nevertheless, this is a result that has not previously been observed, and so holds out considerable hope for further success with additional testing. Given the importance of hand hygiene in many public health contexts, including pandemic control, we believe this result should attract widespread interest.S1 FileData the 9 households where sensors recorded each time the device was used for at least part of the trial period.(XLSX)Click here for additional data file.