Soil-transmitted helminthiasis (STH) is among the most common of parasitic infections, affecting vulnerable populations in tropical/subtropical areas globally. In endemic countries, children, a high-risk population, require treatment and preventive interventions. Mebendazole, a WHO-recommended medicine, originally formulated as a tablet that was often crushed for administration to young children unable to swallow it, was reformulated as a chewable tablet. Acceptability is a key aspect for treatment effectiveness in pediatrics. Herein, we used a validated data-driven approach to investigate the acceptability of the 500-mg mebendazole chewable tablet in children aged 2 to 4 years in Peru. Observer-reported outcomes were collected for 182 medicine intakes. Acceptability was scored using the acceptability reference framework: a three-dimensional map juxtaposing “positively accepted” and “negatively accepted” profiles. Results found that the 500-mg mebendazole chewable tablet was classified as “positively accepted” in children aged 2 to 4 years. Acceptability increased with age and some acceptability issue remain for the younger children. Nevertheless, this formulation was considerably better accepted than the conventional tablets regardless of treatment in young children. This chewable formulation appears to be an appropriate alternative to the hard tablet of mebendazole for treatment of STH and preventive interventions in children aged 2 to 4 years. Soil-transmitted helminthiasis (STH) is the most prevalent parasitic infection among the neglected tropical diseases (NTD), with chronic impacts on health, social, and economic development of affected populations ,2,3. STHA total of 24% of the world’s population, or approximately 1.5 billion people, are infected by soil-transmitted helminths. Some 267 million preschoolers and more than 568 million school-age children live in areas with high infection rates, requiring treatment and preventive interventions [A. lumbricoides (roundworm) and T. trichiura (whipworm) was established in children aged 1 to 16 years [Mebendazole and albendazole are the two World Health Organization (WHO)-recommended treatment drugs . Althoug16 years , as well16 years . Only th16 years . This mu16 years .In the Americas, Peru is among the countries with the greatest prevalence of helminthiasis. Since 2017, Peru’s National Ministry of Health has provided mass drug administration (MDA) of antiparasitic (deworming) medicine to all people 2 years of age and older as a public health measure to control intestinal parasitic infection. In this context, a study on acceptability of 500-mg mebendazole chewable tablet in children was conducted by INMED Andes in 2019. Unpublished results showed that the acceptability of the chewable tablet by children increased with age, although it appeared to not be related to sex, population type , or ethnic group. Children from 5 to 11 years of age were found to sufficiently accept the chewable tablet. These results were in line with the study of Palmeirim et al. . An impoThis multicentric, cross-sectional, and observational study was conducted in Peru between May and June 2021. The objective was to generate evidence on the acceptability of the 500-mg mebendazole chewable tablet (Vermox™) among young children aged 2 to 4 years, and investigate the potential influence of age, sex, and population type .Subjects were enrolled in selected sample preschools in urban (Yarinacocha) and rural (Manantay) districts of the jungle region of Ucayali, where STH infection is endemic, during Peru’s first national deworming campaign of 2021.According to local regulations, the present study was approved by the Ethics Committee of the Ucayali Health Ministry on 21 May 2021 (FUT 0082452).Receiving or received other anthelmintic treatment in the last 6 months;Receiving metronidazole for any other diagnosed illness in the last 15 days;Known allergies to the compounds in 500-mg mebendazole;Presence or history of important systemic or chronic diseases, diagnosed by a doctor;Fever at the time of observation;Acute respiratory disease;Acute or chronic diarrheal disease;Children, parents, or people who live within the child’s household currently diagnosed with COVID-19;Congenital malformation of the mouth or palate that hinders oral administration of medication;Participating in another clinical or similar study concurrently.Subjects aged 2 years (≥24 to <36 months), 3 years (≥36 to <48 months), and 4 years (≥48 to <60 months) were treated for STH with the 500-mg mebendazole chewable tablet during the deworming campaign, with a written informed consent obtained from the parent(s)/legal representative. Children were not included in the study if they met one of the following exclusion criteria:A minimum of 30 evaluations of medicine intake were necessary to get an acceptability score with a satisfactory precision using the CAST methodology. Samples of 60 subjects  per age group  were targeted for participation.Health personnel responsible for drug administration during the deworming campaign and specially trained for the acceptability study administered the chewable formulation to children in their homes or local facilities (due to COVID-19 related school closures). The tablets remained in their original packaging until they were administered to the children. Health personnel recorded the following objective measures on field data forms, including events/behaviors that were observed during the intake of the medicine under investigation:Results of intake ;Patient reaction during the administration using a 3-point facial hedonic scale ;o Prepare the dose ,o Administer the required dose of medication (from a required dose of medication ready to use to