Periodic patterning is widespread in development and can be modelled by reaction-diffusion (RD) processes. However, minimal two-component RD descriptions are vastly simpler than the multi-molecular events that actually occur and are often hard to relate to real interactions measured experimentally. Addressing these issues, we investigated the periodic striped patterning of the rugae (transverse ridges) in the mammalian oral palate, focusing on multiple previously implicated pathways: FGF, Hh, Wnt and BMP. For each, we experimentally identified spatial patterns of activity and distinct responses of the system to inhibition. Through numerical and analytical approaches, we were able to constrain substantially the number of network structures consistent with the data. Determination of the dynamics of pattern appearance further revealed its initiation by ‘activators’ FGF and Wnt, and ‘inhibitor’ Hh, whereas BMP and mesenchyme-specific-FGF signalling were incorporated once stripes were formed. This further limited the number of possible networks. Experimental constraint thus limited the number of possible minimal networks to 154, just 0.004% of the number of possible diffusion-driven instability networks. Together, these studies articulate the principles of multi-morphogen RD patterning and demonstrate the utility of perturbation analysis for constraining RD systems.The people behind the papers’ interview.This article has an associated ‘ Highlighted Article: Going beyond classic two-component Turing Reaction-Diffusion, this study uses inhibitors, modelling and developmental dynamics to define the FGF-Hh-Wnt-BMP network driving striped rugal patterning in the oral palate. The generation of anatomy by self-organisation remains one of the most important subjects in the study of biology. It has acquired new importance as a guiding feature of regenerative medicine and the modelling of disease processes by the creation of self-organising organoids from stem cells . SettingOne aspect of self-organisation that has been set aside hitherto is that, as we now know, large proportions of the genome and proteome are devoted to regulation. Consequently, minimal systems must be expanded if they are to capture this complexity where it is functionally relevant. The extension of RD systems to include multiple morphogens and even non-diffusible components  opens upTaking this level as our motivating principle [while acknowledging that it is a first approximation (see Discussion)], we contemplate how far one can get in defining a model (i.e. a network topology) of key interactions that can capture the behaviour of the system, when considering multiple signalling pathways. We analyse the periodic pattern that generates the transverse ridges on the roof of the mouse palate, the rugae, a striped pattern that we previously showed to exhibit RD system behaviour . Using cShh) gene in the mouse mid-gestation palate depend on the Hh pathway itself as an ‘inhibitor’ and the FGF pathway as an ‘activator’ .We previously showed that the periodic stripes of expression of the sonic hedgehog . Shh expression stripes. Through careful quantification , we could demonstrate that although some inhibitors changed the number of Shh stripes, none of the inhibitors changed the position of the stripes (and therefore the wavelength of the pattern) relative to controls . Rather, inhibition changed the width and Shh staining intensity of stripes . Specifically, dorsomorphin intensified and broadened the stripes similar to what we previously reported for cyclopamine, whereas IWP-2 narrowed and weakened them. FGF inhibition by SU-5402 also weakened the stripes, as previously reported . In some cases, posterior and anterior stripes differed in the magnitude of their response. On cyclopamine or SU-5402 treatment, posterior rugae appeared to widen more than anterior rugae, in some cases to the point of fusing with their neighbours , whereas on IWP-2 treatment, anterior rugae decrease in staining intensity more than posterior rugae, and in some cases to below the detection threshold leading to the apparent loss of stripes . However, although the magnitude of response could vary along the palate, the direction of response was the same across all Shh stripes . These results confirm that all four pathways contribute to the formation of the ruga pattern, and their inhibition changes the width and intensity of stripes but not the wavelength of the pattern.We used the stripes of reported , but carFig. S3C,D), which were already established before the addition of the inhibitor  and which we previously demonstrated were maintained by an RD mechanism through their ability to bifurcate on the removal of a neighbouring stripe . Using pathway targets as the operational measure of pathway activity avoids the complications of multiple ligands, receptors and transduction components as discussed above. We found that each of the pathways had periodic outputs. Where available, additional target markers were tested and gave the same periodic pattern . As expected, Hh target expression was in the same spatial phase as Shh in both epithelium and mesenchyme. Surprisingly, FGF signalling was in-phase with the Shh stripes in the mesenchyme but out-of-phase in the epithelium. This reconciles our previous description of FGF activity as being in-phase with the Shh stripes . Any pair of morphogens could potentially support a two-component system .We then investigated how four or potentially five components can be wired together in a regulatory network that can generate the observed spatial pattern. One potentially simplifying approach is to consider whether the topology of this system  could effectively be described as a classical two-component RD system, which would consist of two ‘master morphogens’, with the other components serving as intermediates [so-called ‘mediators’ ] betweenFig. S8) indicated that although the pattern of waves could be lost under strong inhibition, the series of waves could be preserved (including the wave number) if inhibition strength was reduced . However, upon inhibition, either the amplitude or the absolute level of the waves was shifted up or down. It should be noted that in some instances, the waves appeared bounded. For example, if a wave shifted up, only the troughs of the wave moved up, whereas the peaks did not move noticeably, and vice versa if the wave shifted down.  In these bounded cases in particular, the upward or downward shift in wave position was associated with a stripe widening or narrowing, respectively . The maintenance of wave number along with changes in width and intensity was reminiscent of the changes in Shh stripes seen in our inhibitor-treated explant cultures.We therefore turned to perturbation analysis as a way of constraining possible network topologies. Using simple linear equations, we modelled the effects of perturbing either component in both classical two-component AI and SD systems. We investigated how a series of waves, generated by a two-component RD network from noisy initial conditions, would respond to the inhibition of each component. Preliminary investigations , ensuring that wave number was maintained after inhibition (Table S1). We confirmed that, for almost all parameter sets, the change in mean component level appeared to be the result of a shift in wave amplitude or absolute level, as illustrated in Fig. S8F,G . We then compared the results with the effects on established spatial patterns of experimentally inhibiting Wnt, BMP or Hh itself, considering first all four possible two-component systems containing the Hh pathway (our readout) and excluding FGF [where two nodes are inhibited simultaneously requiring a different analysis (see below)]. We found that for both Wnt-Hh and BMP-Hh systems, one of the two possible two-component models was consistent with the data . This showed that our system  cannot be accurately described as falling into either one of the two classical AI or SD classes.As the behaviour of the waves upon inhibition could be described as upward or downward shifts, we could capture it as shifts in the mean level of each component. Therefore, we generated 1000 random parameter sets consistent with the conditions for DDI for both AI and SD networks, and only considering parameter sets that could produce a stable series of waves, we determined the change in the mean component level upon inhibition  and each interaction can be positive, negative or zero . Considering that all components have some degradation rate, and therefore have some level of negative self-interaction, we only considered the distinction between topologies with positive self-interactions and those without them. This reduces the total number of possible topologies to 23×36=5832. . Inhibition of the production of a component (e.g. IWP-2) was achieved by proportionately reducing the production term of that component by a factor (1–α) . For all parameter sets, by successively reducing the strength of the inhibition parameter α by a factor of 0.25 from complete inhibition (α=1), we determined empirically for each pathway the maximum perturbation αmax that still allowed patterning but did not perturb the number of whole wavelengths or fail to achieve a stable amplitude. The test statistic for perturbation effects was the mean level for each component relative to an unperturbed run. To confirm that this readout accurately reflected an upward or downward shift in the wave, as was seen in the experimental system, the shift in the troughs and peaks of the waves, respectively, were also recorded: if an increase in mean was associated with an increase in the position of the wave troughs, or a decrease in the position of the wave peaks, it was determined that the wave had shifted upwards or downwards, respectively .Scaled parameter sets were simulated as described above, with all grid positions initially drawn at random from a uniform distribution between −0.01 and 0.01 around the equilibrium point of the reaction term matrix of the RD system. For parameterisations in which a stable spatial pattern of 2 to 4.5 complete waves (to avoid discretisation) was established in the RD system, components were inhibited in one of two different ways. Inhibition of the receptor of a given morphogen  was implemented as an equal proportional reduction of all interaction coefficients representing the response to that morphogen [so if the response to component aij were varied linearly through seven values spread evenly around 0 to give a total of 40,353,607 parameter sets. Diffusion parameters were initially set as two fast and one slow or vice versa, although this requirement [based on the longstanding but recently overturned idea (Appendix S1, section 2, were applied.For the three-component RD system, parameter space was systematically sampled to identify parameter sets and topologies capable of giving DDI. Each of the nine reaction parameters ned idea  that thi


Maternal embryonic leucine zipper kinase (MELK) is an aberrantly expressed gene identified as a target in AML. The MELK inhibitor OTS167 induces cell death in AML including cells with FLT3 mutations, yet the role of MELK and mechanisms of OTS167 function are not understood. OTS167 alone or in combination with tyrosine kinase inhibitors (TKIs) were used to investigate the effect of OTS167 on FLT3 signaling and expression in human FLT3 mutant AML cell lines and primary cells. We describe a mechanism whereby OTS167 blocks FLT3 expression by blocking FLT3 translation and inhibiting phosphorylation of eukaryotic initiation factor 4E–binding protein 1 (4E-BP1) and eukaryotic translation initiation factor 4B (eIF4B). OTS167 in combination with TKIs results in synergistic induction of FLT3 mutant cell death in FLT3 mutant cell lines and prolonged survival in a FLT3 mutant AML xenograft mouse model. Our findings suggest signaling through MELK is necessary for the translation and expression of FLT3-ITD, and blocking MELK with OTS167 represents a viable therapeutic strategy for patients with FLT3 mutant AML.Internal tandem duplication (-ITD) mutations of Fms-like tyrosine kinase 3  FLT3) gene characterized by an internal tandem duplication (ITD), point mutations within the juxtamembrane (JM) domain, or point mutations in the tyrosine kinase domain (TKD)1. Constitutive signaling mediated by mutant FLT3 results in anti-apoptotic and pro-growth signals that are critical for the progression of FLT3 mutant AML6. Patients with FLT3 mutant AML typically exhibit worse clinical outcomes although recent phase III data demonstrate a survival improvement when a targeted kinase inhibitor (midostaurin) is added to combination chemotherapy, followed by allogeneic transplant in first complete remission9. Targeting the FLT3 kinase, midostaurin has been approved for the treatment of newly diagnosed FLT3 mutant AML; gilteritinib has been approved recently for relapsed/refractory (R/R) AML11. Results from the ADMIRAL Phase III study (NCT02421939) show significantly longer overall survival (OS) and higher