Additionally, we present results on the DOS in the bandgap of amorphous AgIn-doped Sb2Te, which has not been subject to high-resolution DOS spectroscopy before. We find experimental evidence for clearly non-constant capture coefficients among a continuous spectrum of localised states in both studied materials. According to this observation especially in AgIn-doped Sb2Te, where no pronounced defect can be detected as main channel for carrier emission, we point out the necessity of modifying the current Poole-Frenkel-based transport modelling.As phase-change materials are poised to play a key role in next-generation computing systems, improving the current understanding of electrical transport in their amorphous phase can further strengthen their technological competitiveness. Even though the interaction of charge carriers with disorder-induced localised states largely affect the field-dependent conductivity, a clear link between electrical transport and specific features of the electronic density of states (DOS) could not be established yet due to a lack of knowledge of the capture characteristics of trap states. Here, we address this knowledge gap and employ modulated photocurrent spectroscopy (MPC) to investigate localised states in the frequently studied amorphous phase of Ge The most promising SCM candidate to reshape the memory landscape is phase-change memory (PCRAM)4 as a representative of resistively switching memories, which has been shown to be technologically mature and commercially viable since the introduction of 3D-XPointTM by Intel and Micron Technology in 20155. In 2014, the large potential of PCRAM as SCM was attested, when a prototype PCI-e card including PCRAM chips combined with flash memory and DRAM provided 275 times higher speed compared to a traditional SSD6. PCRAM is also finding applications in emerging non-von Neumann computing paradigms such as brain-inspired neuromorphic computing8.Driven by the explosive growth of data and the speed-gap between the processing unit and conventional storage systems, memory industry introduced the storage class memory (SCM) as next generation, non volatile memory class, satisfying key requirements such as speed, reliability and efficiency at a minimum cost-per-bit ratio9. Switching to the set state is decisively facilitated by the so-called threshold switching event, which occurs as breakdown of electrical resistivity at elevated electrical fields and allows for sufficient Joule heating in the otherwise low-conductive amorphous phase. Preceding the threshold switch, a highly non-linear current-voltage characteristic can be observed, which plays a key role in switching PCRAM devices fast and energy-efficiently10. Even though subthreshold electrical conduction in the semiconducting amorphous phase has been extensively studied so far, the underlying mechanism of its non-linear field dependence is still debated12.PCRAM encodes logical information by means of the electrical resistance of the memory device, utilising that so-called phase-change materials (PCM) can be switched reversibly between a high-resistive amorphous (reset) and a low-resistive crystalline (set) state. Resetting a PCRAM cell is commonly obtained by melt-quenching a portion of the previously crystalline PCM, while the set state results from bringing the amorphous PCM up to a temperature regime of high atomic mobility and thus fast crystallisation14, field-dependent conductivity in amorphous GeTe was attempted to be understood by investigating the interaction between charge carriers and localised states in the bandgap of amorphous semiconductors15. In 2007, Ielmini and Zhang16 proposed a widely recognised model for electrical transport in melt-quenched amorphous Ge2Sb2Te5 PCRAM devices by invoking the Poole-Frenkel (PF) emission of trapped charge carriers21. However, the Ielmini-Zhang-model of charge carriers hopping with a constant travelling distance between neighbouring states is incompatible with the well-known band transport concept11. Le Gallo et al.22 addressed this shortcoming by proposing a PF-based transport model that describes field-dependent conductivity in amorphous Ge2Sb2Te5 and GeTe remarkably well without assuming immediate re-trapping of emitted carriers. Nonetheless, PF-based transport modelling so far only includes trap states attributed to a single energy level, which is commonly attributed to a structural defect at a specific energetic distance to the band edge24. This approach appears plausible at least for PCM containing germanium such as amorphous GeTe, where under- or over-coordinated Ge-atoms have been associated with electronic defect states in the bandgap27. However, in germanium-free PCM, which might lack an appropriate defect as main channel for PF assisted charge carrier emission, the applicability of current PF-based transport modelling is questionable. This presumption is strengthened by the fact that previous PF-based transport modelling on amorphous Ag4In3Sb67Te26 yields an unreasonably large intertrap distance12.Already in the 1970s24. The capture characteristic of a localised state with respect to a certain type of charge carrier is typically expressed by the capture coefficient c = σ · υth as product of thermal velocity υth and capture cross section σ29. As such, c measures the efficacy of a localised state for capturing a charge carrier. In p-type conductivity materials such as amorphous PCM30, the capture coefficient for holes cp is of predominant importance for electrical transport.Recently, Kaes and Salinga made clear by linking subthreshold electrical transport and the density of electronic states (DOS) that more precise knowledge on the density of localised states, but also on capture characteristics of such states is needed to better understand electrical transport in amorphous PCM31, MPC spectroscopy has been used for numerous investigations of the DOS in a large variety of different materials, from amorphous silicon to lead sulfide quantum dot arrays36. Other methods to examine the electronic structure of amorphous PCM films such as x-ray or ultraviolet photoemission spectroscopy typically do not offer a sufficient energy resolution to observe distinct features of the bandgap39. The working principle of MPC spectroscopy is based on intensity-modulated illumination with photons, which generates free charge carriers in the material leading to a modulated photocurrent. During the experiment, the photocurrent amplitude and phase shift with regard to the sinusoidally modulated illumination are recorded as function of modulation frequency. These experimental data can be linked to the density of localised states by interpreting the measured amplitude and phase shift in terms of free charge carriers that are trapped by localised states and released back to the band after some delay. In particular, it has been commonly agreed upon that the phase shift between excitation and modulated photocurrent predominantly originates from the thermal emission of trapped charge carriers (e.g.41). In this picture, photons are solely generating free charge carriers, they are not expected to contribute to the measured MPC signal in any other way, e.g. via optically induced transitions involving localised states.A key technique to obtain experimental input on both density and capture coefficient of localised states in the bandgap of amorphous materials is modulated photocurrent spectroscopy . Decisively shaped by works of Brüggemann, Longeaud and otherset al.42 and Luckas et al.44 to gain remarkable insight into the DOS of amorphous GeTe and Ge2Sb2Te5. According to their MPC studies, the bandgap of these PCM exhibits Gaussian-shaped peaks of localised states related to structural defects. In addition to these pronounced peaks, disorder-induced spectra of localised states were found with exponentially increasing density towards both band edges. Especially with respect to these spectra of localised states, up to now the analysis of MPC results has been performed under the restrictive assumption that the investigated states exhibit one common capture coefficient (cp = const.)44. This simplification makes it much easier to analytically link experimental MPC data to the DOS, since cp may then be assumed to be independent of energy29.So far, MPC spectroscopy was used in studies by Longeaud 2Sb2Te5 represents the probably most popular and widely studied PCRAM material45. By additionally choosing amorphous AgIn-doped Sb2Te (more precisely Ag4In3Sb67Te26), we provide very first insights into the DOS in the bandgap of this germanium-free PCM that stands out due to its drift characteristics, a property relevant for storing multiple levels in a single PCRAM element47.In the present work, we challenge this assumption of a constant capture coefficient based on precise experimental input for the density and capture characteristics of localised states. We suggest crucial improvements to the already established method of MPC spectroscopy, including both experimental and analysis-related aspects. Our study focuses on high-quality MPC measurement of two materials. Amorphous Ge2Sb2Te5 and Ag4In3Sb67Te26. Due to the experimentally demonstrated p-type conductivity of amorphous PCM30, the MPC signal is most likely dominated by trap and release processes of holes42. These holes interact with localised states within the bandgap at energy levels below the Fermi level, which is located in the lower half of the bandgap near the bandgap center. Contributions to the MPC signal from electrons interacting with localised states above the Fermi level can be neglected. This implies that only localised states below the Fermi level can be probed by MPC spectroscopy. Apart from this, it is a common constraint in MPC analysis that exact values for quantities such as the DOS at the band edge μext,p are not known. Consequently, instead of plotting the pure DOS N as function