the end of the intake);Time needed to:Dividing the intake of a prescribed dose of the medication which cannot be taken as a whole ;o Modifying the dosage form ,o Using another route/mode of administration;Altering the use, such aso The prescribed dose of the medication had to be mixed with unintended drink or food ,o The child had to take drink or food just before or after the dose administration ;Using food/drinkUsing a device not provided with the medication ;Promising a reward;Using restraint .For each method used to help with taking the medicine (the last six bullet points) the observers were requested to specify any further information in a text field . In addition, health personnel reported patient characteristics , context of use , and any remarks on medicine use. The field data were entered into the electronic database system by the health personnel coordinators. Thereafter, data were reviewed by the data manager and data collection lead group. The principal investigator clarified any discrepancies with health personnel and their coordinators.In the data table, each row corresponded to one evaluation of the intake of 500-mg mebendazole chewable tablet by a specific patient, and each column represented one of the nine aforementioned observational variables  describing the many aspects of acceptability, with an observed measure  being entered into each corresponding cell. The sum of preparation and administration times was transformed into a categorical variable and classified as short (1 min and less), medium (from 1 to 2 min and 30 s), or long (longer than 2 min and 30 s). In addition to the nine active variables included in the multivariate analysis, each row was associated with supplementary variables describing the patient and the context of use.In this study we used the acceptability reference framework: a three-dimensional map (3D-map) juxtaposing two distinct acceptability profiles—"Positively accepted” and “Negatively accepted”—materialized by green and red areas on the 3D-map, respectively. This intelligible tool was developed based on 1778 standardized evaluations, without missing observed measure, of the intake of multiple oral/buccal medicines in children under 12 years of age. These evaluations were previously collected in eight countries  using the standardized questionnaire. As previously described ,16,17,18The evaluations collected in this study were not included in the dataset of 1778 standardized evaluations that gave rise to the acceptability reference framework. They were included in the multivariate analyses as supplementary information, with no influence on the factorial method. Thus, the evaluations of 500-mg mebendazole chewable tablet from this study were plotted on the 3D-map, allowing for scoring process implementation.The 500-mg mebendazole chewable tablet was positioned on the reference framework at the barycenter of all its evaluations collected in this study and plotted on the 3D-map. Confidence ellipses surrounding the barycenter for all dimension pairs defined an area containing its true position with 90% probability if the experiment was to be repeated. If the barycenter, along with the entire confidence ellipsis surrounding it, belonged to the green area of the map, the medicine was classified as positively accepted.No evaluation of the intake of the mebendazole hard tablet–original form–in children was collected during this study. The acceptability score of 500-mg mebendazole chewable tablet was thus compared to the average score of conventional hard tablets regardless of treatment in the age group of interest—children aged 2 to 4 years—to obtain a relative acceptability evaluation. This acceptability score was determined relative to previous evaluations from the dataset that gave rise to the acceptability reference framework. If confidence ellipses around distinct barycenters did not overlap on the map, acceptability scores were significantly different.Subsequently, the evaluations of 500-mg mebendazole chewable tablet were partitioned into three subgroups according to the age of participating children . Similarly, the influence of sex and population type  was investigated.Pearson’s chi-squared test and Fisher’s exact test were used to determine whether there were statistically significant differences for the observational variables as well as the supplementary variables between the subgroups of children being compared.© (RStudio Team (2016). RStudio: Integrated Development for R. RStudio, Inc., Boston, MA, USA). The R package FactoMineR [Data analyses were performed using R version 1.0.136ctoMineR  was usedIn this study, 182 evaluations of the intake of 500-mg mebendazole chewable tablet in children aged 2 to 4 years were documented. According to the acceptability reference framework, the 500-mg mebendazole chewable tablet was classified as positively accepted in children aged 2 to 4 years considered as a whole . AlthougAcceptability score differences between age groups reflected differences among observer-reported outcomes. The findings indicate that the 500-mg mebendazole chewable tablet was similarly accepted by boys and girls  as well According to the acceptability reference framework, the 500-mg mebendazole chewable tablet was considered as accepted in Peruvian children aged 2 to 4 years as a whole: the medicine was fully located in the green zone of the model defining the “positively accepted” profile. Although only the chewable formulation was administered in children during the deworming campaign, the acceptability reference framework allowed a relative acceptability evaluation: comparing the chewable formulation with conventional tablets regardless of treatment as a surrogate for the original pharmaceutical