response rates with gilteritinib vs salvage chemotherapy in patients with R/R AML. Yet, the median OS for patients in the gilteritinib arm is less than one year, thus additional therapeutic options are required12. Despite the availability of these tyrosine kinase inhibitors (TKIs) for the treatment of FLT3 mutant AML, clonal evolution or drug resistance may result in failure of TKI activity. The acquisition of additional FLT3 mutations during treatment represents a mechanism by which patients acquire resistance to TKIs14. Therefore, combination therapies offer a strategy to contend with multiple mutations and other mechanisms of TKI resistance in mutant FLT3 leukemia.Approximately 30% of cases of acute myeloid leukemia (AML) aberrantly express a mutated Fms-like tyrosine kinase 3  for patients with relapsed/refractory AML18. Here, we investigate the antileukemia activity of OTS167 in FLT3 mutant AML alone or in combination with TKIs. We describe a mechanism whereby MELK inhibition targets FLT3 mutant AML through inhibition of FLT3-ITD signaling and downregulation of FLT3-ITD expression. We further demonstrate the synergy of OTS167 with FLT3 kinase inhibitors in FLT3 mutant AML cell lines and in a xenograft mouse model of FLT3 mutant AML.Maternal embryonic leucine zipper kinase (MELK) is a serine/threonine protein kinase that is aberrantly expressed in many tumor types and demonstrated to be important for the formation and maintenance of cancer stem cells2 for 10–14 days. All types of colonies were counted and labeled as #CFU. Experiments were done in duplicate or triplicate. Synergy analysis using Combination Index (CI) for different dose combination cell viability percentages (Fa) was calculated using CompuSyn20. A combination index value below 1 represents synergistic induction of cell death.Cell viability assays were performed on cells incubated for 48 h in the presence of increasing concentrations of single or combination drug treatments. Cell viability was assayed using Cell Counting Kit-8 (CCK-8) added to the cell culture for the last 4 h, and quantitated using a Bio Tek Synergy H4 plate reader using Gen5 software (SCR_017317). The half-maximal inhibitory concentration (IC50) for cell viability assays was calculated using nonlinear best fit [Inhibitor] vs. response—variable slope (four-parameter) in GraphPad Prism v.8.0 (RRID: SCR_002798). Colony forming cell (CFC) assay was performed by plating 5E4 primary blasts in 0.9% MethoCult . Cultures were incubated at 37degC in a humidified atmosphere of 5% CO21. Briefly, for MV4:11 xenograft experiments, NOD.Cg PrkdcscidIL2rgtm1Wjl/SzJ (NSG) mice  were serially engrafted via tail vein injection with 10E6 MV4:11, then 5E6 splenocytes from the initial engraftment (first generation), before engrafting 1E6 cells (second generation) for pharmacodynamic and survival experiments. 10 days after engraftment, engrafted animals were treated for the indicated times on a 5 days treated/2 days off schedule. For MOLM-14 xenograft experiments, NSG mice were engrafted directly with 1E6 MOLM-14 cells for pharmacodynamic and survival experiments. 7 days after engraftment, engrafted animals were treated for the indicated times on a 5 days treated/2 days off schedule. Inhibitors in powder form were suspended in 0.5% methylcellulose (Sigma #M0512) fresh each day for delivery via oral gavage.Animal studies were approved by the University of Chicago Institutional Animal Care and Use Committee and carried out with adherence to all appropriate guidelines and using a complication scoring system to minimize animal suffering. Xenograft experiments were performed as previously describedt test. Survival outcomes were compared using the Kaplan–Meier method and the log-rank test. Replicates per group for survival analysis were based upon sample size calculations ; alpha = .05, beta=0.8, hazard ratio of 0.237)—10 animals per group were proposed. P values <0.05 are considered statistically significant. P value scheme: ****P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.05, ns-not significant.Statistical analysis was conducted using GraphPad Prism, v8.0. Grouped data are presented as mean ± SD. Differences between the two groups were assessed using a two-tailed unpaired Additional methods and figures can be found in Supplemental Materials.17. To determine whether MELK plays a role in regulating FLT3 expression, FLT3-ITD-expressing cell lines MV4:11 or MOLM-14 were treated with OTS167 and assayed for FLT3 protein expression at 8 and 24 h. Western blots of untreated cells expressing FLT3-ITD protein reveal two protein bands corresponding to a simply glycosylated 130 kDa form (p130) retained in the endoplasmic reticulum (ER) and a complexly glycosylated 160 kDa form (p160) found on the cell surface, each regulating distinct signal transduction pathways22. Treatment of MV4:11 or MOLM-14 cells with 50 nM OTS167 resulted in a loss of the p130 form of FLT3 at 8 h and loss of both forms by 24 h  decrease in FLT3-ITD detection at 24 h after OTS167 treatment  with 50 nM OTS167 also resulted in loss of FLT3 protein expression at 24 h  loss of nascent FLT3-ITD protein expression  decreased by treatment with OTS167, reflecting an inhibition of translation versus a loss of mRNA for which the ratio would not change  decreased amount of FLT3 mRNA at 4 h, confirming a rapid inhibition of FLT3-specific mRNA translation  facilitates translation of certain mRNA transcripts, and has previously been identified as a target for MELK activity31. eIF4B-mediated translation activity is positively regulated by phosphorylation of its ser406 and ser422 residues34. To determine if the block in translation of FLT3-ITD may be due to inhibition of 4E-BP1 or eIF4B, MV4:11 or MOLM-14 were treated with 50 nM OTS167 or gilteritinib. Phosphorylation levels of 4E-BP1 and eIF4B were assayed at 8 h by western blot  and ser65 (***P < 0.001) phosphorylation of 4E-BP1. Whereas treatment with gilteritinib resulted in a partial non-significant loss of thr37/46 and a significant (**P < 0.01) loss of ser65 phosphorylation. Inhibitors of PIM kinase (AZD 1208) or mTORC1 (KU-0063794) were also tested for their ability to inhibit 4E-BP1 phosphorylation , while treatment with gilteritinib resulted in a loss of ser422 phosphorylation (****P < 0.0001), but only a 50% decrease (**P < 0.01) of ser406 phosphorylation. To determine if inhibition of ser406 and ser422 phosphorylation occurs prior to FLT3-ITD downregulation, MV4:11 and MOLM-14 were treated with 50 nM OTS167 and assayed for eIF4B phosphorylation over time  loss of FLT3-ITD expression, although the effect was more dramatic in MOLM-14 (FLT3-ITD+/-) than MV4:11 (FLT3-ITD+/+)  decrease in FLT3-ITD expression in MV4:11 or MOLM-14 cells at 48 h, though the decrease was more pronounced in MOLM-14  in MOLM-14 cells may be more sensitive to disruption of MELK activity than in MV4:11.Eukaryotic initiation factor 4E–binding protein 1 (4E-BP1) binds eukaryotic translation initiation factor 4E (eIF4E) based on the phosphorylation status of several 4E-BP1 sites including thr37/46 and ser65, and regulates the availability of eIF4E for cap-dependent mRNA translationlot Fig.  – graphsion Fig. . Neitherion Fig. . For eIFime Fig. . Inhibitime Fig. . To dete/+) Fig.  – graph. 35.Fig.  To deter-14 Fig.  – graph.-14 Fig. . MOLM-1438. Inhibition of phosphorylation of FLT3-ITD by FLT3 kinase or PIM kinase inhibitors results in an increase of the 160 kDa complex glycosylated form of FLT3-ITD due to maturation of the 130 kDa form in the Golgi Apparatus, and greater sensitivity to FLT3 inhibitor-induced cell death39. An increase in the 160 kDa form of FLT3 observed 8 h after OTS167 treatment in FLT3 mutant cell lines . For MOLM-14 an IC50 of 1.93 nM for the combination of OTS167 and gilteritinib was lower, but not statistically significant from 3.18 nM and 14.18 nM for OTS167 or gilteritinib alone, respectively. In MV4:11 cells treated with submaximal doses (5 nM) of drug, staining with Annexin V demonstrated that the cell death observed was due to apoptosis. An increased percentage of Annexin V positive events was seen after combination treatment with 5 nM OTS167 and 5 nM gilteritinib (36.0%) versus 5 nM OTS167 (21.1%) or 5 nM gilteritinib (20.8%) alone  induced. Fa values between 0.2 (20% cell viability) and 0.8 (80% cell viability) represent the linear (non-plateau) portion of the dose response curves and demonstrate a range of combination doses. CI values <1 represent a synergistic drug combination. In MV4:11 cells the CI values below 1 through the Fa range indicate a consistent synergism between OTS167 and gilteritinib to induce cell death  mice then treated with different doses of OTS167, gilteritinib, or a combination of OTS167 and gilteritinib based on the cell line tested. Flow cytometry and histological analysis of mice injected with MV4:11 cells (hCD45 + ) demonstrate engraftment of the MV4:11 cells in both bone marrow and spleen in untreated control mice  compared to mice treated either with single-agent 10 mg/kg OTS167  or 10 mg/kg gilteritinib  compared with mice treated with single-agent OTS167  or gilteritinib Fig. . The incays)Fig. . Mice enays)Fig. .Fig. 6OTFLT3 mutant AML. OTS167 is currently being evaluated in an early phase trial for patients with advanced myeloid malignancies. Efficacy of OTS167 against FLT3 mutant cell lines suggested that OTS167 might act on mutant FLT3 through a MELK-dependent mechanism. Here we report two previously unrecognized effects of OTS167: (1) inhibition of FLT3 translation and (2) inhibition of FLT3-ITD kinase activity and downstream signaling. Recently OTS167 has been shown to inhibit the MELK-dependent phosphorylation of eIF4B in human breast cancer cell lines, which resulted in downregulation of anti-apoptotic factor myeloid cell leukemia 1 (MCL-1) expression30. In human multiple myeloma (MM), diffuse large B cell lymphoma (DLBCL) and mantle cell lymphoma (MCL) cell lines, MCL-1 expression was also inhibited by OTS167, although by an unknown mechanism41. Here we show OTS167 inhibits phosphorylation of eIF4B in human FLT3 mutant cell lines, and in these cell lines loss of eIF4B expression results in the loss of FLT3-ITD expression. Cooperativity between eIF4B, eIF4A, and DEAD-box protein Ded1 for efficient translation of longer mRNA species supports the requirement of eIF4B for efficient expression of FLT331. eIF4B has been shown to be phosphorylated on ser406 and ser422 via a combination of PI3K, MAP Kinase, Stat5, and MELK-regulated pathways, positively regulating activity42. By demonstrating the ability of OTS167 to inhibit multiple pathways mediated by FLT3-ITD activity, and the subsequent phosphorylation of eIF4B, we reveal the role eIF4B plays in FLT3-ITD protein translation. Our results show that eIF4B phosphorylation on both ser406 and ser422 was effectively inhibited by OTS167. These data support a model in which MELK contributes to phosphorylation of ser406 together with mTORC1, with additional mTORC1-mediated phosphorylation of ser422, culminating in activation of eIF4B33. In agreement with its ability to inhibit FLT3-ITD-mediated signaling pathways, OTS167 also inhibited phosphorylation of 4E-BP1. eIF4E, bound by hypo-phosphorylated 4E-BP1, is released and active after phosphorylation of 4E-BP1 first at thr37/46, then ser70 and ser6529. Interestingly, OTS167 was more effective than gilteritinib, a potent FLT3 kinase inhibitor, at inhibiting phosphorylation of 4E-BP1 at the two sites examined despite similar inhibition of PI3K signaling by the two drugs.In this study, we examined the antileukemic activity of the small molecule MELK inhibitor OTS167 in FLT3 point mutations43. Different juxtamembrane (JM) or activation loop mutations in the FLT3 gene result in varying levels of constitutive FLT3 kinase activity, thus driving diverse levels of persistent signaling by the STAT5, PI3K, and MAPK pathways45. To deter resistance mutations, second-generation TKIs effective against both JM and activation loop mutations such as crenolanib, quizartinib, and gilteritinib are currently being tested in clinical trials, and gilteritinib has recently received FDA approval for treatment of relapsed FLT3 mutant AML47. OTS167 inhibited the kinase activity of recombinant FLT3-ITD in vitro, and in AML cell lines. While the ability of OTS167 to inhibit FLT3 kinase activity and signaling was shared with other TKIs, only OTS167 is capable of downregulating FLT3 expression, circumventing resistance mechanisms including increased FLT3 or FLT3L expression and signaling in AML, or increased WT FLT3 expression in other hematologic malignancies such as MLL(KMT2A)-rearranged acute leukemias48.Known potential mechanisms of TKI resistance include up-regulation of FLT3L or FLT3 expression, activation of parallel pro-survival pathways, modulation of anti-apoptotic pathways, and additional 49. OTS167 treatment resulted in the downregulation of mutant FLT3 expression due to inhibition of FLT3 translation. Although not investigated here, additional effects of MELK inhibition may contribute to the induction of leukemia cell apoptosis, as observed in non-FLT3 mutant cell lines17. Furthermore, additional off-target effects of inhibitor OTS167 on other cellular pathways cannot be ruled out. While the mechanism of synergy is not fully understood, the ability of OTS167 to inhibit FLT3 kinase activity and downregulate FLT3 expression make it an attractive potential therapeutic option for FLT3 mutant AML. Results from our FLT3 mutant xenograft model demonstrate a synergy between OTS167 and gilteritinib in vivo, suggesting potential therapeutic value for OTS167 and a path forward for future dual agent targeting in FLT3 mutant AML. Currently, dose-escalation in the OTS167 clinical trial is proceeding to determine a maximum tolerated dose and biologically effective dose that could be tested in combination therapy using OTS167 with gilteritinib in FLT3 mutant AML.The synergy between OTS167 and TKIs in combination treatment experiments in vitro and in vivo suggests that OTS167 may induce cell death in a manner other than through cooperative FLT3 kinase inhibition. Drugs targeting translation initiation factors eIF4A, eIF4E, 4E-BP1, as well as translation elongation have been developed and show encouraging results in several cancer models and clinical trialsSupplemental Material