of probed energy Eωp, usually a so-called MPC DOS being proportional to N is plotted against energy (see methods).Figure cp of the probed localised states, because only then the aggregation of individual MPC scans yields a coherent DOS. Considering that cp itself depends on temperature T as in cp(T) = kc · T1/2 due to the thermal velocity of charge carriers29, we suggest to introduce the factor kc. Thereby, the product Looking at Fig. During this process of determining the capture characteristics of localised states, it is crucial to consider that charge carriers trapped at energy levels closer to the bandgap center (and thus measured at lower modulation frequencies) have an increasing probability of recombining instead of being re-emitted to the band. Since the MPC analysis employed in the present work can only handle an MPC signal dominated by trap- and release processes, segments of MPC scans corresponding to energy levels dominated by recombination traffic must not be used to form a coherent MPC DOS and are therefore excluded from further analysis  of 27.3 ± 0.8 meV (Ge2Sb2Te5) and 35.9 ± 0.7 meV (Ag4In3Sb67Te26) close to values previously found in MPC spectroscopy studies on amorphous PCM42.At first sight, for both materials MPC scans seem to form a somewhat coherent MPC DOS as constant capture characteristics . A precise value for MATLAB. According to this algorithm, MPC DOS data points that are recorded at different temperatures and found in the same MPC DOS bin are scaled to meet at a common energy level (see methods).When compared to existing MPC studiescp = const. Since no MPC scans are excluded due to the appearance of a defect in the case of Ag4In3Sb67Te26, the amount of MPC DOS data being scaled by overlapping is considerably larger compared to the Ge2Sb2Te5 data set. Apart from this, in both materials the MPC DOS of the continuous spectrum of localised states is still found to decrease exponentially towards the bandgap center  = kc · T1/2 of the probed localised states. This striking observation quantitatively confirms the above mentioned indication for a non-constant capture coefficient along the continuous spectrum of localised states.Figure 2Sb2Te5 and in general amorphous PCM44, which has always been restricted to the assumption of cp = const. and in which MPC data has given no reason to hypothesise locally varying capture coefficients. Compared to existing studies (e.g.50), the newly introduced quantitative matching algorithm in our study benefits from the high signal-to-noise ratio, large data point density in the frequency domain and small temperature step size between MPC scans. Moreover, the present work proves the necessity to include a flux-dependent analysis of the MPC signal as preceeding step, which is not given for previous studies.This result clearly conflicts with existing MPC spectroscopy work on amorphous Gecp is treated as energy-independent constant29. Since this assumption turns out to be invalid in the present case, the results shown in Fig. With respect to the compelling experimental evidence for non-constant capture characteristics in the continuous spectrum of localised states, it has to be noted that the currently applied MPC analysis still relies on a framework in which cp for the continuous spectrum of localised states would be included in the analysis. This task could be approached by identifying an underlying mechanism of charge carrier trapping and emission that has been absent in MPC studies so far and that implies inhomogeneous capture characteristics. The involvement of multiple phonons in the transitions to and from localised states as described by Mott and Davis is a potential candidate for such a mechanism51. According to related studies by Baranovskii et al. on glassy semiconductors54, the multiphonon nature of charge carrier transitions between bands and localised states causes the probability for such transitions to decrease exponentially with increasing energy distance. This exponential energy dependence could be considered as additional factor in the capture coefficient cp. In principle, the current MPC analysis framework could be extended towards such an exponential energy dependence of cp, but establishing an analytical link between the DOS and the experimental MPC data becomes less straightforward  and attribute them to structural defects involving germanium atoms26. In the scenario of germanium-free Ag4In3Sb67Te26 apparently lacking such a singular defect as main channel for charge carrier emission, a realistic electrical transport model would need to account for trap- and release processes of charge carriers with a continuous spectrum of localised states. Such a revised transport model would also have to consider our finding of non-constant capture coefficients among the continuous spectrum of localised states, which indicates that some states in the bandgap interact more strongly with charge carriers than others. These conclusions on subthreshold electrical transport in amorphous Ag4In3Sb67Te26 should also be of importance for the technologically highly relevant threshold switching in amorphous PCM. While popular threshold switching models so far rely on trap centers at a single energy level57, it appears likely in view of the above that not only subthreshold conduction, but also threshold switching in Ag4In3Sb67Te26 demands a different explanation accounting for the presence of a continuous spectrum of localised states. Ultimately, a transport model that is able to explain the typical current-voltage characteristics of amorphous PCM as a consequence of a continuous spectrum of localised states within the bandgap should be applicable also to PCMs like GeSbTe and might thus render the existence of sharp defect levels less relevant for the observed electrical properties.Even though a revision of the current MPC analysis seems necessary before quantitative DOS characteristics can be extracted from our MPC results, we can already report two significant findings regarding the DOS of amorphous PCM. These are a) the observation of a non-constant capture coefficient for a spectrum of localised states in the bandgap of both studied materials and b) the apparent lack of a structural defect at a specific energetic distance to the band edge below the Fermi level in amorphous Ag4In3Sb67Te26 or Ge2Sb2Te5) deposited on a substrate in between two tungsten electrodes. To prevent the fully as-deposited amorphous PCM from oxidising, it was capped in-situ with a 10 nm layer of (ZnS)80:(SiO2)20. We recorded experimental data by homogeneously illuminating a complete electrically contacted sample of amorphous PCM. No further areal segmentations of the sample were applied. The active volume of amorphous PCM between the two electrodes (100 nm by 1.2 mm by 40 μm) is large compared to potential spatial fluctuations in the density of localised states. Hence, the measurements are of intrinsically averaging character.Lateral devices were fabricated for MPC spectroscopy measurements, with 100 nm thick PCM material  and is fed into the cryostat chamber (further details are listed in the Supplement Section 41), the HP 3562 A performs a single-point FFT analysis of the input signal, offering a remarkable measurement speed when measuring the amplitude of the modulated photocurrent |Iac| and its phase shift ϕ with respect to the excitation signal. This allows for conducting the flux-dependent MPC spectroscopy described below in a time-efficient manner.Electrical measurements were conducted in a Janis ST-500-2UHT cryogenic probing station, evacuated to pressures ω from 10 Hz to 40 kHz, recording the amplitude |Iac|(ω) and phase shift ϕ(ω) of the modulated photocurrent at various light fluxes (see below) at a specific temperature. This procedure was repeated for various temperatures (in 5 K steps), as long as a sufficient signal-to-noise ratio was encountered. Photoconductivity in amorphous PCM commonly exhibits a peak at around 200 K (AgIn-doped Sb2Te) and 250 K (Ge2Sb2Te5), respectively59. MPC scans for temperatures above or below this peak suffer from decreasing signal-to-noise ratio, additionally impaired by exponential increasing device resistance for decreasing temperature.Before starting the MPC measurement, the optimum fibre position for a most homogeneous sample illumination was determined following an automated procedure. Subsequently, the signal analyser performed a frequency sweep (MPC scan) for the modulation frequency et al.29, an analytical expression to relate |Iac|(ω) and ϕ(ω) to the density of localised states can only be obtained if the probed energy levels are unaffected by recombination. Following work of Taylor and Simmons (e.g.28) and also Shockley and Read60 on occupation statistics in the non-equilibrium steady state, increasing light flux widens the so-called recombination zone around the bandgap center. We therefore propose an experimental procedure to identify, which light flux should be used to record data for |Iac|(ω) and ϕ(ω) with as large as possible signal-to-noise ratio and unaffected by recombination.As described in the original MPC analysis based on trap and release processes by Longeaud ω and probed energy level Eωp is only made in the subsequent analysis as described below. Further details on this measurement procedure and exemplary raw data for flux-dependent MPC scans can be found in the Supplement Section Recording a first MPC scan with the highest available light flux implies that probably all probed energy levels are affected by recombination due to a very large recombination zone. Repeatedly recording MPC scans at decreasing flux shrinks the recombination zone and leads to more and more energy levels  being completely unaffected by recombination. Eventually, the MPC output is independent of further lowering the flux, indicating which particular MPC scan segment can be used for further MPC analysis. Note