form of mebendazole. In children aged 2 to 4 years, the chewable formulation of mebendazole was significantly better accepted than hard tablets regardless of treatment. Indeed, the barycenter of the 71 evaluations of tablet intake in this age group from the dataset that gave rise to the acceptability reference framework, and the confidence ellipses surrounding it, were fully located in the red zone of the model defining the “negatively accepted” profile. The chewable formulation improved acceptability significantly for all of the objective measures observed during the medicine intake: the required dose was significantly more fully taken, in a shorter time, with less negative reaction and fewer recourses to methods for achieving administration. Considering the difficulty, or even inability, experienced by young children attempting to swallow SODF ,14, suchResults found that the acceptability increased as children grew older. The 500-mg mebendazole chewable tablet was significantly better accepted in children 4 years of age, while the barycenter for children aged 2 were closer to the negative area. The medicine was positively accepted in children aged 3 and 4 regardless of studied subpopulations. For children aged 2 years of age, a limited part of the confidence ellipses fell in the negative area of the 3D-map. Nevertheless, the chewable formulation was considerably better accepted than hard tablets in children aged 2 to 4 years, even in the younger children aged 2 years, due to an improvement in the different observational variables. According to the summary of product characteristics (SmPC) the chewable formulation should be to chewed completely before swallowing. For younger children, who have difficulty chewing the tablet, an alternate method of administration by first dissolving the tablet in clean water was permitted according to the SmPC. In this study, the alternate method was used for 30% of children aged 2 years, 15% of children aged 3 years, and 5% of children aged 4 years. Although using this permitted alternate method has an impact on the medicine use , drug effectiveness is ensured by the SmPC. For conventional tablets, manipulations are often off-label and may result in critical issues regarding dosing accuracy and bioavailability beyond acceptability issue ,34,35,36There was no influence of sex of children on acceptability. While certain aromas may have contrasting effects on girls and boys, previous study on acceptability of oral antibiotics in children indicated that strawberry flavoring was positively accepted by both girls and boys . In the In this study, we focused on children aged 2 to 4 years. As the 500-mg mebendazole chewable tablets could be used in children aged 12 months and older ,37, invePreventive interventions for STH infections are crucial as those common infections affect deprived communities in tropical/subtropical areas worldwide. Medicine acceptability is at the utmost importance to ensure their effectiveness in pediatrics, reaching a high-risk population. Generating evidence on the acceptability of the new chewable formulation of mebendazole in young children, this study demonstrated that it is an appropriate alternative to the conventional mebendazole hard tablet in preventive chemotherapy for STH in children aged 2 to 4 years.


Severe acute respiratory syndrome coronavirus (SARS-CoV-2) infections result in the temporary loss of smell and taste (anosmia and dysgeusia) in about one third of confirmed cases. Several investigators have reported that the viral spike protein receptor is present in olfactory neurons. However, no study has been published to date showing the presence of viral entry sites angiotensin-converting enzyme 2 (ACE2), neuropilin1 (NRP1), and TMPRSS2, the serine protease necessary for priming the viral proteins, in human nerves that are responsible for taste sensation . We used immunocytochemistry to examine three postmortem donor samples of the IXth  and Xth  cranial nerves where they leave/join the medulla from three donors to confirm the presence of ACE2, NRP1 and TMPRSS2. Two samples were paraffin embedded; one was a frozen sample. In addition to staining sections from the latter, we isolated RNA from it, made cDNA, and performed PCR to confirm the presence of the mRNAs that encode the proteins visualized. All three of the proteins required for SARS-CoV-2 infections appear to be present in the human IXth and Xth nerves near the medulla. Direct infection of these nerves by the COVID-19 virus is likely to cause the loss of taste experienced by many patients. In addition, potential viral spread through these nerves into the adjacent brainstem respiratory centers might also aggravate the respiratory problems patients are experiencing. Since the start of the Covid19 virus pandemic two years ago in 2019 more than 250 million people have been infected and over 5 million have died worldwide. Information about the virus has grown at an amazingly fast pace. The expectation that the number of infected people might be 50–80% of the world’s population suggests that the overall number of patients with neurological disease could become rather significant . Data reAnonymized postmortem human brain samples were obtained from the Human Brain Tissue Bank, Semmelweis University, Budapest, Hungary. The brains were dissected and specific brainstem areas with cranial nerves were isolated. The samples were either flash frozen or embedded in paraffin after formalin fixation. The study conformed to European ethics regulations (TUKEB #189/2015).The paraffin-embedded sections were deparaffinized with SafeClear II  and rehydrated in decreasing concentrations of ethanol followed by heat-induced epitope retrieval (HIER) in 10 