Ice-building up on the leading edge of wings and other surfaces exposed to icing atmospheric conditions can negatively influence the aerodynamic performances of aircrafts. In the past, research activities focused on understanding icing phenomena and finding effective countermeasures. Efforts have been dedicated to creating coatings capable of reducing the adhesion strength of ice to a surface. Nevertheless, coatings still lack functional stability, and their application can be harmful to health and the environment. Pulsed laser surface treatments have been proven as a viable technology to induce icephobicity on metallic surfaces. However, a study aimed to find the most effective microstructures for reducing ice adhesion still needs to be carried out. This study investigates the variation of the ice adhesion strength of micro-textured aluminum surfaces treated using laser-based methods. The icephobic performance is tested in an icing wind tunnel, simulating realistic icing conditions. Finally, it is shown that optimum surface textures lead to a reduction of the ice adhesion strength from originally 57 kPa down to 6 kPa, corresponding to a relative reduction of ~90%. Consequently, these new insights will be of great importance in the development of functionalized surfaces, permitting an innovative approach to prevent the icing of aluminum components. Atmospheric icing includes all meteorological phenomena in which ice is accreting and accumulating on surfaces. As a consequence of icing, serious issues in different industrial sectors result, such as in telecommunications where transmission lines and towers can collapse due to the increased weight, leading to power outages . The effMost of the technical surfaces today consist of aluminum alloys, which, due to their light weight and high strength, are employed, e.g., on the leading edge of the wings ,8,9. TheIn the last decades, increasing interest in a novel technological concept, the hybrid ice protection systems (HIPS), was observed. HIPS includes an engineered surface combined with an ice protection system (IPS) . AlternaA novel approach to target these objectives are micro-textured functional surfaces with icephobic properties. An icephobic surface has per definition the following properties: (i) they delay the solidification time of the water or reduce ice accretion while the heterogeneous nucleation is hindered  and (ii)A fast and cost-efficient way to directly fabricate functional surfaces is laser surface texturing (LST). In LST, microstructures are generated on a surface by using laser-based methods like direct laser writing (DLW) or direct laser interference patterning (DLIP), among others . In DLW,z adds an advantageous effect on reducing ice adhesion [Another laser-based technology that can be applied for producing textured surfaces is direct laser interference patterning (DLIP). This method requires the use of at least two laser beams, which are superimposed on the substrate. This superimposition leads to an interference-generated modulation of the laser intensity distribution, which allows fabricating different periodic micro-patterns. The feature size of the pattern is usually in the range between ~500 nm and 10–20 µm ,37. In padhesion . By usinadhesion ,45. HoweIn this study, various single- and multiscale patterns are fabricated on aluminum 2024 by means of DLW and DLIP. By combining both laser methods, complex hierarchical structures are introduced. The wetting characteristics are analyzed by static water contact angle and sliding angle measurements. The icephobic property is analyzed in relation to the adhesion of the formed ice on the laser-textured surfaces. Therefore, the terms “ice-repellent” and “icephobic” are to be understood in such a way that it requires reduced strength to release the ice from its surface. The performance regarding the dynamic ice adhesion is examined by interfacial shear stress experiments for rime, glaze, and mixed icing conditions using a lab-scaled icing wind tunnel. Hence, in these experiments, it is also feasible to take into account the wind speed and the droplet size, which allows a significantly more realistic view of the icing and deicing processes of laser-textured aluminum substrates.3. They were polished to a surface roughness Rz of 150 nm ± 40 nm. Prior to the laser processing, the samples were cleaned from contamination using isopropanol in an ultrasonic bath for 10 min. After the laser process, the structured samples were spray-coated with a hydrophobic agent  and stored under atmospheric conditions of 22 °C room temperature and 55% relative humidity. The non-flammable agent is composed of a chemically active perfluoropolyether compound dissolved in a fluorinated solvent, which is clear colorless and has an atmospheric lifetime of 0.77 years for a temperature range between −50 °C and +150 °C [The samples used in all experiments consisted of Al2024  in the shape of a cantilever with dimensions of 125 × 13 × 1 mm +150 °C . The use +150 °C . The com +150 °C .2 by controlling the laser power. The laser beam was scanned along the surface of the sample with a constant speed of 250 mm/s. In order to achieve sufficient material removal, the structuring process was repeated 10 times in all cases. The mesh-like surface structures processed by this setup consisted of 3 sequentially formed line-like structures, whose orientation was each time rotated by 60°. The distance between the parallel lines was fixed to 50 μm, which is, therefore, defining the spatial period of the structures. Previous works using a similar texturing approach showed that this texture provided remarkable results in terms of water repellence and static icing properties and was, therefore, considered for the dynamic icing tests. A detailed description of the fabrication process was already published elsewhere [Two different structuring methods, direct laser writing (DLW) and direct laser interference patterning (DLIP), were used for fabricating the microstructures. For DLW processing, a fiber laser was used because the pulse duration could be adjusted and optimized for aluminum processing. For the DLIP experiments, however, a solid-state laser was used since it is especially characterized by its pulse stability for ultrashort laser pulses (ps range). Moreover, under determined texturing conditions, nanosecond laser pulses can create deeper micro-tranches on metal surfaces than picosecond ones due to the displacement of molten material . The DLWlsewhere ,35,49.4 laser  with 10 ps pulses and maximum output power of 10 W. The workstation is equipped with a commercially available DLIP optical head , which uses a diffractive optical element (DOE) to split the initial laser beam into two sub-beams  with an accuracy of ±2.5 μm. By moving the sample in the y-direction, the pulse-to-pulse overlap can be controlled, which finally determines the number of laser pulses, influencing the ablated structure depth. To obtain the pillar-like structures, it is necessary to rotate the samples by 90° and repeat the process.For processing the samples with DLIP technology, a two-beam configuration was used. The used configuration utilizes an IR (1064 nm) solid-state Nd:YVOub-beams b. These ub-beams c. In thiThe hierarchical structures, consisting of the DLW and DLIP, were manufactured by combining both strategies successively. A detailed description of the fabrication processes, the laser process windows, and the choice of the processing parameters was already published elsewhere ,35,49.z was measured with a benchtop stylus profiler , following the standard DIN ISO 25178 and DIN EN ISO 4287, respectively [To analyze the surface topography, scanning electron microscopy  at an operating voltage of 15.0 kV was used. The wettability of the surfaces was characterized via static contact angle (SCA) and sliding angle (SA) measurements using the sessile drop method with a video-based optical goniometer . This method was chosen due to its suitability for reproducible measuring. Deionized water droplets of 10 µL volume were deposited in atmospheric conditions for all measurements. The wetting tests were repeated 6 times to ensure statistical significance. Additionally, the real-to-projected area ratio Sdr was measured using a confocal microscope  with a 150× objective, resulting in vertical and lateral resolution of 2 nm and 140 nm. The surface roughness RTo analyze the ice adhesion behavior for four different ice conditions, the interfacial shear stress was measured using a circular closed-loop wind tunnel iCORE  based on several publications. It is worth mentioning that there were slight differences in the Eice for the different icing conditions. Nevertheless, our assumption allowed comparability of the ice adhesion data within each ice type. For simulating atmospheric in-flight conditions, four different ice conditions  were investigated by applying five testing parameters. These are the icing conditions to which an aircraft mostly can be exposed during a flight. They result from the varying flight altitude, which is associated with different air temperatures and ice-water mixtures. For instance, the glaze ice condition usually occurs at an altitude around 2000 m at a temperature range between 0 °C and −10 °C. Contrarily, the rime ice condition appears at altitudes above 3000 m and at a temperature of at least −20 °C. Furthermore, different ice conditions are characterized by varying densities and hardnesses [The calculation was performed under the assumption of a constant Young’s modulus of 9 GPa for each type of ice  . The infz of ~43.1 µm and an average structure depth of 35.7 µm. Significantly less melting occurred on the DLIP-treated samples due to the ps-based micro-processing. Applying a spatial period of 7.0 µm, perfectly ordered pillar-like microstructures were fabricated, and a roughness Rz of ~4.1 µm was reached hydrophobic character of a surface. Therefore, the sliding angle (SA), denoted as the tilting angle where the droplet starts to slide on the surface, is an additional parameter that has to be considered . In thisIt is important to state that the determination of the wetting contact angle is representative of the macroscopic wetting behavior of the treated surfaces, and it is not sufficient to describe the wetting phenomena involved during in-flight (icing) conditions . In factIt is worth mentioning that the durability of the laser-generated structures and their chemical composition when exposed to consecutive icing/de-icing cycles was examined in a previous study . It was It is well-known that superhydrophobic aluminum leads to delayed ice-formation due to the reduced contact area between a single water droplet and the surface . However3 cantilevers, which were placed afterward in an icing wind tunnel in order to investigate the ice adhesion. Four icing conditions were analyzed, which simulated flight conditions encountered in different atmospheric regimes. These generated four qualities of ice were rime, mixed rime, mixed glaze, and glaze ice. After that ice was accreted on the surface of the cantilever, the icing cloud was turned off, and an oscillation was induced on the cantilever through a piezoelectric transducer. A linear frequency sweep was applied, letting this increase with time and causing an increase in the amplitude of the oscillation when the resonance frequency of the oscillating body was reached [The microstructures in this study were fabricated on 125 × 13 × 1 mm reached . Since tThe amplitude of the strain recorded right before the interfacial crack represented the limit at which the interfacial shear stress matched the adhesion strength of the ice to the surfaces. Exceeding this value triggered a crack at