that the explicit assignment between excitation frequency et al.29 and were used also recently for MPC DOS spectroscopy on amorphous PCM43, serve as point of departure for our DOS spectroscopy analysis:Two essential expressions, which have been developed by Longeaud Iac|(ω) and ϕ(ω)) to the DOS N at the probed energy level Eωp, with cp denoting the capture coefficient for holes of the probed states, μext,p the extended-state mobility of holes, q the elementary charge, ξ the applied electrical field, kB the Boltzmann constant, S the current cross section, Gac the modulated component of the photogeneration rate and T the sample temperature during the MPC scan. It is important to note that the MPC signal in equation Equation 29, this assumption of cp = const. is also essential to linking DOS and experimental MPC data in equations 61, the product S · Gac can be substituted by l · Fac with l denoting the electrode length and Fac the modulated component of the light flux.The term on the left side of equation ω to the probed energy level with respect to the valence band edge EV, originating from the fact that these states are probed, whose emission rate of trapped carriers is equal to the excitation frequency29. The attempt-to-escape frequency for holes νp is related to the capture coefficient cp and the effective density of states at the valence band edge NV,eff via νp = cp · NV,eff. In amorphous semiconductors the latter is commonly assumed as 62, EV. Eventually, it is noted that equations 29. This assumption appears to be justified for modulated photoconductivity data recorded for the present work  of the probed states (see e.g.43). Hence, the proportionality constant linking the actual DOS N(Eωp) to the MPC DOS on the left side of equation Due to the logarithmic frequency dependence in equation N, we bring cp to the right side of equation cp = const. might not be valid. As cp is linked to νp used as matching parameter in equation νp = cp · NV,eff. Thus equation cp, but also by NV,eff resulting in νp in the denominator of the right hand side of the equation.For the present study, we propose some modifications of equations μext,p and the capture coefficient cp are expected to have a certain temperature dependence: μext,p = kμ · 1/T  and cp = kc · T1/2 , with kμ and kc constant in T.We also take into account that various quantities have implicit temperature dependencies, which is problematic for the collective analysis of MPC scans measured at various temperatures. Besides the already mentioned T5/2 term. Also, the whole equation is multiplied by the Boltzmann constant kB, which was introduced through NV,eff before. Our modified MPC DOS (left side of equation N(Eωp) normalised by the DOS at the valence band edge kμ, which is treated as unknown in the present case and might be determined by Hall measurements62, remains as the constant of proportionality between this normalised actual DOS and the derived MPC DOS.While the temperature dependencies on the left side cancel each other out, all temperature terms on the right can be combined in a single νp used for matching MPC scans from different temperatures has been replaced by νp was varied to match overlapping scans, now the product cp and νp explicit and dividing equation cp, the matching parameter is no longer hidden within the MPC DOS, but instead appears together with all other quantities derived from the MPC measurements on the right side of equation kμ, in contrast, cannot be derived from the MPC data and therefore remains within the MPC DOS  = EG(T = 0 K) − δ(T) with δ(T) = αT2/(T + β), we follow the argument that the temperature-induced shrinking of the energetic distance between valence and conduction band edge by a factor r(T) = (1 − δ(T)/EG(T = 0 K)) translates into the same relative shrinking of all energetic distances between states within the bandgap.Here, we indicated also that the energetic distance between probed energy level Eωp − EV is transferred to the right side of equation Eωp − EV at T = 0 K:α and β in δ(T) we used the values determined experimentally in our earlier publication47. While results in Fig. As the process of matching MPC scans recorded at different temperatures demands that both derived quantities, i.e. MPC DOS and energy scale, are temperature-independent, the mentioned temperature dependency of entclass1pt{minimaγ separately for thin slices of the MPC DOS. Considering one of these MPC DOS bins, an algorithm programmed in MATLAB checks for data points that describe the same MPC DOS (at least within the sufficiently narrow bin width). The found data points are most likely not scaled precisely to the same energy level by the initial γ has to be chosen in a way that the MPC data points found in this bin are scaled to the same energy level, Thereby, changing the product Eωp − EV](T = 0 K)), but also affects the y-axis (γ per bin lies in the general order of 102. Further details on this MPC DOS composition procedure can be found in the Supplementary Section Starting from an initial value for the product Supplement to: Localised states and their capture characteristics in amorphous phase-change materials


We solve an open problem on some majorization inequalities involving the cyclic moving average. We first recall two definitions.n-tuples of real numbers. (i)x is said to be majorized by y S-convex function) ifLet For fixed For example:In 2006, I. Olkin, one of the authors of the book , wrote aIn 2010, Shi [For any For proving our main results, we need the following lemmas.LetandThenif if entclass1pt{minima □For any From the proof of Lemma Letand letFor anythere existandsuch thatandLetand(i)Ifthen(ii)Ifthen(iii)Forifthen(iv)Ifthen(v)Forifthen(vi)Forifthen(i)By Lemma ality of  we have(ii)By Lemma ality of  we get\(iii)By  we get\(iv)By  we get\(v)By  we have(vi)By  we get\ □Letand(i)Ifthen(ii)Ifthen(iii)ForifthenWe only prove (i). Using a similar method, we can obtain (ii) and (iii).By Lemma If mentclasspt{minima then by  the righmentclasspt{minimaion with .If  then by  we have her with , we getmentclasspt{minimaNext, we prove that Therefore h2=r. So  holds. □Letandand let(i)Forwe have(ii)Forwe have(iii)Forandmust be one of the following two cases:or(i)If }m=1, by  we have(ii)If  then by  we have(iii)By Corollary If by Lemma (iii) we  side of  includesion with .If  then by  we have  side of  must incNext, we prove that Thus In a similar way as in Corollary Letand(i)Ifthenmust be one of the following two cases:or(ii)Ifthenmust be one of the following two cases:orLetandand letIfthen we have: (i)ifthen(ii)ifthenmust be one of the following two cases:orLetandand letIfthenBy a simple calculation we obtainmai). By  we haveans that  holds. □Letandand letIfthenNote that1ai). By  we haveans that  holds. □We are now in a position to prove our main results  in two cFor anywe haveIt is clear that  holds if(i)If (ii)If m2≤0. So  holds. □For For anyandwe haveIt is clear that  holds foNext, let (i)If (ii)If k+1), so  holds. □For any In the theory of majorizations, there are two key concepts, majorizing relations and Schur-convex functions. Majorizing relations are weaker ordered relations among vectors, and Shur-convex functions are an extension of classical convex functions. Combining these two objects is an effective method of constructing inequalities.In the theory of majorization, there are two important and fundamental objects, establishing majorizing relations among vectors and finding various Schur-convex functions. Majorizing relations deeply characterize intrinsic connections among vectors, and combining a new majorizing relation with suitable Schur-convex functions can lead to various interesting inequalities; see –13.


CNOT1 de novo missense mutation, GenBank: NM_016284.4; c.1603C>T (p.Arg535Cys), resulting in a syndrome of pancreatic agenesis and abnormal forebrain development in three individuals and a similar phenotype in mice. CNOT1 is a transcriptional repressor that has been suggested as being critical for maintaining embryonic stem cells in a pluripotent state. These findings suggest that CNOT1 plays a critical role in pancreatic and neurological development and describe a novel genetic syndrome of pancreatic agenesis and holoprosencephaly.We report a recurrent  PTF1A [MIM: 615935], PDX1 [MIM: 260370], GATA6 [MIM: 600001], GATA4 [MIM: 600576], HNF1B [MIM: 137920], and RFX6 [MIM: 615710]) have been reported to severely affect pancreatic development and result in pancreatic agenesis.PTF1A, PDX1, or RFX6 results in pancreatic agenesis. In contrast, while haploinsufficiency of GATA6 is a common cause of pancreatic agenesis in humans,Gata6 knockout does not result in abnormal pancreatic development.Discovering genes with mutations causal of pancreatic agenesis is crucial to identifying factors needed for pancreatic development. To date, pathogenic variants in six genes   and exocrine (pancreatic enzymes) replacement therapy within the first 6 months of life—and identified a mutation in a known gene in 98 of them . To iden (n = 7) .CNOT1  in three individuals with pancreatic agenesis. The variant had arisen de novo in two of them and was not present in the DNA sample from the 3rd individual’s father  (C. elegans)  predicted the variant to have a deleterious effect on protein function (We identified a heterozygous missense mutation in testing)  and S3. testing) . The p.Aelegans) B. All thfunction .Figure 1CNOT1 p.Arg535Cys variant had strikingly similar clinical features . P01 and P02 also had gallbladder agenesis, a clinical feature frequently associated with pancreatic agenesis.The three individuals who were heterozygous for the ures see . In addiures see , a disorde novo CNOT1 variants in three individuals with developmental delay (two missense—p.Leu2323Phe and p.Arg623Trp—and a nonsense—p.Gln33∗—variant) but none of them had holoprosencephaly or diabetes. Since our three case subjects were all heterozygous for the same novel missense CNOT1 variant and none of the DDD participants with heterozygous de novo CNOT1 variants had pancreatic or neurological structural malformations, we hypothesized that a mutation-specific mechanism rather than loss of function was responsible for the phenotype seen in our case subjects. We therefore generated a mouse line harboring the Cnot1p.