mM citrate buffer at pH 6.0 in a microwave oven. Slides were placed in the oven lying flat in a plastic container and covered with the citrate buffer. They were brought to boil at high power (700 W), and then incubated for 5 more minutes at 50% power (350 W). After HIER, the slides were allowed to cool to room temperature in the buffer. Next, Bloxall  dual endogenous enzyme blocking solution was applied to the sections for 15 min before the primary antibodies were added. Primary antibodies used: ACE2 (rabbit), Abcam ab15348; NRP1 pre-conjugated to Alexa-488; Neurofilament (chicken), Abcam ab4680; TMPSSR2 (rabbit), Novus Biologicals NBP3–00492; MBP pre-conjugated to Alexa-647; fibronectin (rabbit) gift from Dr. Ken Yamada . Lung RNA purchased from Ambion  was used as a positive control. The cDNA was diluted to 25–50 ng/μl for downstream applications. PCR analysis of the ACE2R, NRP1, TMPRSS2, and ACTB (housekeeping) cDNAs were performed using the Bio-Rad T100 and REDtaq polymerase, . The primers used are listed in the table below. The PCR products were run on a 2% agarose gel along with a 100 bp ladder .Preparation of cDNA and PCR analysis was performed as follows: Before it was fixed (to perform ICC) a small piece of the frozen sample of the IXth and Xth cranial nerves was broken off the block and placed in Trizol . RNA was prepared according to the manufacturer’s protocol. After quantitation by Nanodrop 1.0 μg of RNA was used to prepare cDNA using a BioLinkTwo immortalized cell lines were used to validate the ACE2 receptor, TMPRSS2 and NRP1 assays: Human oligodendroglioma cells  were obtained from Millipore Sigma-Aldrich  and cultured in DMEM-high glucose medium  with 10% fetal bovine serum and antibiotics. Human primary brain vascular fibroblasts (Cat# H-6076) from Cell Biologics, Inc . These cells were cultured in complete fibroblast medium  that included all required supplements. For immunostainings the cells were put in chamber slides at 35,000 cells/well and fixed with 4% paraformaldehyde. ICC was performed as above and using two step simple fluorescent immunostaining without microwaving and tyramide amplification. Controls were run with secondary antibodies only.Immunostainings of samples from all three donors showed a very similar picture between the two paraffin embedded and the frozen samples of the IX/X cranial nerves. Also, all sections of the cranial nerve samples examined had similar staining patterns; the branches of both nerves had the same distribution of antigens.We first used antibodies to identify the two known entry sites for the virus SARS-Cov-2: ACE2 and NRP1, a widely expressed transmembrane glycoprotein that was found to be a co-receptor, facilitating the entry of the virus ,11. In aACE2, the primary viral binding protein, was present in all structures of the nerves, such as nerve sheaths, connective tissue inside the bundles, small vessels, axons and myelin. We then looked at the overlap of ACE2 and NRP1 by staining cross and longitudinal sections of the nerves and found that both labeled connective tissue and the nerve fibers within nerve bundles ,B. ACE2 Finally, we colocalized ACE2 –H and NRUsing small pieces of the frozen human postmortem, we performed PCR to confirm the presence of mRNA of the viral entry sites and the TMPRSS2 protease. We used human lung tissue as a positive control and no template as a negative control. We used two primer pairs for both TMPRSS2 and ACE2 based on literature data ,15. All As described in the In the last two years information about the SARS-CoV-2 virus has expanded dramatically. The “neuroinvasive capacity” of the virus was described and in human organoids this invasion was prevented by ACE2 antibodies as well as by CSF isolated from Covid patients  At the sIn spite of the fact that the loss of taste is just as prominent as the loss of smell in COVID-19 infections, no studies of human tissues have been published that shed light on the mechanisms responsible for ageusia – most likely because of the difficulty of obtaining appropriate cranial nerve samples. One hypothesis is that the loss of taste is secondary to the loss of smell, but most people can distinguish these problems from one another. Furthermore, the gustatory (taste) pathway is very different from the olfactory one . Taste cIn our study we looked at the vagal and glossopharyngeal nerves in human postmortem samples (non SARS-CoV-2 infected) at the point where the nerves leave (or connect to) the medulla. We found that both of the binding sites for the SARS-CoV-2 viral spike proteins, ACE2 and NRP1, are widely expressed in the nerve bundles, in myelin sheaths as well as axons. Vascular walls also contain these entry sites. Cranial nerves contain a large number of connective tissue cells  that forGiven these results, we tried to imagine how all these cellular elements might play a role in the loss of taste in infected individuals. The gustatory pathway originates in the oral cavity where ACE2 has been shown to be present in human taste buds  and the It should be clear that there is still a lot to learn about how the SARS-Cov-2 virus affects the peripheral and central nervous system. Demonstration of the expression of molecules that it can use as an entry site is just the beginning. We need to find out how it choses cells to infect, how it highjacks neuronal transport mechanisms (anterograde and retrograde), how it uses non-neuronal (fibroblast and immune) cells, and how it evades immune surveillance within the nerve and CNS. We know that eventually a healthy immune system eliminates the virus, because taste usually returns within a matter of weeks, but it will be important to learn more about viral entry to stop CNS infections completely.