the ice-surface interface. The Euler-Bernoulli beam theory could be used to correlate the strain to the maximum interfacial shear stress  between ice and surface, which is expressed in Equation (1) :(1)τint=nterface . The eccense ice ,64. As aense ice . In geneense ice . It is wense ice . Moreoveense ice .In contrast to that, the results of the ice adhesion tests for the seven samples (two references and five micro-structured cantilevers) by means of the retrieved interfacial shear stress under four different icing conditions are summarized in Each measurement was repeated 4 times for each surface type and for each ice condition . The average value of one measurement series defines the height of the bar, while the error bars result from the standard deviation of each measurement series. The term reference refers to the untextured sample, and it provides average interfacial shear stress of around 36 kPa, whereas, for mixed rime, mixed glaze, and glaze conditions, the stress was slightly increased to around 50 kPa. Besides that, some trends were observed, which are discussed in the following.z of 43.15 µm, 4.16 µm, and 38.16 µm  resulting from the coarse feature sizes of 50 µm (DLW) or 7 µm (DLIP) provided a favorable topography for the droplets to attach and interlock. Note that the mean effective volume droplet diameter (MVD) of the impinging droplets was ~20 µm and thus in the order of magnitude of the investigated microstructures (see The DLW structure (50 µm), the pillar-like DLIP structure (7 µm), and the hierarchical pattern showed interfacial shear stress between 48 and 64 kPa, which was similar (for mixed glaze ice) or even significantly higher (up to 107 kPa for rime and mixed rime ice) compared to the reference. It could be assumed that the roughness Rures see .In contrast, the DLIP structures with a spatial period of 2.6 µm showed a significantly lower interfacial shear stress. The stress for the smallest examined texture was between 27 and 38 kPa (DLIP line-like 2.6 µm) and even lower between 6 and 10 kPa (DLIP cross-like 2.6 µm) for the icing condition of rime, mixed rime, and mixed glaze. This amounted to a shear stress reduction of ~90% compared to the reference. This behavior could be explained with a synergic effect of high hydrophobicity and low surface roughness.Besides that, it is worth mentioning that both references did not show superhydrophobic characteristics for all icing conditions. This property is fundamentally necessary since impacting droplets will not adhere to the surface. The droplet rolls off the surface because the anti-wetting capillary pressure is greater than the dynamic and water hammer wetting pressures .In fact, the texture size produced on the “DLIP cross” sample is low enough to both repel micrometer-sized water droplets and to prevent the formation of large anchor points for the ice adhesion and accretion . AlthougTo further investigate the influence of the surface peak-to-peak-roughness, the interfacial shear stress was plotted as a logarithmic function of the real-to-projected area ratio Sdr in %, for the four ice adhesion characteristics . The corz of 0.96 µm, and resulted in the detachment of the ice from the aluminum surface at interfacial shear stress below 10 kPa. However, a direct correlation of the individual textures regarding their ice adhesion, as shown in Although the experimental data did not show a clear trend, it was evident that a defined Sdr ratio was favorable in order to reach low interfacial shear stress under rime, mixed rime, and mixed glaze conditions. The differences of the Sdr values for the 50 µm DLW and 2.6 µm DLIP textures could be ascribed in-depth and the aspect ratios of the textures. Since the 2.6 µm textures were a few hundred nm deep, the aspect ratio was consequently low and in the range of ~0.1. In contrast, the DLW textures showed a structure depth of ~35.7 µm, which resulted then in an aspect ratio of ~0.7. As a result, the DLW textures provided a significantly higher Sdr ratio. The Sdr ratio was in the range of ~13 (for the DLIP cross 2.6 µm textures), corresponding to a surface roughness RThese findings were supported by previous studies reporting the systematic reduction of ice adhesion strength with the decrease of surface roughness regardless of surface wettability ,73. In-s3  compared to the reference. Consequently, it can be stated that microstructures with feature sizes smaller than 5 µm and a particular structure type (line-like and cross-like) are especially suitable for designing icephobic surfaces. Using the results of these experiments, the field of the perfect icephobic texture for real dynamic icing conditions can be further narrowed down, and future research can build on this knowledge. The results obtained and the methodology developed in this work can be further adapted to other alloys and even polymers, unlocking completely new application examples in the challenge facing icing surfaces in the fields of aerospace technology.In this work, five different periodic patterns were textured on AA2024 clad cantilever samples using direct laser interference patterning, direct laser writing, and a combination of both methods. Additionally, two untextured reference samples were considered. The spatial period of the textures ranged from 2.6 µm to 50 µm, showed a line-, cross-, pillar-, or mesh-like pattern, and covered a corresponding roughness R


Canis familiaris) often cohabite at interfaces shared by humans and wildlife, interacting with wild canids as predators, prey, competitors and reservoirs of several multi-host pathogens, such as canid-borne micro and macro parasites that could impact on wildlife, livestock and public health. However, spatio-temporal patterns of indirect interactions as promoters of pathogen transfer between domestic and wild canids are largely unknown. In this study, we used camera traps to describe the activity patterns and habitat use of dogs, chilla  and culpeo  foxes and identify the local-scale factors that may affect the frequency of dog-fox interactions through an anthropization gradient of the Coquimbo region, Chile. We assessed local-scale variables that may predict the number of interactions between dogs and foxes, and compared the time interval between dog-culpeo and dog-chilla interactions. Our findings suggested that closeness to urbanized zones predicts the frequency of indirect interactions between dogs and foxes. We found higher number of dog-fox interactions (60 interactions) at a periurban site adjacent to two coastal towns (Tongoy and Guanaqueros), compared to other two more undisturbed sites (12 interactions) increasingly distanced from urbanized areas. We showed that dogs interacted more frequently with chilla foxes (57 interactions) than with culpeo foxes (15 interactions), and the first interaction type occurred almost exclusively at the periurban site, where dogs and chillas were more frequently detected than in the other sites. We detected a marked temporal segregation between dogs and foxes, but dog-chilla interactions resulted in shorter time intervals (2.5 median days) compared to dog-culpeo interactions (7.6 median days), suggesting a higher potential risk of pathogen spillover between the first species pairing. Based on previous studies, we suggest periurban zones may constitute a potential focus of pathogen exposure between dog and fox populations in the study area. Our research contributes to improving the knowledge on the spatio-temporal patterns of interspecific contact between invasive and native carnivores within the context of multi-host pathogen dynamics. Our outcomes will inform theoretical epidemiological models designed to predict and minimize the contact risk between domestic and threatened species, guiding effective control strategies at the wildlife-domestic interface.Domestic dogs ( Interspecific interactions are relevant behavioral factors that modulate population and community dynamics at multiple ecological levels , 2. FromCreated by the encroachment of human activities into natural landscapes, wildlife-domestic interfaces represent critical boundaries where humans, vectors, and reservoir hosts  coexist, thus increasing contact and the risk of cross-species transmission and emergence of pathogens –9. DurinCanis familiaris) is one of the most ubiquitous and damaging invasive carnivore worldwide, often cohabiting at the interface between human and wildlife communities  and culpeo  foxes  foxes . In the s) foxes , 35, wits) foxes , 35. Hows) foxes , and whes) foxes , 38.Camera traps constitute an increasingly popular non-invasive and cost-effective monitoring tool for cryptic and crepuscular species , 40, allThe study was conducted in the coastal zone of the Coquimbo region in north-central Chile  . The stuAtriplex nummularia) and pioneer native vegetation. Rural economy has been historically based on livestock production, mostly focused on small ruminant farming ; (2) Rural, a private 31 km2-site inserted into the “El Tangue” ranch, a farm of nearly 45,000 ha situated 10 km south of Tongoy. The property is characterized by a mosaic of agricultural fields, grasslands, and a mixture of exotic and native shrubs, with small properties scattered along the westward border distance up to 3 km of the field site; and (3) Wild, a 22 km2-site characterized by a xeric-mesic ecosystem into the BFJNP, located at 43 km south of Tongoy. Geographical distancing across sites was considered to ensure independence of dog-fox interactions and sites were arbitrarily chosen based on the hypothesis of higher opportunities of dog-fox interactions and pathogen spillover in proximity to urban zones compared to rural and undisturbed zones. High dog densities have been reported across towns in the Coquimbo region . To. To42)].n is the number of camera stations per site and σ is the standard distance. Previously, depending on the raster surface to create, we weighted the standard distance by either the number of culpeo, chilla, dog or interaction events captured per camera at each site.where 1 and Δ4, which ranges from 0 (no overlap) to 1 (complete overlap), which indicate whether dog-fox pairings overlap in their use of diel cycle periods . Coefficients Δ1 and Δ4 have been reported to perform appropriately if the sample has <50 and >75 photos, respectively. The precision of both coefficients was determined by the calculation of confidence intervals as percentile intervals from 1,000 bootstrap samples  site ; (b) year; (c) season; (d) distance from each camera to the nearest known inhabited house, village or city (hereafter human settlements); (e) distance from each camera to the nearest main road; and (f) an indicator of seasonal productivity index (see below). Distances to human settlements and roads were calculated based on human made features within a 2-km buffer around each sampling site, based on the mean maximum distance of foray activity exhibited by dogs from households according to Sepúlveda et al. . SpatialU-test to assess whether the time interval of dog-fox interactions differed between types of interaction . All the statistical tests used α = 0.05 for determination of statistical significance.Given indirect interactions counts between dogs and foxes were over-dispersed , we specified models with a negative binomial distribution for the following statistical analyses. First, we used generalized linear mixed-effects models to test whether the number of indirect interactions between dogs and foxes was related to the defined environmental variables. Because cameras sampled repeatedly the same locations during different seasons, we included the camera ID as random effect in all the models. To account for variations in trapping effort across cameras, we included the log of CTN at each camera as an offset variable in all the models , 42, 68.Our study produced a total of 2,161 independent photos (hereafter photos) of wild and domestic canids over a total 16,979 CTN . The culpeo fox was the most frequently detected canid species with 1,426 photos, followed by the chilla fox (626 photos) and the domestic dog (109 photos) through the entire study .Culpeo fox was more frequently detected in the wild site compared to the other sites, resulting in a trapping success three times higher than in the rural site . We totaled 0.5 culpeo's photos/100 CTN in the periurban site. On the contrary, chilla fox displayed a higher intensity of habitat use at the periurban site, with a trapping success more than double than in the rural site . We did not detect chillas in the wild site. Dogs were more frequent at the periurban site, with a trapping success almost six times higher than in the rural site . We only obtained 0.1 dog's photos/100 CTN in the wild site .n = 57) were more frequently recorded than dog-culpeo fox interactions (n = 15). In the periurban site, we totaled 60 interactions (55 dog-chilla and 5 dog-culpeo). In the rural site, we detected 9 interactions (7 dog-culpeo and 2 dog-chilla), while we found only 3 dog-culpeo interactions in the wild site , which were documented by 19, 6, and 3 cameras deployed over the periurban, rural and wild sites, respectively. Dog-chilla fox interactions  of cameras, occurring as several hotspots nearby the southeast border of the site . Chilla 1 = 0.76 with 95% CI ] and rural [Δ4 = 0.87 with 95% CI ] sites. Thus, we pooled both fox species photos for further comparative analyses with dog temporal patterns. In overall, we found a low temporal activity overlap between dogs and foxes in the periurban  and rural  sites, and both groups exhibited significant differences in temporal patterns within both sites  (U2-test had p < 0.01); thus, we only performed statistical comparisons between both canid groups' activity patterns at the site level. While we found a temporal activity overlap of Δ1 = 0.60 between dogs and culpeo foxes in the wild site, the coefficient precision was comparatively lower than the other sites , due to the smaller number of dog photos (n = 4).In overall, both culpeo and chilla were mainly crepuscular/nocturnal and exhibited prolonged nighttime activity . Domestic dog activity peaked around 1,000 h, displaying a diurnal activity mostly concentrated between 0700 and 1,500 h . We found high degree of activity overlap between both fox species in both periurban . Fo. Fo38)].ulations . We expeThe raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.Ethical review and approval was not required for the animal study because a non-invasive technique  to estimate indirect interactions between domestic and wild canids was used.FAH and GAJ conceived the ideas, designed the study, analyzed the data, and led the writing of the manuscript. All authors carried out the field work, collected the data, revised the manuscript, contributed critically to the drafts, and approved the final version for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Ulva ohnoi and provides an initial overview of their mode of action. The ability of Ulva protease inhibitors to inhibit digestive proteases of three marine fish species, as well as their capacity to hamper the hydrolysis of a reference protein by those fish proteases, were assessed. In addition, thermal stability and the mode of inhibition on trypsin and chymotrypsin were also studied. Dose-response inhibition curves and in vitro protein hydrolysis assays revealed a noticeable inhibition of fish enzymes when Ulva concentration increased in the assay. The thermal treatment of Ulva reduced markedly the inhibitory effect on fish digestive protease. Finally, Lineweaver–Burk plots indicated that trypsin and chymotrypsin inhibition consisted of a mixed-type inhibition mechanism in which the inhibitory effect depends on Ulva concentration. Overall, the results confirmed the presence of protease inhibitors in Ulva, though heat treatment was enough for inactivating these compounds.This piece of research evaluates the presence of protease inhibitors in the macroalga  Anti-nutritional factors (ANFs) can be defined as substances that, by themselves or through their metabolic products, can exert negative effects on food utilization and interfere with the normal growth, reproduction and health of fish . From a In general, ANFs have been mainly related to plant-derived feedstuffs, and they comprise a wide variety of compounds, such as protease inhibitors, phytohemagglutinin, lectins, phytic acid, saponins, phytoestrogens or antivitamins ,5. AlthoUlva have been successfully evaluated as a dietary ingredient in some farmed fish species, such as gilthead seabream (Sparus aurata)  assayed. Lineweaver–Burk curves, 1/v versus 1/[S], were plotted and the Michaelis constant (Kp < 0.05) were determined using Tukey’s multiple comparison test. Data with nonparametric distribution were analyzed by using Kruskal–Wallis test, and significant differences were determined using box-and-whisker plot graphs. All statistical analyses were performed using the Statgraphics Plus 4.0  software.The results were expressed as mean ± standard deviation. In order to test data normality and variance homogeneity, the Kolmogorov–Smirnov test and Levene’s F-test were used, respectively. Data with parametric distribution were analyzed using one-way analysis of variance (ANOVA), and the significant differences between treatments (Ulva able to inhibit digestive proteases of commercial fish species. The inhibitory capacity was dose-dependent. From a physiological point of view, high dietary inclusion of crude Ulva would be requested to achieve higher inhibition values. The thermal treatment during feed processing is high enough to inactivate the inhibitors from U. ohnoi; hence, it can be used efficiently as potential sustainable ingredient for aquafeeds.This work showed the presence of protease inhibitors in


Winter activities on ice are culturally important for many countries, yet they constitute a high safety risk depending upon the stability of the ice. Because consistently cold periods are required to form stable and thick ice, warmer winters could degrade ice conditions and increase the likelihood of falling through the ice. This study provides the first large-scale assessment of winter drowning from 10 Northern Hemisphere countries. We documented over 4000 winter drowning events. Winter drownings increased exponentially in regions with warmer winters when air temperatures neared 0°C. The largest number of drownings occurred when winter air temperatures were between -5°C and 0°C, when ice is less stable, and also in regions where indigenous traditions and livelihood require extended time on ice. Rates of drowning were greatest late in the winter season when ice stability declines. Children and adults up to the age of 39 were at the highest risk of winter drownings. Beyond temperature, differences in cultures, regulations, and human behaviours can be important additional risk factors. Our findings indicate the potential for increased human mortality with warmer winter air temperatures. Incorporating drowning prevention plans would improve adaptation strategies to a changing climate. Winter activities on ice are a defining component of winter culture and identity in northern countries, contributing food through ice fishing, winter transportation on ice roads, and recreational activities, such as ice skating . Winter Falling through the ice is one of the major risk factors associated with winter ice activities, resulting in fatal and non-fatal winter drownings. Globally, drowning is the third leading cause of unintentional injury deaths , and in The complex nature of interacting climate factors makes it difficult to predict the safety of ice conditions. Over the past century, lakes, rivers, and seas are experiencing earlier ice break-up, later ice formation, and shorter seasons of ice cover –16. MoreHere, we explore whether there are more winter ice drownings in regions with warmer winters, because air temperature is a major factor for ice stability , 17. We This study is a multi-centered, multi-nation retrospective review of climate and fatal drowning event data for winter seasons, over 26 years. We used non-linear mixed effects models to characterize the relationship between winter air temperatures and winter drownings.Drowning is defined as “the process of experiencing respiratory impairment from submersion/immersion in liquid." The outcomes can be fatal and non-fatal. Non-fatal drowning outcomes can be with or without morbidity . We collData were consolidated by month and region to conform to winter months and ice-covered regions. The Canadian North is represented by the Northwest, Yukon, and Nunavut Territories. Newfoundland, New Brunswick, Nova Scotia, and Prince Edward Island constitute the Canadian Atlantic. The Canadian Prairies consist of Alberta, Saskatchewan, and Manitoba. Italy is represented by the northern Trentino-South Tyrol region and Japan is represented by the Nagano prefecture. The United States is restricted to Alaska, Connecticut, Iowa, Maine, Massachusetts, Michigan, Minnesota, Montana, New Hampshire, New York, North Dakota, South Dakota, Vermont, and Wisconsin.We were unable to acquire data on non-fatal drownings, which may occur at much greater numbers, and thus our results provide a conservative estimate of the overall consequences associated with winter drownings. Full details on how the data were acquired from each country are provided in We acquired mean air temperature and precipitation data for each month corresponding to the time period and specific areas encompassed by the drowning records averaged for each region from the University of East Anglia’s Climatic Research Unit (CRU TS4.02). For example, for the United States, we averaged the winter climate data for the regions within the 14 northern states which contain lakes that typically experience winter ice cover. The climate data are available on a 0.5° latitude/longitude grid based on interpolations of air temperatures and precipitation measured at meteorological stations . We calchttps://www.cia.gov/library/publications/the-world-factbook/; https://www.gutenberg.org/wiki/CIA_World_Factbooks_(Bookshelf)). For provincial and state level population estimates, we obtained official national census data within each country. For the Trentino-South Tyrol region of Italy, we obtained the official national census data in 2001 and 2011 from the Italian National Statistics office (http://dati.istat.it/). For the Nagano prefecture in Japan, we obtained official census data every year for the period 2006 to 2018 (https://tokei.pref.nagano.lg.jp/). For Canada, we obtained annual provincial population numbers from Statistics Canada from 1992–2014 (https://www150.statcan.gc.ca/t1/tbl1/en/tv.action?pid=1710000501).We obtained population data for all countries from the CIA World Factbook website for 1990–2017. The US Bureau of the Census provides an annual estimate in July of the population for each country from population censuses and vital statistics registration systems , utility terrain vehicle (UTV), off highway vehicle (OHV), and heavy vehicle  and other vehicles). The code and data used in the analysis are available at: We documented over 4000 winter drownings , which cGenerally, winter drownings rapidly increased as warmer winter air temperatures approached 0°C . At wintWinter drownings were more likely to occur towards the end of winter, generally coinciding with periods when ice is more likely to be weak as air temperatures warm and the sun is higher in the sky . Most drAs a case study, we used detailed data from Minnesota, USA to illuminate additional risk factors for winter drowning, including providing details on the age and activity of the victim at time of drowning. The most vulnerable age groups for winter drowning were children and young adults . ChildreIn addition to victim’s age, certain activities were more closely associated with drowning. Drowning while driving a light vehicle was more common than drowning while driving a heavy vehicle owing to the popularity of snowmobiles . SpecifiOur results underestimate the true consequences of through-ice drownings as we were unable to collect data on non-fatal drownings. Cold-water immersion is associated with unique and rapid physiological consequences that that can increase the likelihood of drowning. Humans immediately experience the cold shock response (gasping and uncontrolled breathing) for up to 2 minutes, and cold incapacitation which increases from 2 to 20 minutes; this can cause drowning very quickly. If the airway is protected during immersion, mild hypothermia occurs after 30–60 minutes, and unconsciousness, cardiac arrhythmia and death can occur after 2 or more hours; this will occur earlier with children due to their decreased body mass , 10. SurDrowning requires immediate intervention, so prevention strategies are more effective for reducing drowning than rescue or treatment  Strong iThe potential for increased winter drownings is a previously unrecognized consequence of warmer winters. Excess winter deaths have been difficult to correlate to cold temperatures because of the influence of seasonal factors such as influenza, respiratory infections, and cardiac risk . HoweverOur results suggest that milder winter air temperatures will have implications for winter drownings in countries that have historically had ice cover on inland waters. The complex nature of changing winters including warming temperatures, rain on snow, and freeze-thaw events could decrease the stability of ice , 20, 40,S1 FigWinter drownings are shown as a percentage of total annual drowning deaths with median quartile, range, and extremes for the time period during which we collected records for all countries. The number of years of data collected with monthly drowning data is included above the box plot for each county.(DOCX)Click here for additional data file.S1 TableFor each of the 11 countries, we provide details on the types, sources and time periods of data that we used in this study. ICD Diagnosis codes are as follows: W69 , W70 , and W71 .(DOCX)Click here for additional data file.