(Arg535Cys) mutation using CRISPR  in pancreatic development.High-resolution episcopic microscopy (HREM) highlighted a significant reduction in the size of the pancreas in homozygous embryos in addition to several other abnormalities H and S3.Cnot1p.(Arg535Cys)/p.(Arg535Cys), and Cnot1p.(Arg535Cys) embryos showed a significant increase of Shh expression in homozygous embryos, with decreased expression in Pdx1, Ins, Hnf1b, and Ptf1a /p.(Arg535Cys) E14.5 mouse embryos are consistent with the pancreatic agenesis and holoprosencephaly observed in the three case subjects, confirming that the de novo CNOT1 mutation is indeed the cause of their disease. Mice required a homozygous mutation in Cnot1 to display a pancreatic and brain phenotype while a heterozygous CNOT1 mutation resulted in the phenotype in three individuals in our cohort. This has been described with other pancreatic developmental genes  and supports the hypothesis that the early stages of pancreatic development are not identical in mice and humans.The pancreatic and neurological phenotypes observed in In vitro studies have proposed that CNOT1 plays a critical role in maintaining human and mice embryonic stem cells in a pluripotent state by inhibiting primitive endoderm factors.CNOT1 expression peaks in undifferentiated human induced pluripotent stem (iPS) cells compared to subsequent stages of in vitro differentiation toward pancreatic endocrine cells,The CNOT1 protein has not previously been suggested to have a role in pancreatic development; it is known to act both as scaffold of the CCR4-NOT complex and as an independent factor. As such, it mediates transcriptional repressionp.(Arg535Cys)/p.(Arg535Cys) embryos would be consistent with a model in which the CNOT1 p.Arg535Cys mutation results in embryonic stem cells being maintained in an undifferentiated state through SHH-mediated inhibition of differentiation. SHH is a key developmental factor that is known to be crucial for pancreatic and brain development. Heterozygous loss-of-function mutations in SHH cause holoprosencephaly (MIM: 142945) and studies in both mouse and human embryos have shown that SHH expression needs to be repressed in the dorsal foregut endoderm for successful differentiation toward dorsal pancreas.Shh and decreased expression of Pdx1, Ins, Hnf1b, and Ptf1a detected in RNA extracted from pancreatic tissue in the E14.5 Cnot1p.(Arg535Cys)/p.(Arg535Cys) embryos would support this hypothesis. However, Gata4 expression could not be assessed as the assay specificity was too low and Gata6 expression was not found to be reduced. It is possible that Gata6 activity is actually inhibited earlier during development and then re-activated by a different pathway  or could be inhibited by a different mechanism that does not result in reduced expression. The CNOT1 p.Arg535Cys mutation also affects neurological development in both our case subjects and mouse embryos. It is possible that this mutation results in ectopic SHH expression during brain development. This would be consistent with previous reports of Shh ectopic expression impairing midline development.CNOT1 mutation on SHH signaling differs between the brain and the pancreas, resulting in a reduced expression in the developing brain and increased expression during pancreatic development. Further experiments, ideally on younger embryos and human iPS cells, are needed in order to elucidate the mechanism by which the CNOT1 p.Arg535Cys mutation results in impaired pancreatic and neurological development.The increased expression of Shh in pancreatic tissue extracted from Cnot1CNOT1 p.Arg535Cys mutation as the genetic cause of a rare syndrome of pancreatic agenesis and holoprosencephaly, highlighting a previously unsuspected role of CNOT1 as a key factor in both pancreatic and neurological development. This is the 7th gene causative of pancreatic agenesis described so far and the first pancreatic agenesis gene that is thought to be important for maintaining stem cells’ pluripotency. These findings suggest a new mechanism by which impairment of the very early stages of development result in pancreatic agenesis and abnormal brain development.Our study identifies a spontaneous The authors declare no competing interests.


Testing whether the films can benefit from encapsulation revealed that this can provide extra bending stability and prevent contacts deterioration in the long term.Aluminium doped zinc oxide (AZO) films were grown by Atomic Layer Deposition (ALD) on yellow Kapton and transparent Kapton (type CS) substrates for large area flexible transparent thermoelectric applications, which performance relies on the thermoelectric properties of the transparent AZO films. Therefore, their adhesion to Kapton, environmental and bending stability were accessed. Plasma treatment on Kapton substrates improved films adhesion, reduced cracks formation, and enhanced electrical resistance stability over time, of importance for long term thermoelectric applications in external environment. While exposure to UV light intensity caused the films electrical resistance to vary, and therefore their maximum power density outputs (0.3–0.4 mW/cm This work demonstrates the stability of AZO films deposited by ALD on Kapton when exposed to external elements – humidity, UV light and bending, – in view of its potential use in large area flexible electronic devices over extended periods of time.1, energy harvesting devices in large smart windows2 and thermoelectric touch sensors3. Kapton is an adequate substrate since it is transparent and has softening temperatures (Tg > 360 °C) that allow ALD4. Fabrication of metal oxide films by ALD has emerged in the last decade as their properties are competitive with others made by a variety of processes7. Films made by stacking ZnO and Al2O3 layers grown by ALD revealed similar electro-optical properties to AZO grown by sputtering and do not require post-annealing9. Recently, H.-W. Park deposited conformal high quality ZnO/Al2O3 stacked multilayers on transparent nanostructures with improved optical and electrical properties10. Hence, and because of the similar properties ALD ZnO/Al2O3 multilayered films are often and hereon referred to as AZO films. Prior to the AZO stack deposition, a 10 nm thick Al2O3 layer is used as an interface to avoid diffusion of chemical species to and from the polymeric substrates, improve adhesion and reduce cracks formation. Alumina adhesion to polymer substrates is remarkable due to the covalent bonding11. Nonetheless, a plasma treatment of polymeric substrates is also frequently used to improve adhesion and performance of the grown films12. In this work, plasma treatment of some Kapton substrates was performed prior to the ALD deposition, aiming at further improving adhesion. As it will be shown in the following, the long term performance and stability of the films under UV-light was concurrently improved. Moreover, the ALD AZO films proved to have low sensitivity to humidity and bending. Encapsulation did not hinder the performance of the devices but improved stability while protecting the electrodes .Atomic layer deposition (ALD) is a gas phase thin film deposition method, known to be good for surface conformity and precise atomic level control of film composition and thickness through surface reactions, providing uniform dopant distribution. The deposition can be done in large areas and at relatively low temperatures (200 °C or lower), making it compatible with polymeric substrates (e.g. as Kapton polyimide) commonly used for e.g. Organic Light-Emitting Diodes (OLEDs)2O3 coated 25 μm thick Kapton substrates (CS and Yellow types), with and without previous plasma treatment. The Al2O3 coating was grown at 50 °C to a thickness of 10 nm. The functionalization of Kapton surfaces prior to ALD deposition aimed at improving adhesion of deposited thin layers through two type of plasma treatments: He/O2 (T1) and He/N2 (T2), both performed on a GRINP PLAFilm HT 600 machine that creates plasmas at atmospheric pressure by dielectric barrier discharge (DBD). Mixed gas-plasmas of He/O2 and He/N2 were produced at room temperature with a mixing ratio of 4:1 and of 4:3, respectively. The source-to-sample distance was 1 mm and the scan speed 33 mm/s, with the plasma power set to 2000 W.AZO films were grown by ALD onto Al2 atmosphere at a pressure inferior to 1 mbar and a substrate temperature of 200 °C. The multilayered films were grown on top of the 10 nm Al2O3 interfacial layer in the sequence: nominal 22 nm thick ZnO followed by 0.5 nm Al2O3 until the desired nominal thickness, of 100 nm or 460 nm was reached. Hence, the number of precursor cycles for the referred thicknesses and an Al content of 1.5–4%, totalized 4 and 20 master cycles, respectively. The precursors used for deposition of Al2O3 and ZnO layers were trimethyl-aluminum and diethyl-zinc with H2O. The composition and thicknesses of the AZO films deposited by ALD on Kapton substrates were measured by Rutherford Backscattering Spectroscopy (RBS) using a 2 MeV 4He+ beam. RBS spectra were collected at 140° and 165° scattering angles by means of Si-diode detectors of 16 keV energy resolution, and analysed with the help of the RUMP code13. Some key features of the RBS technique applied to thin films are the ability to determine elemental composition and thickness of the layers. Briefly, the position of the spectral features of a RBS spectrum correlate with the masses of the scattering centres (and so with the contributing elements), its height with the