Structural and electronic differences compared to bulk SMO result mainly from undercoordination at the film surface. The changed crystal field leads to a depletion of subsurface valence-band states and transfer of this charge to surface Mn atoms, both of which strongly affect the defect chemistry in the film. The result is a strong preference of oxygen vacancies in the surface region compared to deeper layers. Finally, for metastable oxygen vacancies in the substrate, we predict a spatial separation of the defect from its excess charge, the latter being accommodated in the film but close to the substrate boundary. These results show that surface and interface effects lead to significant differences in stability and electronic structure of oxygen vacancies in thin-film geometries compared to the (strained) bulk.Complex oxide functionality, such as ferroelectricity, magnetism or superconductivity is often achieved in epitaxial thin-film geometries. Oxygen vacancies tend to be the dominant type of defect in these materials but a fundamental understanding of their stability and electronic structure has so far mostly been established in the bulk or strained bulk, neglecting interfaces and surfaces present in a thin-film geometry. We investigate here,  3 thin film grown on a SrTiO3 substrate is affected by crystal-field effects at the very surface and the induced electrostatic potential.The oxygen vacancy formation energy in an SrMnO Ferroelectricity and magnetism can, for example, be enhanced4 or even introduced in the thin-film material.5,6 Moreover, interfaces between the substrate and the film or between different layers of a heterostructure have emerged as an avenue to generate rich and novel electronic phases.3Transition-metal perovskite oxides represent an extremely versatile class of materials that can host a large range of functional properties such as ferroelectricity, magnetism or superconductivity.7,8 Oxygen vacancies (VO) are particularly abundant in perovskite oxides under typical synthesis conditions. Depending on their charge state and the localization of the excess charge, VO may lead to changes in oxidation state and local distortions that affect the ferroelectric and magnetic properties.9–11 Point-defect engineering could thus be a route to tailor properties for a given application. Our understanding of point defects and their formation energetics and electronic structure is, however, currently mostly limited to idealized bulk or strained bulk systems, neglecting the effect of the substrate–film interface and the film surface, except for select cases like the LAO/STO interface.12 Therefore, realistic models containing both surfaces and hetero-interface with the substrate are crucially needed to accurately assess defect-induced phenomena in thin-film systems.Depending on the synthesis conditions, complex oxides typically contain point defects that can strongly affect conductive, ferroelectric or magnetic properties useful for applications in electronics. While often detrimental to functional properties, defects were also shown to induce novel functionalities in specific cases.3 (SMO) thin film grown on a typical (001) TiO2-terminated SrTiO3 (STO) substrate to study the formation and electronic structure of VO in a thin-film geometry. This model system has recently been experimentally realized, showing excess charge accommodation in the film but close to the interface.13 The thermodynamic ground state of SMO is a hexagonal phase,14 but it is synthesizable in an orthorhombic perovskite structure when grown on a perovskite structured substrate,8,15,16 possibly adopting ordered Brownmillerite phases at high oxygen deficiency.17 SMO adopts a G-type antiferromagnetic order with Mn atoms of alternating spin arranged in a 3D checkerboard pattern. Alternatively, this magnetic order can be seen as alternating spins on adjacent Mn-containing (111) planes. VO were previously studied in both materials separately. In SMO and related manganites such as CaMnO3 and BaMnO3, the three 3d electrons in Mn4+ fully occupy the t2g orbitals of majority-spin in the G-type AFM order. Upon VO creation, the eg orbitals of Mn adjacent to the defect are stabilized and accommodate the two excess electrons, resulting in a reduction of both Mn from Mn4+ to Mn3+.8,18,19 This is energetically much preferred over the reduction of a single Mn to Mn2+. We note that the oxygen vacancy in this setup is doubly positively charged with respect to the O2− lattice site and charge compensated by the reduction of the two adjacent Mn4+ to Mn3+. It was also shown that magnetic order and polar distortions can affect the formation energy of VO in these materials.19,20 In STO, a large variability in the experimental and theoretical literature reveals that states with excess charge accommodation in the t2g conduction band or in shallow eg defect states are close in energy, results depending also on the crystal structure  and the size of the simulation cell.21 For electrons in the t2g conduction band, the vacancy is doubly positively charged with respect to the O2− lattice site and charge compensated by free electrons, while the vacancy with electrons in eg defect states forms an F-center with the same charge as the O2− lattice site. We note that for SMO, on the other hand, charge localization around an