IgE-binding monocytes were analyzed monthly in Culicoides hypersensitive horses and nonallergic horses living together with natural exposure to Culicoides midges. The phenotype and frequency of IgE-binding monocytes remained consistent in all horses regardless of Culicoides exposure. All horses upregulated IgE-binding monocyte CD16 expression following initial Culicoides exposure. Serum total IgE concentration and monocyte surface IgE densities were positively correlated in all horses. We also demonstrated that IgE-binding monocytes produce IL-10, but not IL-4, IL-17A, or IFN-γ, following IgE crosslinking. In conclusion, we have characterized horse IgE-binding monocytes for the first time and further studies of these cells may provide important connections between regulation and cellular mechanisms of IgE-mediated diseases.Human IgE-binding monocytes are identified as allergic disease mediators, but it is unknown whether IgE-binding monocytes promote or prevent an allergic response. We identified IgE-binding monocytes in equine peripheral blood as IgE+/MHCII Culicoides (Cul) hypersensitivity. This disease is also frequently called insect bite hypersensitivity (IBH), summer eczema, summer seasonal recurrent dermatitis, or sweet itch [Cul midges [The most prevalent, naturally occurring allergy in horses is known as eet itch –9. Allerl midges ,10. Reacl midges . Exposurl midges ,18.2) and is lacking the β chain, which is part of the tetrameric receptor (αβγ2) on basophils and mast cells [2 FcεRI is involved in antigen recognition [A variant of FcεRI is expressed on human antigen-presenting cells including monocytes ,20. The st cells –18,21–24st cells ,26. In hognition ,27 whereognition –30.Cul allergens are inherently innocuous [Cul hypersensitivity [Monocytes exhibit highly plastic functions and are responsible for rapid migration towards sites of inflammation, phagocytosis of invading pathogens or allergens, and presentation of foreign epitopes to T cells in regional lymph nodes ,32. Imponnocuous –36, the sitivity . The exaCul hypersensitivity. We also developed a high purity sorting protocol of IgE-binding monocytes that enabled the characterization of intrinsic properties of these cells. We asked the following questions: (1) What surface and secreted proteins characterize IgE-binding monocytes in horses? (2) Do cell numbers and/or does cell surface protein expression change during seasonal environmental exposure to Cul in IgE-binding monocytes of allergic and nonallergic horses? (3) What cytokines are produced by IgE-binding monocytes in response to IgE-dependent stimulation?In humans, IgE-binding monocytes are present in all individuals but express higher densities of FcεRI in atopic dermatitis patients, compared with healthy controls ,20,38. CCulicoides (Cul) hypersensitivity. Allergic (n = 7) and nonallergic (n = 7) groups both consisted of sex-matched Icelandic horses (6 mares and 1 gelding per group). The two geldings were 6-years and the mares were ages 6–15 years. Peripheral blood mononuclear cells (PBMC) from these horses were used for all flow cytometry experiments and RNA extractions. In addition, PBMC from allergic (n = 8) and nonallergic (n = 8) groups composed of one stallion (6 years), two geldings (7 and 8 years), and 13 mares (6–16 years) were used for cytokine secretion experiments. All horses lived together in the same geographic location and environment in Ithaca, NY for at least two years prior to this study. They were kept on large pastures 24/7 with free access to water, mineral salt blocks, grass in the summer and grass hay in the winter months. All horses were annually vaccinated against rabies, tetanus, West Nile virus, and Eastern and Western Encephalitis virus. They were also dewormed twice a year. Vaccination and deworming were performed for all horses on the same days.Horses studied for this approach were either clinically healthy or were affected with Cul exposure before and also during the study. Cul-specific hypersensitivity was confirmed by intradermal skin testing with Cul whole body extract  in comparison to saline and histamine controls as previously described [Cul WBE. Nonallergic horses never exhibited clinical signs scoring above 3 . Both heparinized blood samples and serum samples were collected once a month for one year (April 2018 to March 2019).Clinical scores  were assescribed ,17. Alle above 3 , and alsAll animal procedures were carried out in accordance with the recommendation in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The animal protocol was approved by the Institutional Animal Care and Use Committee at Cornell University (protocol #2011–0011). The study also followed the Guide for Care and Use of Animals in Agricultural Research and Teaching.PBMC were isolated from heparinized blood of all horses by density gradient centrifugation of the cell-rich plasma . For flow cytometry analysis, heparinized blood was allowed to settle at room temperature for 1–2 hours before collection of cell-rich plasma. For cell sorting, heparinized blood sat at room temperature for 2 hours to allow for maximal separation of neutrophils and erythrocytes from the other cells in the cell-rich plasma. Following density gradient isolation, PBMC were washed in PBS and either used immediately for cell sorting or fixed in 2% (v/v) formaldehyde (SigmaAldrich) for 20 minutes at room temperature prior to cell surface staining and flow cytometry. PBMC for RNA isolation were isolated as described above, pelleted, and snap frozen in liquid nitrogen.Fixed PBMC were incubated for 15 minutes at room temperature with multiple antibody master mixes  in PBS-Bhigh/CD14low) in the monocyte/large lymphocyte forward (FSC) and side scatter (SSC) were analyzed quantitatively. CD16 and CD163 expression of IgE+/MHCIIhigh/CD14low cells was also calculated. Median fluorescence intensity (MFI) of anti-IgE (AlexaFluor 488) was calculated on IgE-binding monocytes (IgE+/ CD14low).For sample analysis, 100,000 events per sample were recorded on a FACS Canto II flow cytometer  and data analysis was performed with FlowJo version 10.4 . Compensation was calculated with input from single stained UltraComp beads (ThermoFisher). IgE-binding monocytes  [2 to label the mouse-anti-horse CD14 mAb . 1x10one 105)  in 1.2 mhigh/CD14low/CD16-/CD163- cells were collected into cell culture medium (DMEM supplemented with 1% (v/v) non-essential amino acids, 2 mM L-glutamine, 50 μM 2-mercaptoethanol, 50 μg/ml gentamicin, 100 U/ml penicillin, 100 μg/ml streptomycin , and 10% FCS  at room temperature. Following sorting, IgE-binding monocytes were counted and tested for viability using trypan blue exclusion. All samples resulted in >95.5% viable (average 99.2% live cells).For FACS following CD14 enrichment, the CD14+ fraction was further incubated with mAbs  and RNA was extracted using a RNeasy Mini kit (QIAGEN) and DNase digestion (QIAGEN). cDNA was generated using random hexamers  and gene specific primers were used to amplify unique regions of each equine FcεRI subunit gene with a PlatinumTM PCR SuperMix High Fidelity master mix (Invitrogen). The three equine genes and primer pairs were: FCERIA , FCERIB , FCERIG . The FcεRI gamma chain (i.e. common Fc receptor gamma chain) is conserved in FcεRI and FcγRs, and therefore served as a positive control.Aliquots of 1x105 cells/well) were stimulated with 3 μg/ml anti-equine IgE clone 134 (IgE mAb 134) which induces crosslinking of the IgE receptor [Culicoides whole body extract , or 3 μg/ml anti-equine IgE clone 176 (IgE mAb 176) as a negative control which does not induce crosslinking [2 for 24 and 48 hours before supernatants were collected and stored at 4ºC until analysis. Cytokine production was measured using a fluorescent bead-based multiplex to simultaneously detect equine IL-10, IL-4, IL-17, and IFN-ɣ. Assay was set up as previously described [CD14+ MACS enriched cells  were plated in 35 mm MatTek No. 1.5 coverslip dishes (MatTek Corporation) and incubated in cell culture media or with 3 μg/ml IgE mAb 134 for 24 hours. A final concentration of 10 μg/ml Brefeldin A  was added during the last 4 hours of incubation before cells were fixed in MatTek wells in 2% (v/v) formaldehyde (SigmaAldrich). Following fixation, cells were incubated with mAbs described above in For visualization of IL-10 production by IgE-binding monocytes, CD14+ MACS enriched cells  following the recommended manufacturer’s protocol and as previously described [4 beads/ml. Native equine IgE purified from supernatant of equine B cell/murine X63-Ag8.653 myeloma cell hetero-hybridoma clone 37–1 was used as standard for quantification [Equine IgE mAb 176  was coupescribed . The totescribed . Brieflyfication . Concenthttp://www.luminexcorp.com). The data were reported as median fluorescent intensities (MFI) and total IgE concentration was calculated with a standard curve fit and logistic 5p formula (y = a+b/(1+(x/c)d)f) (Luminex 100 Integrated System 2.3).Millipore Multiscreen HTS plates  were soaked with PBS-T (PBS supplemented with 0.1% (w/v) BSA, 0.02% (v/v) Tween 20 and 0.05% (w/v) sodium azide) using an ELx50 plate washer  for 3 minutes. PBS-T was aspirated from wells and replaced with 50 μl serum samples diluted 1:50 in blocking buffer or 50 μl of each diluted concentration standard. 