amount of the contributing elements present (i.e. the corresponding concentrations) and its width or energy extent with the physical thickness that those elements take.The ALD was performed with PICOSUN P-300B ALD system under NCharacterization of the surface morphology was performed with a Hitachi S2400 Scanning Electron Microscope (SEM) operated in Secondary Electron Imaging (SEI) mode and with the Atomic Force Microscope (AFM) of WITec Alpha 300 RAS confocal spectrometer. The cantilever was operated with an Al coated probe in AC mode at 75 kHz and constant load of 2.8 N/m. Lateral and depth resolutions were 1 nm and 0.3 nm, respectively.AZO films were submitted to variable relative humidity (%) inside a glove box equipped with a cold steam sprayer and a RS Pro 408–6109 humidity electronic sensor, providing an uncertainty of ±5% in the range 25% to 80%, and ±10% out of that range. The relative humidity inside the glove box was first raised from 50% to 100% in steps of 5% (cycle 1) and next lowered by venting the box (cycle 2). Cycles 1 and 2 lasted for ∼104 min and ∼120 min, respectively. The influence of UV-light exposure was carried out in a UV-light box with a lamp emitting a dominant wavelength of 288 nm (4.31 eV), enabling photo excitation of AZO electrical carriers. For comparison, the AZO films were also submitted to sunlight UV radiation.14, imposing a thermal gradient (ΔT) in the plane of film and substrate using two TEC1–12707 Peltier modules, connected to independent power sources. The Seebeck coefficient was determined by imposing a ∆T that was monitored by a FLIRA310 thermal camera, while the thermoelectric voltage (ΔV) was measured using an Agilent 34420 A nano-voltmeter, using in-plane Al electrodes deposited by thermal evaporation on top of the AZO film, with a size of 3 × 6 mm2 and 3 mm separation. Seebeck coefficient was obtained from the slope of the plot ΔV vs. ΔT shown in . The AZO films resistance over time was continuously monitored by a Keysight 34972A LXI data acquisition unit connected to a computer with home-made software.Electric and thermoelectric characterizations were performed with a home-made setup in Fig. , subsequThe transmittance measurements were performed in a JASCO V-770 spectrophotometer, in the wavelength range 190–2500 nm, in steps of 0.5 nm. The bending tests were performed with a home-made machine that measures the film electrical resistance as a function of the bending radius, calculated according to the International Electrotechnical Commission standard 47/2199/NP draft. Hence, a continuous resistance measurement profile is taken for each sample while bending.13, allowing to extract the overall elemental compositions and thicknesses. These simulations were generated starting from a base model of a layered structure of equal Al2O3/ZnO double-layers with thicknesses and compositions equal to the nominal values of deposition. The substrates are composed of Carbon, Hydrogen and Nitrogen. Fluorine was also detected – its contribution to the RBS spectra is clearly pointed at, – a finding that is not totally unexpected, since DuPont Kapton is often coated with a fluoropolymer resin layer (e.g. FEP) for imparting heat sealability, moisture barrier protection, and chemical resistance enhancement4. The RBS spectra of the AZO samples show a structure consisting of Zn, Al and O profiles of similar energy widths, with two thin Al peaks separated in energy by a difference consistent with the energy widths of the Zn, Al and O profiles  belonging to a common ALD run, indicating that the ALD process is reproducible and uniform in small areas without visible coating defects or folds. The AZO films thickness of (86 ± 4) nm measured through RBS is within the thickness range of 85–95 nm measured by optical ellipsometry.Representative RBS spectra of AZO films deposited by ALD on untreated (KNA) and plasma treated Kapton CS substrates with T1 and T2) are shown in Fig.  and T2 a16. For instance, hydroxyl and carboxyl groups  for the He/O2 treatment. Such chemically active groups help forming affinity or chemical bonds with a coating material and thus adhesion is improved. Elemental composition of plasma-treated kapton film surface was previously addressed by N. Inagaki et al. and it was demonstrated that the plasma treatment led to incorporation of oxygen atoms rather than nitrogen atom17. The O/C atomic ratio of the plasma treated kapton film, determined by XPS in this work, revealed to be 1.9–2.3 times higher than that of the untreated Kapton.The plasma treatment is known to change the surface morphology and create chemically active functional groups and radicals that are able to enhance the wettability and surface energy of Polymers2 (T1) or He/N2 (T2) as shown in the SEM images of Fig. The morphology of AZO films deposited onto 25 μm thick Kapton CS were assessed by SEM and AFM. The AFM images, shown in Fig. R0 and R are the film resistances measured respectively before exposure to the UV-light and at time 0 values for thin and thick films vary in the ranges 300–650 Ω and 50–200 Ω, respectively. The UV spectrum of the lamp used is shown in Fig. R0 as UV is switched OFF. This photo-response to UV-light is well reported for AZO films18 and it is attributed to the combination of two phenomena: photoconductivity and oxygen photo-detachment. When an electron-hole pair in conduction and valence bands is generated following the UV-photon absorption, supply of photo-generated electrons to adsorbed oxygen can also promote photo-detachment thereby liberating trapped electrons, the electrons trapped at these oxygen sites, that add to the photocurrent. Therefore, both effects may cause an increase in the photocurrent and thereby, a decrease in the baseline resistance, as well reported in the literature for ZnO films doped with Al and with a band gap of 3.4 eV. ALD deposited AZO films revealed to have the same band gap, and as such those effects may also explain the photo response of ALD AZO films. Figure 2 or H/N2 plasmas enhance the long term stability of the UV-triggered variation of electrical resistance. This maybe related with the fact that, plasma treatment promotes a better interaction of ALD precursors with the substrate surface leading to better adhesion and seed layers from where ALD films growth with improved lattice crystallinity. Therefore, the virgin, untreated, Kapton substrates may have a smoother surface compared to the plasma treated surfaces providing lower adhesion to the AZO films. Across this weaker binding interface, there should develop a larger discontinuity in electronic density that may introduce localized defect states (“surface-like” electronic states) that potentially absorb UV photons and affect the absorption spectra. The resulting enhanced UV absorption is a source of photo-electrons injection in the conduction band, increasing the photocurrent and causing larger baseline variation.Figure 19, as such the electrical resistance, Seebeck coefficient, power factor and power curves for thin and thick AZO films was measured before and during UV illumination.In ZnO the increase in photo-induced carriers due to the UV-light exposure contribute to the thermoelectric transport2 to ∼302 μW/mK2 . During UV exposure the films resistance decreases and as a result the power density output increases, specially for thicker films as shown in Table 19. Table The Seebeck coefficient (S) is not affected by plasma treatment or UV exposure, but is enhanced from ∼75–80 μV/K to ∼105–150 μV/K, when the film thickness is changed from ∼100 nm to ∼460 nm, respectively. Likewise, the power factor  also increased, from ∼71 μW/mKe.g. Al and Ag) and changing relative humidity. Figure 21) 25 μm thick and ARALDITE epoxy adhesive were selected to test as they match the mentioned conditions. As shown in Fig. The influence of humidity on AZO films deposited on flexible substrates was evaluated by monitoring the films resistance over time in the dark while using different electrode materials . The critical bending radius Rc, is the radius of curvature below which the resistance exceeds 5% of its original value (R0) as illustrated in Fig. Rc of encapsulated AZO films is smaller than the Rc of uncoated samples, indicating that the protective coatings selected do positively affect the bendability of the films. From those, ARALDITE was chosen to make repetitive bending tests, as it produced the lowest critical bending radius Rc. Moreover, SEM images  was around zero for all samples. The surface morphology of encapsulated samples after 5 and 20 cycles is shown in the SEM images of Fig. Figure This work demonstrates that AZO films deposited on flexible substrates retains good thermoelectric properties with great stability under humidity environment and bending. Therefore, ALD is a viable and practical technology to produce AZO films consistently and reproducibly with the mechanical and electrical properties that make these suitable for large flexible transparent thermoelectric generator applications.2 (T1) or H/N2 (T2), prior to the atomic layer deposition (ALD) of Al doped Zn oxide (AZO) films enhances the stability over time of their electrical and thermoelectric properties, and reduces films cracking in subsequent handling. The highest Seebeck coefficient (∼120 μV/K) and power factor (∼300 μW/mK2) were obtained for 460 nm thick AZO films, and the power output density for a small volume element of these films (3 × 6 mm2 of area and ∼460 nm of thickness) and a temperature difference of 10 °C is ∼0.3 mW/cm3. This increases to 0.4 mW/cm3 during exposure of the films to UV-light, due to the photo-thermoelectric effect. The electrical resistance of the films proved to be stable under humidity, even when exceeds 75%. Nevertheless, it was shown that electrodes moisture absorption and retention can be avoided through their covering with a protective layer such as TESA and ARALDITE. These are highly transparent keeping the original transmittance of the AZO film in the visible wavelength range and did not increase the films critical bending radius, which in the case of ARALDITE it was even further reduced, from 19 mm to 14 mm. Continuously bending of films down to 100 mm over 20 cycles, led to films resistance variations near zero, demonstrating the films durability under mechanical stress. These observed changes in critical bending radii and electrical resistance upon repeated cycling may be explained by a proposed mechanism, based on the immediate generation of extended defects that collapse to micro-cracks capable of cyclically interrupting charge transport, along with continuing increase in point like defects which upon scattering and/or trapping charge carriers increase the overall resistance.The work further demonstrates that plasma treatment of Kapton substrates, with He/OSupplementary Information