oxygen vacancy does not significantly depend on the cell size and that oxygen-vacancy formation energies computed for one vacancy per 8 formula-unit, 40 atom cell in the related CaMnO318 agree well with those observed in experiment.22In the present work, we use the model system of a SrMnOU calculations show that crystal-field changes of the under-coordinated surface atoms lead to a charge transfer towards the surface and hence surface Mn3+ species. This asymmetric structure leads to an electric field in this nominally non-polar interface, which strongly affects the defect chemistry. We find that vacancies more easily form at the surface and that the formation energy increases in a near linear fashion with increasing distance from the surface. For oxygen vacancies in the STO substrate, we predict separation of the defect and the charge, the latter residing on Mn atoms at the interface, which leads to a marked reduction in formation energy compared to bulk STO. For our model system, the formation of oxygen vacancies is, therefore, greatly different compared to either of the bulk materials.For the thin-film geometry, our DFT+223,24 at the PBE+U level of theory25,26 with Hubbard U calculated self-consistently27–29 as 4.26 and 4.48 eV for the Mn and Ti 3d orbitals respectively.20,21 All atoms are represented by ultrasoft pseudopotentials30 with Sr, Mn, O and Ti valence electrons. The cutoff for the plane-wave basis was 70 Ry for the kinetic energy combined with 840 Ry for the augmented density.DFT calculations were performed using Quantum ESPRESSOPnma structure with a G-type antiferromagnetic order,31 which we use to initialize all calculations throughout this work. The structure is close to the ideal cubic structure with small octahedral tilts and rotations found computationally but not yet observed experimentally.32 The STO substrate undergoes, around 105 K,33,34 a transition from a high-temperature cubic (space group Pm3̄m) to a tetragonal antiferrodistortive  phase, where TiO6 octahedra rotate around the c-axis with out-of-phase rotations in consecutive layers (a0a0c− in Glazer notation35). Since our DFT calculations are performed at 0 K, we use the relevant AFD phase of STO.We model a SMO thin film, grown epitaxially on a (001) STO substrate. Unstrained perovskite SMO has a paraelectric 2 terminated 80-atom supercell slab that has 2 × 2 × 4 dimensions compared to the 5-atom cubic cell. The Pnma SMO film also has 2 × 2 × 4 dimensions compared to the 5-atom cubic cell and is MnO2 terminated. We note that one VO per 160-atom supercell results in a defect concentrations slightly smaller than observed in typical experiments of manganite films.18,22 Larger lateral dimensions of the simulation cell lead to negligible changes in formation energies and electronic structure  was calculated as described in ref. EOtot,V and Etot,stoi are the total energies of the defective and stoichiometric supercells, respectively and μO is the oxygen chemical potential for which we assume the oxygen-rich limit, evaluated from total energies of water and hydrogen (μO = E2OH − E2H) to avoid the well-known overbinding of O2 in DFT.39 Since only neutral oxygen atoms were removed from the simulation cell, no terms accounting for the electron chemical potential nor potential alignment are required.The Brillouin zone of this thin-film system is sampled using a 6 × 6 × 1 Monkhorst–Packg. S4 ESI, under c33.140 This change in lattice parameters is accommodated by changes in Mn–O bond lengths and Mn–O–Mn bond angles  dimensions of SMO are expanded, while the out-of-plane (OP) dimension shrinks according to Poisson's ratio.gles see . In part41–43 as well as at surfaces,44 while their effect in a thin film geometry was not yet analyzed. The concomitant lowering of Mn 3d energies leads to more favorable electron accommodation at the surface compared to Mn sites in other layers. This change is visible in the layer-resolved projected density of states (2g peak visible around 3 eV in lower MnO2 layers (circled in red) is significantly destabilized and a small minority-spin (down-polarized Mn) peak is visible just above the Fermi energy (circled in blue). This change in the electronic structure leads to a ferrimagnetic surface layer (see 3 and SrTiO3).The truncation of Mn–O bonds at the film surface changes the crystal field of surface Mn atoms from octahedral to approximately square pyramidal. Changes in crystal field were previously shown to affect defect stability in the bulk3+, while Mn atoms in all other layers retain their nominal Mn4+ oxidation state. We verified that the change in oxidation state is not an artefact of the geometry. As shown in Fig. S3  in the different layers of the thin film. Within each layer, multiple symmetry inequivalent VO positions exist, the variation in formation energy of which is however negligible compared to the effect of the distance from the surface. We observe an increasing trend (by more than 1 eV) in formation energy from the surface to the hetero-interface, which continues into the substrate. This implies that VO will have a tendency to be formed in the surface region, respectively to migrate there, if oxygen mobility is sufficiently high. As indicated by horizontal dashed lines in O formation energy of bulk SMO. The VO formation energy in the substrate, on the other