50 μl of diluted IgE mAb 176 coupled #35 bead solution was added to each well and incubated on a shaker at room temperature in the dark for 30 minutes, and then washed with PBS-T. Biotinylated IgE mAb 134 was diluted to 8 μg/ml in blocking buffer and 50 μl was added to each well, incubated for 30 minutes as above, and washed with PBS-T. Streptavidin-phycoerythrin  was diluted to 10 μg/ml in blocking buffer, 50 μl was added to each well, incubated for 30 minutes as above, and washed with PBS-T. Beads were resuspended with 100 μl/well blocking buffer and analyzed in a Luminex 200 instrument .low/MHCIIhigh/CD16-/CD163- cells  subunits was analyzed in monocytes to identify if IgE on equine monocytes is bound via FcεRI. Viable IgE+ monocytes were purified  using magnetic and subsequent fluorescence activated cell sorting . Sort pulow, see 2) and that these cells are IgE-binding monocytes.In addition, low affinity IgE receptor FcεRII, also named CD23, expression on IgE+ monocytes was determined via cell surface staining and flow cytometric analysis of unsorted PBMC. While IgE+/CD14- cells were FcεRII/CD23+, IgE+ monocytes  compared to April  when Cul were not present  between serum total IgE and IgE on the surface of monocytes was 0.384. . We observed that IL-10 production by IgE-binding monocytes varied between seasons with or without Cul exposure. Future exploration of IL-10 production throughout changes in allergen exposure may highlight a temporal importance for IL-10 production leading to allergen tolerance, or may increase the importance of looking for more robust indicators of immune regulation in Cul hypersensitivity. It is also possible that additional regulatory factors may contribute to IgE-binding monocyte mediated allergen tolerance in nonallergic horses, but not allergic horses.The stimulation experiments in this study were conducted during the end of Our IL-10 production results stand in contrast to human studies, which showed increased IgE-mediated IL-10 production only in humans with atopic disease . IL-10 has canonically been attributed to the development of immune tolerance ,62. HoweCul hypersensitivity. One possible explanation for the differences between prior human studies and our horse study could be due to uncontrolled variables inherent to human cohorts. Similar uncontrolled variables, differences in housing environments, and seasonal variation between sample collection timepoints also applied to previous horse studies on Cul hypersensitivity [Cul exposure between all horses. Human atopic disease is a highly heterogeneous condition that develops in response to a wide range of inducing allergens [Cul hypersensitivity is an allergic reaction against known and characterized allergens [While human studies present multiple differences between IgE-binding monocytes in healthy and allergic individuals that could serve as predictive biomarkers of disease, the results in our equine model do not confirm IgE-binding monocytes as biomarkers of sitivity ,12,16–18llergens , while Cllergens –36. As aWe also detected upregulation of CD16 on IgE-binding monocytes in all allergic and nonallergic horses during the summer months. CD16 (FcγRIII) is an IgG receptor that is primarily found on monocytes and large lymphocytes in horses, but some CD16+ granulocytes and lymphocytes are also present . FcεRI aThe initial characterization of IgE-binding monocytes in the horse, the second species besides humans in which these cells have now been identified, will enable us to further explore mechanistic and regulatory functions of IgE-binding monocytes specifically in response to allergen and IgE stimulation. In the horse model this can be performed with fewer confounding variables than in humans. In order to further characterize the mechanistic roles of equine IgE-binding monocytes during allergic responses, immunologic, genomic and proteomic approaches will provide valuable unbiased insight into the inflammatory or anti-inflammatory bias of IgE-binding monocytes in response to IgE signaling.low/MHCIIhigh cells. Overall, IgE-binding monocyte characteristics and functions were similar in both clinically healthy and allergic horses. They bind IgE via an αγ variant of FcεRI lacking the β chain. Monocyte surface IgE was moderately positively correlated to serum total IgE in all horses throughout the year. Following environmental Cul exposure, IgE-binding monocytes upregulated CD16 expression. In all horses, IgE-binding monocytes secreted IL-10 upon IgE crosslinking.IgE-binding monocytes can be identified and characterized in peripheral blood of horses as IgE+/CD14S1 Figlow/MHCIIhigh/CD16-/CD163-), converted to cDNA, and amplified with gene specific primers for equine FcεRI α, β, and γ mRNA. RNA samples were also amplified as a negative control. Samples were loaded onto a 1% agarose gel, run at 90V/400A and imaged with GelRed. All cells came from the same horse on the same day. PCR reactions and gels were run simultaneously. White boxes denote cropped images included in RNA was extracted from snap-frozen cells Click here for additional data file.S2 FigIndividual percentages, median fluorescent intensities and concentrations for each horse that were used to generate figure graphs are compiled in labeled data tables. (A) Percentage of IgE+ monocytes out of total cells in unsorted, MACS sorted and MACS+FACS sorted samples from 18 different horses in (XLSX)Click here for additional data file.S3 FigCD14+ MACS sorted cells were incubated in MatTek coverslip wells in the presence of IgE mAb 134 for 24 hours at 37ºC. Cells were fixed and incubated with fluorescently coupled mAbs against CD14 and IL-10. 16-bit images were taken at 65x magnification under (A) brightfield, (B) 488 nm laser excitation of CD14 mAb staining, and (C) 633 nm excitation of IL-10 mAb staining. White boxes denote cropped images included in (TIF)Click here for additional data file.


We find that the resistivity of VO2(A) and VO2(B) is widely tunable by doping them with tungsten ions. When x < 0.1 in V−x1WxO2(A), the resistivity decreases drastically by four orders of magnitude with increasing x, while that of V−x1WxO2(B) shows the opposite behaviour. Using spectroscopic ellipsometry and X-ray photoemission spectroscopy, we propose that correlation effects are modulated by either chemical-strain-induced redistribution of V−V distances or electron-doping-induced band filling in V−x1WxO2(A), while electron scattering induced by disorder plays a more dominant role in V−x1WxO2(B). The tunable resistivity makes correlated VO2(A) and VO2(B) appealing for next-generation electronic devices.Applications of correlated vanadium dioxides VO Therefore, VO2 polymorphs have been extensively studied for a range of interesting applications. VO2(M1) and VO2(R) have attracted wide interest for electronic devices since they show heat-, light-, electric field-, and chemical-induced reversible metal-insulator transitions near room temperature (TMI = 340 K in bulk)7. On the other hand, VO2(A) and VO2(B) have been mainly used for energy applications, including redox-flow batteries, ion batteries, solid oxide fuel cells, hydrogen storage devices, and catalysts8. Compared to the many studies on electronic devices using VO2(M1) and VO2(R), however, the scarcity of published methods to tune the electrical properties of correlated VO2(A) and VO2(B) has limited their potential applications in electrical devices.Since the level of correlation effects between neighbouring electrons in the outermost 32(M1) since broad tunability of electrical properties is valuable for electrical devices. In addition to metallization induced by pressure application9, hydrogen doping11, or ionic liquid gating13, cation doping has been widely used as a method with high efficacy. Chromium doping can result in transition of the dimerization of V−V chains in the VO2(M1) phase into partial dimerization in the VO2(M2) phase14. Substituting a small amount of tungsten for vanadium in VO2(M1) causes notable changes in its electrical property15. For convenience, V−x1WxO2 will be used herein to denote VO2 with tungsten doping of x × 100%. As x increases, the resistivity of V−x1WxO2 films decreases by several orders of magnitude, and TMI also rapidly decreases at a rate of dTMI/dx = 2,100–2,800 K. For 0.08 < x < 0.09, the V−x1WxO2 epitaxial films have a metallic ground state for a wide temperature range of 50–400 K. When x is increased beyond this range, V−x1WxO2 reenters its insulating state.Several methods have been suggested for tailoring the correlation effects of VO2(A) and VO2(B) would not only enable their usage in electrical devices but also promote rich functionalities for energy devices. Motivated by research on correlated VO2(M1), here, we investigate the effects of tungsten doping on the resistivity of correlated VO2(A) and VO2(B). We organize this paper as follows: first, we describe how, as the tungsten concentration increases, chemical strain increases the lattice parameters of both VO2(A) and VO2(B). Next, we find that tungsten doping is effective for tuning the resistivity of correlated VO2(A) and VO2(B) over a broad range. For x < 0.1–0.15, VO2(A), an insulator in the pure phase, exhibits a monotonic decrease in resistivity of four orders of magnitude with increasing x. VO2(B), a semiconductor in the pure phase, shows a monotonic increase in the resistivity of two orders of magnitude. Finally, to understand these opposite dependences, we explore the systematic evolution of the electronic structures and vanadium oxidation states by performing spectroscopic ellipsometry and X-ray photoemission spectroscopy (XPS), respectively.Exploring tuning knobs of the electrical properties of VO−x1WxO2(A) and (001)-oriented V−x1WxO2(B) epitaxial films on (011)SrTiO3 and (001)LaAlO3, respectively, for x from 0 to 0.25. We provided details of the deposition conditions in our previous reports18 as well as in the Methods section. Figure θ − 2θ scans of V−x1WxO2(A) and V−x1WxO2(B), respectively . We clearly observed (600)VO2(A) diffraction peaks for x < 0.1 and (002)VO2(B) diffraction peaks for x < 0.15, indicating preservation of the crystal structures for x < xc ≈ 0.1–0.15. However, for x > xc, these XRD intensities significantly decreased, and some peaks disappeared, indicating that heavy doping of tungsten could lower the quality of our epitaxial VO2(A) and VO2(B) films  were additionally required to resolve the XPS spectra of V−x1WxO2(A) . It should be noted that the V3+ peaks were stronger with increasing x. This similar observation between V−x1WxO2(A) and V−x1WxO2(M1) indicates that electron-doping-induced band filling also plays an important role in tungsten-doped metallization30.As an alternative mechanism for V(A) Fig.  with V4+3+ XPS peaks in V−x1WxO2(B)  became more insulating with increasing x. Therefore, we hypothesize that another mechanism, different from those for V−x1WxO2(A) and V−x1WxO2(M1), plays an important role in the tunable properties of V−x1WxO2(B) (x < xc). It should be noted that the correlation effects in VO2(B) and VO2(R) are weaker than those in VO2(A) and VO2(M1), considering their lower resistivities and narrower bandgaps2. Therefore, we suggest that the increase in resistivity in V−x1WxO2(B) originates from disorder-induced electron scattering. Since more dopants will scatter more electrons, the resistivity will increase with heavier doping33.Although we also found significant evolution of the V(B) Fig. , it is q2(A) and VO2(B). XRD revealed that the crystal structures of V−x1WxO2(A) and V−x1WxO2(B) expanded and were well preserved for x < 0.1–0.15. At this low doping concentration, the resistivity of V−x1WxO2(A) decreased, similar to that of V−x1WxO2(M1), with increasing tungsten concentration; in contrast, the resistivity of V−x1WxO2(B) increased, similar to that of V−x1WxO2(R). Spectroscopic ellipsometry revealed that tungsten doping resulted in a redshift of the unoccupied t2g orbital, the creation of an in-gap state in V−x1WxO2(A), and a slight blueshift of unoccupied t2g