The robust, potentially universal approach for the synthesis of large-size transition metal dichalcogenides bilayer single crystals is highly-promising for fundamental studies and technological applications.Epitaxial growth of atomically thin two-dimensional crystals such as transition metal dichalcogenides remains challenging, especially for producing large-size transition metal dichalcogenides bilayer crystals featuring high density of states, carrier mobility and stability at room temperature. Here we achieve in epitaxial growth of the second monolayer from the first monolayer by reverse-flow chemical vapor epitaxy and produce high-quality, large-size transition metal dichalcogenides bilayer crystals with high yield, control, and reliability. Customized temperature profiles and reverse gas flow help activate the first layer without introducing new nucleation centers leading to near-defect-free epitaxial growth of the second layer from the existing nucleation centers. A series of bilayer crystals including MoS Epitaxial growth of the two-dimensionally thin material flakes with effective layer number and shape calls for precise control over temperature and carrier gas. Here, authors report controlled epitaxial growth of the second layer vertically for MoS2, WS2, MoWS and MoWSSe compounds by reverse hydrogen gas flow chemical vapor epitaxy. Monolayer TMD crystals10 have small state densities which may limit their practical device applications. Few-layer, especially bilayer (2L) TMD crystals, are particularly attractive for applications in thin film transistors11, logic devices12, and sensors13 owing to their high density of states, carrier mobility and stability at room temperature14. Moreover, bilayer materials are important basic structures for studying atomic interaction between single layers. Despite considerable efforts and limited success to date, reliable synthesis of large-size high-quality TMD bilayer single crystals remains a significant challenge, in part due to major issues in controllable epitaxial growth of the second monolayer from the first monolayer.Atomically thin two-dimensional (2D) crystals of transition metal dichalcogenides (TMDs) have been studied with great attention in view of their unique properties and potential applications in electronic and optoelectronic devices13 and fundamental studies20 are mainly derived through micromechanical exfoliation, limited by low yield, poor controllability in layer number and size. Post-treatment using laser21, plasma23, patterning method24, and thermal annealing25 also produces few-layer, bilayer, and monolayer flakes but show same constrains. Contrary to these top-down methods, the bottom-up chemical vapor deposition (CVD) approach is highly regarded as one of the most promising ways to produce TMD multilayers29. However, the as-produced TMD structures are typically polycrystalline with the many randomly oriented domains and domain boundaries, which inevitably diminish their device performance33. Other common issues include co-existence of areas with monolayer, bilayer, trilayer and few-layer structures, small crystal size , and low process yield34.Bilayer or few-layer TMD materials used for applications38. Unfortunately, TMD monolayer crystals are usually too delicate to survive multiple sequential stages required for homogeneous epitaxy of the second monolayer and relaxation of as-formed bilayer crystals. Poor control of chemical vapor during the temperature swing stage between the growth stages often leads to uncontrolled and undesired nucleation centers on the first monolayer. To achieve large size and high quality TMD bilayer single crystals, it is essential to ensure homogeneous epitaxy of the second monolayer from activated nucleation centers of the first monolayer while suppressing new nucleation centers.These issues are particularly daunting for bilayer TMD crystals because CVD growth requires at least two stages with different growing temperatures, adjusted to enable high-order stacking of single layers2) with high yield, large size, and high controllability. The as-grown MoS2 samples exhibit uniform and dominant bilayer domains with AA and AB stacking structures. The maximum lateral size of such bilayer domains can reach 300 μm, well above typically achievable otherwise. A series of advanced characterization tools including optical microscopy, Raman, photoluminescence (PL), atomic force microscopy, and electrical transport measurements are used to evaluate the quality of the as-grown MoS2 bilayer samples. Back-gated field-effect transistor (FET) devices based on the as-grown AA stacking MoS2 bilayer samples show better electronic performances compared to monolayer TMD-based devices. Our reverse-flow chemical vapor epitaxy is also applied for the synthesis of various high-quality TMD bilayer crystals, including WS2, ternary Mox1−WxS2, and even quaternary Mox1−WxSy)2. The synthesis details and challenges in bilayer growth are described in Supplementary Note 2 domains may meet each other and merge together to form a continuous bilayer film as shown in Supplementary Figure 39.Bilayer MoS2/H2 flow during the temperature swing stage can be explained as follows. First, the reverse carrier gas flow can prevent unintended supply of chemical vapor source to eliminate the generation of new nucleation centers on the growth substrate and the as-grown first layer during the temperature rising process40. Zhang et al.8 employed the same idea to prevent uncontrolled homogeneous nucleation and thus enabled highly robust epitaxial growth of diverse heterostructures, multi-heterostructures, and superlattices from 2D atomic crystals. Second, the hydrogen flow could saturate the dangling bonds on the edge and at the surface of the as-grown first MoS2 monolayer crystals, thus blocking the laterally epitaxial growth as reported by Jia et al.41. The surface energy of the edge-terminated structure is considerably higher compared to that of the as-grown flat basal-plane structure43. Consequently, the second monolayer is more likely to deposit on the as-grown monolayer surface. At the same time, H2 can slightly etch away emerging nucleation points on the growth substrate and thereby reduce the wettability of the growth substrate44. As such, the source vapor during the C-D stage can be easier to transfer through the substrate surface and have more possibility to reach the surface of the as-grown first monolayer. With a suitable carrier gas flow rate, the source vapor can have enough kinetic energy to reach the surface center of the as-grown first monolayer where there is an initial nucleation to trigger the growth of the second layer45. Therefore, the second monolayer crystals prefer to grow epitaxially and homogenously on the activated nucleation centers of the first monolayer, finally promoting the growth of MoS2 bilayer crystals, as shown in Fig. The strikingly positive effect of such reverse N2 crystals with triangle and hexagon shapes are dominating over the substrate surface. Also, the optical contrasts of these bilayer crystals are not only quite uniform across the whole substrate surface but also deeper than that of the monolayer MoS2 crystals as shown in Supplementary Figure 2 crystals with different stacking structures, and the results are presented in Supplementary Figure 2 bilayer crystals corresponding to 3R like phase in which the crystal orientation of the upper layer is consistent with that of the bottom layer37, while 800 °C is optimal for AB stacking ones namely 2H phase in which the crystal orientation of the upper layer is opposite to that of the bottom layer37. The schematic diagram of the reverse-flow chemical vapor epitaxy process for bilayer MoS2 crystals is displayed in Fig. From Fig. 2 samples with the maximum lateral size of even up to 300 μm. The representative AFM image in Fig. 2 film characteristic6. Interestingly, the structure of the as-grown AA stacking bilayer MoS2 samples displays a close relationship with the growth time of C-D stage (for the second layer growth), as presented in the optical images of the as-grown AA stacking bilayer MoS2 samples under different growth time of C-D stage  is calculated to be 0.27 nm, in good agreement with the reported values48. The fact that only AA and AB stacking MoS2 bilayer grains were obtained in experiment means that they are the most stable ones, and this is in good agreement with the previously calculated results49 and experimental results50. The detailed mechanism behind this can be found in Supplementary Information using extensive analysis of literature as well as theoretical calculations. Taking the reverse flow epitaxy method a step further, we were also able to grow MoS2 trilayers including AAA, AAB, ABB as well as ABA stacking orders and even multilayers as shown in Supplementary Note Figure 2 grains, and the representative results on big MoS2 bilayer flakes with a size of ~100 µm (including AA and AB stacking) are