hand, is always lower than in bulk STO. This hints at a different electronic structure of the VO compared to the bulk phases.In O and we hence start our discussion with vacancies in the SrO layers, returning to MnO2 layers below. In bulk manganites, VO formation results in a lowering of the eg states of the two Mn atoms adjacent to the vacancy, which become the lowest unoccupied states and accommodate the excess electrons resulting from VO formation.8,18,20 In the topmost SrO layer of our thin film, we observe a different picture. Instead of localizing on one Mn atom each in the surface and first subsurface MnO2 layers, we observe the typical Mn eg defect state only in the first subsurface layer , whereas the other (the spin-up Mn) does not get reduced, the remaining electron filling the empty valence-band states.For Vvia chemical expansion arguments,18,46 expect the VO formation energy to be inversely correlated with the polyhedral volume in the stoichiometric structure. While the strongly expanded truncated polyhedron at the surface is indeed associated with a low formation energy, this trend does not continue towards the interface  would consistently lead to charges in the layer observed by experiment and are a more likely scenario.A previous experimental study3 film grown on top of a SrTiO3 substrate, these effects lead to a metallic and ferrimagnetic surface with potential applications, in particular, in oxide electronics. We expect similar behavior in other perovskite oxide films with multivalent B-site cations, but note that the different d-orbital occupation can strongly alter the crystal-field effects. Detailed studies on other film materials will be a worthy topic for future studies but we stress that gaining a fundamental understanding of defects in oxide thin films will have to involve surface and interface effects in addition to strain effects.Our results show that oxygen vacancy formation in a thin-film geometry can be significantly different from a bulk or strained bulk situation. Not only can the film surface induce subtle changes in the electronic structure that affect excess charge accommodation and stability of oxygen vacancies, but the film can also attract excess electrons from the substrate. These effects are not captured in established strained bulk calculations and are expected to strongly depend on the properties of the film and the substrate. In the present SrMnO10.24435/materialscloud:1y-q7.The main Quantum ESPRESSO pw.x input and output files needed to compute the formation energies in There are no conflicts to declare.CP-024-D1CP04998D-s001


SCN1A gene are associated with several neuro developmental disorders with or without epilepsy, including Dravet syndrome (DS). Conversely, there are few published cases with homozygous or compound heterozygous variations in the SCN1A gene. Here, we describe two siblings from a consanguineous pedigree with epilepsy phenotype compatible with genetic epilepsy with febrile seizures plus (GEFS+) associated with the homozygous likely pathogenic variant (NM_001165963.1): c.4513A > C (p.Lys1505Gln). Clinical and genetic data were compared to those of other 10 previously published patients with epilepsy and variants in compound heterozygosity or homozygosity in the SCN1A gene. Most patients (11/12) had missense variants. Patients in whom the variants were located at the cytoplasmic or the extracellular domains frequently presented a less severe phenotype than those in whom they are located at the pore-forming domains. Five of the patients (41.7%) meet clinical criteria for Dravet syndrome (DS), one of them associated acute encephalopathy. Other five patients (41.7%) had a phenotype of epilepsy with febrile seizures plus familial origin, while the two remaining (17%) presented focal epileptic seizures. SCN1A-related epilepsies present in most cases an autosomal dominant inheritance; however, there is growing evidence that some genetic variants only manifest clinical symptoms when they are present in both alleles, following an autosomal recessive inheritance.Dominant pathogenic variations in the  SCN1A gene encodes the alpha 1 subunit of the sodium channel, Nav1.1, an important protein that makes up the voltage-dependent sodium channels. Pathogenic variants cause a reduction in sodium currents in gamma-aminobutyric acid (GABA)-ergic inhibitory interneurons, leading to hyperexcitability of neuronal network and the appearance of seizures .Peripheral blood samples from individuals and their parents were collected by standard methods for the genetic studies. Genomic DNA was isolated using the Qiacube extractor . DNA purity and concentration were measured using the NanoDrop 8000 spectrophotometer (Thermo Scientific) and a Qubit 2.0 fluorometer (Invitrogen), respectively.Alissa Interpret platform (Agilent Technologies). All disease-causing genes related with epilepsy and/or neurodevelopmental disorders described in different databases were analyzed. To evaluate the clinical impact and to assess the pathogenicity of variants in exome sequencing, we applied the previously described criteria . The libraries were sequenced on an Illumina NextSeq 500 following the manufacturer's protocol to get a mean reading depth of 100 X. Sequence read alignments, variant calling, and annotation were performed in the criteria . All