orbitals in V−x1WxO2(B). Both vanadate films showed evolution of the V3+ oxidation states based on the XPS study. Referring to the mechanisms in correlated V−x1WxO2(M1), we proposed that V−x1WxO2(A) and V−x1WxO2(B) showed opposite dependences due to either chemical-strain-induced redistribution of V−V distances or electron-doping-induced band filling and disorder-induced electron scattering, respectively. We leave further consideration of the mechanisms for future studies.Tungsten doping provided an effective way to tune the resistivity of correlated VO2(A) and VO2(B) enables their use in next-generation electronic devices, as well as energy devices. As we mentioned in the Introduction section, VO2(M1) has shown a reversible resistivity change due to intercalation of hydrogen11 and ionic liquid gating13. The similar doping dependence between VO2(A) and VO2(M1) suggests that these dynamic control methods would enable application of correlated VO2(A) [also VO2(B)] in memories, transistors, and gas sensors, as has been extensively studied for VO2(M1).The extreme tunability of correlated VO2(A) and VO2(B) on either perovskite oxides or Y-stabilized ZrO218. Using pulsed laser epitaxy, we deposited VO2(A) and VO2(B) epitaxial films on (011)-oriented SrTiO3 and (001)-oriented LaAlO3 substrates, respectively. We ablated a tungsten-doped V2O5 target with a KrF (248 nm wavelength) pulsed laser at a rate of 10 Hz and an intensity of 1 J cm−2. For the targets, we mixed WO3 and V2O5 powders in the desired molar ratio and sintered pellets at 650 °C for 12 hours in air. We used this low sintering temperature due to the low melting point (690 °C) of V2O5. We fixed the substrate temperature at 420 °C since VO2(A) and VO2(B) are thermodynamically unstable above 430 °C and transition into VO2(R) above 470 °C29. We used a flow of oxygen gas with a partial pressure, 2(A) and 15 mTorr for VO2(B) since VO2 stably forms in only a narrow range of 5 mTorr <34.We recently optimized the growth conditions for epitaxial films of VO35. We deposited evenly spaced Pt contacts on the middle of the film surface. We applied a small constant current through the outer two contacts and measured the voltage between the inner two contacts. We swept the temperature in the range of 10–400 K. We measured the reflectance, R(ω), spectra in a photon energy range of 0.1–1 eV via a Fourier transform-type infrared spectrometer . We employed an in situ gold overcoating technique to obtain an accurate absolute value of R(ω). We obtained the optical conductivity of the VO2 film via a two-layer model fit of the measured R(ω) with Drude-Lorentz oscillators36. We used spectroscopic ellipsometers  to obtain the complex dielectric constants, σ1(ω), in this energy range can be calculated by 23, where To investigate the electrical transport properties, we used a physical property measurement system (Quantum Design Inc.). We used the four-point probe method, which is the most common method for measuring the resistivity−8 Torr. To remove contamination, the film surface was sputtered with argon ions for 10 seconds16. We used the O 1 s peak at 530.0 eV as the energy reference. We supplied electrons using an electron gun to avoid any charging effect.We investigated the crystal structures via a four-circle high-resolution X-ray diffractometer  using Cu radiation with a wavelength of 1.5406 Å. Using the fringe patterns obtained in X-ray reflectivity measurements, we confirmed that the films had an ~100-nm thickness Fig. . To deteSupplementary information.


Systematic reviews have highlighted that repeated severe wasting after receiving treatment is likely to be common, but standardised measurement is needed urgently. The Council of Research & Technical Advice for Acute Malnutrition (CORTASAM) released recommendations on standard measurement of relapse (wasting within 6 months after exiting treatment as per recommended discharge criteria), regression (wasting within 6 months after exiting treatment before reaching recommended discharge criteria) and reoccurrence (wasting after 6 months of exit from treatment as per recommended discharge criteria). We provide a theoretical framework of post‐treatment relapse and regression to severe wasting to guide discussions, risk factor analyses, and development and evaluations of interventions. This framework highlights that there are factors that may impact risk of relapse and regression in addition to the impact of contextual factors associated with incidence and reoccurrence of severe wasting more generally. Factors hypothesised to be associated with relapse and regression relate specifically to the nutrition and health status of the child on admission to, during and exit from treatment and treatment interventions, platforms and approaches as well as type of exit from treatment . These factors influence whether children reach full recovery, and poorer nutritional and immunological status at exit from treatment are more proximate determinants of risk of severe wasting after treatment, although post‐treatment interventions may modify risks. The evidence base for many of these factors is weak. Our framework can guide research to improve our understanding of risks of relapse and regression and how to prevent them and inform programmes on what data to collect to evaluate relapse. Implementation research is needed to operationalise results in programmes and reduce post‐treatment severe wasting at scale. We propose a theoretical framework to harmonise efforts for reducing relapse and regression to severe wasting after treatment, distinguishing contextual factors with broad influence on risks of wasting from additional factors hypothesised to impact risks of relapse and regression.Factors hypothesised to be associated with relapse and regression relate to the status of the child at admission, treatment interventions and type of exit from treatment. These influence whether children reach full recovery, which determines relapse risk, although post‐treatment interventions may modify the risk.Our framework can guide much needed research on risk factors for relapse and regression and how to prevent them and inform programmes on what data to collect.1z‐score (WHZ) (or weight‐for‐length in children aged under 24 months), low mid‐upper arm circumference (MUAC) and/or the presence of nutritional oedema. The most severe form of wasting, called ‘severe wasting’, is diagnosed by having a WHZ < −3 or MUAC < 115 mm, whereas ‘moderate wasting’ is diagnosed by WHZ < −2 but > −3 or MUAC between 115 and < 125 mm  has recently recommended definitions and use of the following terms related to severe wasting . The development of this framework was informed by technical consultations, involving experts from academic and nonacademic institutions. See the 22.1Relapse and regression to severe wasting after exit from treatment occurs within a broader socio‐economic and ecological environment with contextual factors acting at different levels  Figure . These fNutritional and immunological status at exit from treatment may also be associated with reoccurrence of severe wasting after 6 months. It seems likely, however, that the longer the time interval between exit from treatment and reoccurrence of severe wasting is, the less influential these factors become, and the more influential broader contextual factors are. The focus in this article is on severe wasting within 6 months of exit from treatment.2.22.2.1Risks of relapse and regression to severe wasting are likely related to the extent to which a child treated for severe wasting reaches full recovery, which in turn is influenced by the characteristics of the child before entry into treatment. A cohort study in Ethiopia   for managing severe wasting at the community level  and increased relapse risks were found in a study in India  to provide a comprehensive view on programme performance and identify children at increased risk of relapse and wasting‐related mortality. Treatment of severe wasting should not be considered a standalone intervention but part of a continuum of care for children at risk of morbidity and mortality from wasting. Evaluations of these programme components that ensure continuity of care and their effects on severe wasting after exit from treatment is key and our framework can guide the development and evaluation of post‐treatment interventions. Particularly, CHWs are uniquely positioned to provide this continuum of care, collect information on children after exit from treatment, support households and assess the root causes of wasting in communities and households, so the role of CHWs to track children after exit from treatment and prevent relapse and regression should be further explored and evaluated. Robust trials are needed to identify efficacious interventions to reduce relapse and regression after exit from treatment, and these studies need to have a wide geographical representation to understand how contextual factors influence relapse and interact with interventions. Although, in practice, it may not be possible to differentiate between ongoing episodes of wasting and regression after incomplete recovery, programmes and research studies should, where possible, distinguish relapse from regression. This would improve the identification of determinants of these outcomes and of children most at risk of severe wasting after exit from treatment, which further supports the identification of efficacious interventions to prevent post‐treatment severe wasting. Implementation research needs to build on research studies to operationalise results in programmes. Ensuring these links between research and programmes is critical to reduce post‐treatment relapse and regression to severe wasting at scale. Our framework can be a starting point to stimulate and guide much needed research to improve our understanding of severe wasting after exit from treatment, how to identify children most at risk of relapsing and regressing and thus wasting‐related mortality and morbidity, and how to prevent wasting after treatment exit. The framework should be revisited and revised as needed in light of future evidence in order to most effectively support progress towards global wasting‐reduction targets.The authors declare that they have no conflicts of interest.RS, AM, MMcG and RB developed the first draft of the framework. All authors contributed to development of the final version of the framework. RS prepared the first draft of the article, with input from AM, MMcG and RB. All authors contributed to the final version of the manuscript. All authors have read and approved the final manuscript.Data S1 Supporting InformationClick here for additional data file.