presented in Fig. 2, CVD-grown monolayer MoS2 and mechanically exfoliated bilayer MoS2, respectively. The A1g peak of all the bilayer samples has a distinct blue shift in comparison with that of the monolayer sample due to the interlayer coupling52. The location of both A1g and E12g peaks are almost the same between the as-grown stacking bilayer and the mechanically exfoliated one, suggesting a similar situation of the Van der Waals force interactions between the upper layer and the bottom layer. The frequency difference for both the as-grown AA and AB stacking bilayer is estimated to be 22.2 cm−1, quite similar to that (21.9 cm−1) of the mechanically exfoliated one but larger than that (20.5 cm−1) of the CVD-grown monolayer one53. The Raman peak intensity mappings of E12g and A1g of the as-grown complete AA stacking bilayer MoS2 shown in Fig. We further conducted micro-Raman and micro-PL studies to explore the optical properties of the as-grown bilayer MoS38. All the bilayer samples show a greatly suppressed PL intensity (at least 100 times weaker) compared to the monolayer one because of the transition from a direct bandgap (monolayer) to an indirect one (bilayer)5. For the as-grown complete AA stacking sample, the PL intensity mapping of peak A presented in Fig. 2 with steps also have a strong contrast between the monolayer and the bilayer areas, as shown in Supplementary Figure 2 bilayer flakes (including AA and AB stacking) and the results are shown in Supplementary Figures The PL spectra of all the bilayer samples presented in Fig. 2 crystals with AA or AB stacking. Many correction methods such as DFT-D2 correction54 and DFT-TS correction55 were developed to add the van der Waals interactions. Here, we adopt the Grimme’s DFT-D2 dispersion correction approach to describe van der Waals interactions in all models and the reason can be found in Supplementary Note 2 monolayer, AA stacking MoS2 bilayer, and AB stacking MoS2 bilayer are shown in Fig. 2 monolayer has a direct bandgap, corresponding to the ultra-strong and single PL peak (A), while both AA stacking MoS2 bilayer and AB stacking MoS2 bilayer are of indirect bandgap with three electronic transitions from conduction band to valence band, corresponding to three weak PL peaks namely A, B, and C, respectively. The emergence of peak B can be attributed to the band splitting caused by spin–orbit coupling, and peak C is related to the indirect gap transition40. Figure 2 bilayer crystals, respectively. The AA stacking (3R-like phase) lattice structure  lattice structure  calculation. The van der Waals interaction is very important to describe the interlayer interaction of the bilayer MoSure Fig.  consistsure Fig.  is more 2 samples to measure their electronic characteristics, and four devices have been tested for each group in order to explore consistency in electrical performances. Figure 2 FET devices, respectively. The electrical performances of the other three devices can be found in Supplementary Figures Vg from −60 to 60 V, which is the same as that commonly observed in monolayer one57. It is interesting that the output characteristics (Ids − Vds) of AA stacking devices  measured by switching source and drain electrodes  in a top gate configuration57 or using the four terminal-configurations to evaluate the intrinsic carrier mobility as reported61. The different energy band structures between AA and AB stacking induced by the different stacking order may also result in some discrepancy in mobility values.We further calculate the mobility and on/off ratio values for both groups of devices. The field effect mobility is calculated by the following equation:2, ternary alloy Mox1−WxS2, and quaternary alloy Mox1−WxSy)2 to enable them simultaneously evaporate, we can also achieve large area quaternary alloy Mox1−WxSy)2 and E12g of MoSe2 (282 cm−1). Similar to the cases of monolayer and bilayer MoS2 crystals, the Mox1−WxSy)22222 and heating zone 2 (downstream). The reverse-flow chemical vapor epitaxy process was intentionally divided into two growth stages: A-B stage stands for the growth of first monolayer, C-D stage (relatively higher working temperature) represents the vertically epitaxial growth of second monolayer, while B-C stage corresponds to the growth swing stage between the first and second layers. The system is modified so that the gas flow direction can be switched (reversed) during the growth swing stage (B-C stage). A forward gas flow of 50 sccm N3 powder (99.9%) was put in a ceramic boat in the center of heating zone 2 and excessive S powder  were put in the heating zone 1 (upstream) with a distance of about 38 cm away from the MoO3 powder. The Si substrates with 300 nm SiO2 (SiO2/Si) were placed facing down above the MoO3 powder. The heating zone 2 (MoO3 precursor) was heated but the heating zone 1 (S precursor) was turned off intentionally so that the latter would become hotter as the former was heated up because of thermal radiation. The distance of 38 cm between MoO3 and S powders ensure the simultaneous sublimation of both at growing stages. The heating zone 2 was initially heated to 700 °C in 100 min and then maintained at 700 °C for 10 min for the growth of first MoS2 monolayer (A-B stage). Afterwards, the temperature of heating zone 2 was further increased to 750 or 800 °C in 5 min (B-C stage) and then kept for 20 min for the vertically epitaxial growth of second MoS2 monolayer (C-D stage). Experimental results showed that 750 °C is suitable for AA stacking MoS2 bilayer crystals while 800 °C is optimal for AB stacking ones.Fifteen milligrams of MoO2 bilayer, 25 mg WO3 powder and 100 mg S powder were used as the sources and their distance was fixed at 42 cm to ensure the simultaneous sublimation at growing temperatures. The heating zone 2 was initially heated to 800 °C in 100 min and then maintained at 850 °C for 10 min for the growth of first WS2 monolayer (A-B stage). Afterwards, the temperature of heating zone 2 was further increased to 900 °C in 5 min (B-C stage) and then kept for 20 min for the vertically epitaxial growth of second WS2 monolayer (C-D stage). The other experimental parameters were the same as those in the growth case of MoS2 bilayer.Similar to the case of MoS3 powder was placed at the center of heating zone 2 while 15 mg MoO3 powder at the downstream of heating zone 2 with a suitable distance of 8–10 cm away from the WO3 source. Hundred milligrams of S powder and 25 mg Se powder were put in the heating zone 1 with distances of 42 and 38 cm away from the WO3 powder, respectively. The selected distances between the sources enable the simultaneous sublimation of them at growing stages. The heating zone 2 was initially heated to 900 °C in 100 min and then maintained at 900 °C for 10 min for the growth of first Mox1−WxSy)2(1−Sey2 monolayer (A-B stage). Afterwards, the temperature of heating zone 2 was further increased to 950 °C in 5 min (B-C stage) and then kept for 20 min for the vertically epitaxial growth of second Mox1−WxSy)2(1−Sey2 monolayer (C-D stage). The other experimental parameters were the same as those in the growth case of MoS2 bilayer.Ten milligrams of WOThe Raman and PL spectra characterizations were measured using a Renishaw LabRAM Invia micro-Raman system with 532 nm excitation laser at room temperature and in atmospheric environment. The testing laser spot size was about 1 μm by using a ×50 objective and the laser power was kept below 0.3 mW. The thickness of TMD bilayer and monolayer crystals was measured by AFM (Bruker model: Dimension ICON). The elemental composition of the as-grown TMD bilayer crystals was identified by XPS  in which the Al-ka (1486.6 eV) was used as source. TEM characterizations were performed with the Jeol JEM-ARM200F equipped with a cold field emission electron source and a probe-Cs-corrector at 200 kV accelerating voltage. The electronic characteristics of the TMD bilayer FETs were tested by a Keithley 2612A Source Meter in vacuum (~1 Pa) at room temperature.2 models, the monolayer one, the bilayer ones with AA and AB stacking orders, were calculated through First-principles based on DFT, which were implemented in the Atomistic-ToolKit (ATK) version 2017.0. The Perdew–Burke–Ernzerhof (PBE) of generalized gradient approximation (GGA) was used for studying the band structures. Besides, pseudopotentials of MoS2 using the Hartwingster–Goedecker–Hutter scheme with Tier 4 basis set. The density mesh cutoff was set as 75 Hartree to achieve the energy convergence of 10–5 eV and the force convergence of 0.01 eV/Å on each atom. The Brillouin zone was sampled by 9 × 9 × 1 and 25 × 25 × 1 Monkhorst–Pack K-point mesh for structure optimization and electronic band calculation. A vacuum spacing of 15 Å was used to avoid interaction between the calculated individual structures. In order to prove the validity of this method, we also benchmark our calculated results of monolayer MoS2 with other available publications which can be found in Supplementary Information.The geometry optimizations and the energy band structure calculations of the three kinds of MoS2/Si substrates, 5 nm-thick Ni and 50 nm-thick Au (Ni/Au) patterned by standard electron beam lithography (EBL) were used as the source and drain electrodes. The SiO2 layer with a thickness of about 300 nm acted as the dielectric layer and the P-doped silicon substrate served as the back gate electrode.The field effect transistor (FET) devices were fabricated with the as-grown TMD bilayer samples just on SiOSupplementary InformationPeer Review File