cliPatient 1 is a 4-year-old female born to healthy consanguineous parents see . During Patient 2 is a 35-month-old male, the brother of Patient 1 see . The chiCurrently, psychomotor development is at the lower limit of normality, and the neurological examination is normal. The clinical phenotype of the Patient 2 is compatible with GEFS+.GJB2 gene was considered of relevance. The inheritance pattern of hearing loss due to this gene is autosomal recessive and requires the presence of a second mutation in this gene to confirm the genetic diagnosis of hearing loss. It is possible that there might be a second undetected mutation or that there is another genetic or non-genetic cause of the hearing loss. On the other hand, Patient 2 presents the homozygous variant c.4513A > C in the SCN1A gene as the only clinically relevant result. This variant causes the substitution of the highly conserved amino acid lysine by glutamine in position 1,505 of the protein (p.Lys1505Gln), predicted as likely pathogenic by most of the in silico predictors  in the tors see . This vators see . AccordiSCN1A gene in homozygous state, while the healthy parents of the two patients were heterozygous carriers.Familial segregational study by Sanger sequencing confirmed that Patient 1 shares the variant c.4513A > C in SCN1A gene have been published. The spectrum of the clinical phenotype associated with variations in the SCN1A gene is wide. Numerous studies have attempted to correlate the clinical phenotype according to the genotype  are clinically encompassed by Dravet syndrome (DS), one of them associated acute encephalopathy (AE). Five (41.7%) had a phenotype of GEFS+ of familial origin. Two (16.7%) of the patients presented FS+ and focal epileptic seizures . In TablSCN1A variants with sensorineural deafness, despite the large number of published patients with variants in this gene. For this reason, we think that this finding is probably related to consanguinity of the family rather than to SCN1A gene. The concurrence in a family of two autosomal recessive inherited diseases is not so uncommon when it comes to consanguineous families  and the pore region (S5-S6 segments) of the four homologous domains, as in the case of Patients 3, 4, 7, and 11 of this series . As an eSCN1A . This phA  present a less severe epileptic phenotype. Conversely, those variants found at the pore region  more frequently present a more severe phenotype, with a phenotype of Dravet syndrome in most patients.It is worth noting that heterozygous carrier parents or other relatives are asymptomatic. Therefore, the variants described in this work are probably hypomorphic, both due to their location within the gene and because of their intrinsic properties, and they are only capable of altering the function of the Nav1.1 protein when they are in homozygous state. In any case, it is also remarkable that the phenotypic spectrum of the whole series of patients presenting the autosomal recessive phenotype related to SCN1A is quite similar to the phenotypic spectrum of most patients with autosomal dominant inheritance, as well as a similar genotype-phenotype correlation.SCN1A show that not all patients with symptoms associated with this gene have autosomal dominant inheritance. On the other hand, it is worth noting that the resulting phenotypes do not differ from those usually caused by dominant heterozygous variants. Furthermore, the analysis of the variants found in each patient, correlating it with the phenotype he or she presents, allows us to know a little better the correlation of genotype-phenotype associated with SCN1A. It is possible that hypomorphic heterozygous variants only manifest when affecting the two alleles of this gene.The 12 patients published so far with an autosomal recessive epileptic phenotype associated with The datasets presented in this article are not readily available due to ethical and privacy restrictions. Requests to access the datasets should be directed to the corresponding author.The studies involving human participants were reviewed and approved by Comité de ética del Instituto de Investigación Sanitaria La Fe. Written informed consent to participate in this study was provided by the participants' legal guardian/next of kin. Written informed consent was obtained from the minor(s)' legal guardian/next of kin for the publication of any potentially identifiable images or data included in this article.AM: drafting of the article, clinical examination of the patients, and bibliographic search. AC and SM: substantial contributions to analysis and interpretation of genetic data. MT: revising the article for important intellectual content and contribution to clinical data analysis. FM: analysis and interpretation of genetic data and revising of the article and final approval. All authors contributed to the article and approved the submitted version.This work had received financial support from the CM19/00181 Grant from the Carlos III Institute (AM) and the Mutua Madrileña Foundation (holded by SM). AC was supported by a research grant by Fundación Mutua Madrileña. Support for open access publication was provided by Fundación Síndrome de Dravet (Dravet Syndrome Foundation Spain) (grant number FSD-OASP-XI-III).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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.