Brucella suis, a bacterial infection of nearly worldwide distribution. Brucella suis is also transmissible to humans, dogs and cattle and is considered a reemerging disease of public health concern. To date, there is no effective vaccine for swine. This prompted us to investigate the potential use of the commercially available vaccine for cattle or the live attenuated vaccine candidate S19ΔvjbR. As the first step, we sought to study the safety of the vaccine candidates when administered in pregnant sows, since one of the major drawbacks associated with vaccination using Live Attenuated Vaccines (LAV) is the induction of abortions when administered in pregnant animals. Fifteen pregnant gilts at mid-gestation were divided into four groups and subsequently vaccinated subcutaneously using different formulations containing 2.0 ± 0.508 × 109 CFU of either S19 or S19ΔvjbR. Vaccination in pregnant animals with the vaccine candidates did not induce abortion, stillbirths or a reduction in litter size. Multiple tissues in the gilts and piglets were examined at the time of delivery to assess bacterial colonization and histopathological changes. There was no evidence of vaccine persistence in the gilts or bacterial colonization in the fetuses. Altogether, these data suggest that both vaccine candidates are safe for use in pregnant swine. Analysis of the humoral responses, specifically anti-Brucella IgG levels measured in serum, demonstrated a robust response induced by either vaccine, but of shorter duration (4–6 weeks post-inoculation) compared to that observed in cattle or experimentally infected mice. Such a transient humoral response may prove to be beneficial in cases where the vaccine is used in eradication campaigns and in the differentiation of vaccinated from infected animals. This study provides evidence to support future efficacy studies of both vaccine candidates in swine.Brucellosis in swine is caused by  Among the 12 identified species classified based on preferential host specificity, 3 are highly pathogenic for their preferred host species , as well as humans, and are all associated with significant economic losses in different parts of the world Brucella infection in animals is characterized by abortion and infertility in domestic and wildlife animals Brucellosis is a zoonosis caused by vjbR vaccine candidates in swine. We have previously tested S19ΔvjbR vaccine strain in multiple animal species and have demonstrated an added effect in terms of reduction of inflammatory response or tissue colonization, therefore reducing the possible side effects associated with vaccination of S19 in pregnant animals. Specifically, we describe the safety profile of both vaccine candidates using different delivery systems when inoculated into pregnant sows, since abortion secondary to vaccination is a common and undesired side effect observed when using Live Attenuated Vaccine candidates (LAV) for brucellosis. We also sought to investigate the potential of vertical transmission by performing bacteriological and histopathological analysis of maternal and fetal tissues. Finally, humoral responses induced by the vaccine formulations were characterized as the first step towards understanding immune protection induced by LAVs against brucellosis in swine.The United States (US) is free from the disease in domestic swine in contrast to feral pigs in which the numbers of infected animals are on the rise 22.1American Yorkshire healthy gilts were used for this study and confirmed to be negative for brucellosis by ELISA. Gilts were synchronized and artificially inseminated to generate pregnancies that were at the same gestational age during vaccination. Only pregnant animals, confirmed via ultrasound, were included in the study. All animal procedures were performed under TAMU Institutional Animal Care and Use Committee (IACUC) guidelines.2.2vjbR vaccine strain was engineered and used as a vaccine candidate in a previous study Brucella abortus S19 was obtained from the National Veterinary Services Laboratories . Both strains were grown on tryptic soy agar plates (TSA) for 3 days at 37 °C with 5% (v/v) CO2 and harvested from the surface of the plates using phosphate-buffered saline (PBS), pH 7.2. A dose of 2.0 ± 0.508 × 109 CFU/ animal was used as confirmed by actual viable colony counts of bacterial serial dilutions on TSA plate.The S19 Δ2.3B. abortus S19ΔvjbR was resuspended in 1 ml of MOPS buffer  and mixed with 5 ml of alginate solution in MOPS buffer of pH 7.3. Extrusion of the suspension through a 200-μm nozzle into a 100 mM calcium chloride solution produced capsules under continuous stirring. Capsules were crosslinked in poly-l-lysine and coated with 2.5 mg of VpB (vitelline protein B), followed by the addition of an outer layer of alginate Alginate encapsulation was performed as previously described 2.49 CFU of either (1) S19 (n = 4), (2) encapsulated S19ΔvjbR (n = 4), (3) unencapsulated S19ΔvjbR (n = 4), or (4) empty capsules/control (n = 3).Pregnant gilts were randomly distributed into 4 groups and inoculated at mid -gestation (60–66 days of gestation) subcutaneously (SQ) in the scapular area with a single dose containing 2.0 ± 0.508 × 102.5Prior to vaccination and until delivery, gilts were monitored twice a day for side effects associated with vaccination including abortion, adverse reactions at the injection site, food consumption, vaginal discharges, and fever. Rectal temperature of pregnant gilts was measured daily. A temperature of 38.8 °C (±0.3 °C) was considered the normal body temperature threshold for gilts 2.6Screening for vaginal shedding was performed biweekly on all animals. Vaginal swabs were plated onto Farrell’s agar medium for bacterial isolation. Plates were incubated at 37 °C and monitored daily for up to 30 days.2.7Within the first 3 days post-delivery, all sows were euthanized via pentobarbital overdose and necropsied for the detection of any gross changes associated with vaccination. Tissue sections including spleen, liver, lung, uterus, placenta, pre-scapular, mammary, inguinal and mesenteric lymph nodes were collected and formalin fixed. Paraffin sections were H&E stained and examined by a board-certified veterinary anatomic pathologist.2.82. Bacterial colony forming unit (CFU) was measured by visual enumeration.Colonization of maternal tissues by the different vaccine formulations was assessed by culture after delivery. Spleen, liver, lung, uterus, pre-scapular, mammary, inguinal and mesenteric lymph nodes were removed and weighed. One gram of tissue from each organ was homogenized in 1 ml of phosphate buffered saline, pH 7.2 (PBS) and cultured on TSA/ and Farrell’s agar media and incubated up to 30 days at 37 °C with 5% (v/v) CO2.9Piglets were euthanized and a full necropsy was performed within the first hours of birth. Lung float test was conducted to assess fetal viability upon birth. Tissue sections of spleen, liver, lung and umbilical cord were fixed in 10% (v/v) buffered formalin, paraffin embedded and sections were stained with hematoxylin and eosin (H&E). Histological changes were assessed by a board-certified veterinary anatomic pathologist.2.102. A total of 181 piglets were examined and all cultures were done in duplicate.Within the first 12 h of delivery, sections of spleen, lung, liver, kidney, stomach, and umbilicus were collected from all piglets. Homogenized tissue was cultured on TSA and Farrell’s agar media and incubated up to 30 days at 37 °C with 5% (v/v) CO2.11Brucella antigen was performed . 30 μl of sera from immunized animals were added to an equal volume of Rose Bengal antigen onto a card and mixed thoroughly. A scale was developed to categorize the degree of agglutination as; (1) ++++/+++ strong, (2) ++ mild, (3) + weak and (4) − no agglutination.Serum agglutination against 2.12Brucella abortus lysate (250 ng/well) overnight at 4 °C. Following blocking  tween 20) for 2 h at room temperature, plates were washed and incubated with sow sera samples (diluted 1:500 in the blocking buffer) for 1 h at 37 °C. Following washing, peroxidase-conjugated secondary antibody  was added at a dilution of 1:1000 in blocking buffer and incubated at 37 °C for 1 h. Following washing, horseradish peroxidase substrate was added for 15 min and OD was measured at 450 nm. All assays were performed in triplicate.iELISA was performed at 0, 2, 4- and 6-weeks post-vaccination. 96 well plates were coated with heat killed and sonicated 2.13P values <0.05 were considered significant. The significance of differences between the different clinical parameters including litter size, abortions, CFU and fever were analyzed by ANOVA followed by Tukey post-hoc analysis. The two-way analysis of variance (ANOVA) test was used for the anti-Brucella IgG and IgM experiments followed by Tukey’s multiple comparisons test.All analyses were performed using the GraphPad Prism 6.0 software  and 33.1vjbR induced abortions when given to pregnant animals, sows were vaccinated subcutaneously (SQ) at a dose of 2.0 ± 0.508 × 109 CFU with the different vaccine candidates at mid-gestation and were monitored daily until delivery. No adverse side effects were observed during the course of the study. The duration of pregnancy was 112 to 116 days with no significant differences between groups (Table S1). There was no significant difference in litter size, abortions, mummies or stillbirths among the different groups . No significant changes in body temperature were observed between groups regardless of the vaccine formulation or vaccine strain  at the time of delivery regardless of the formulation and vaccine strain used.3.4Assessment of any gross and histopathological changes associated with vaccination was investigated. Following delivery, all sows were euthanized and a full necropsy was performed. No gross changes associated with vaccination in any major organ, site of inoculation or reproductive tissues was evident. Tissue sections consisting of liver, lung, spleen, uterus, and placenta were histologically normal and resembled the control group .Fig. 1Hi3.53.5.1vjbR remained negative to RBT during the whole period of the experiment but was later confirmed by ELISA to have seroconverted. Serum samples from control animals remained negative throughout the study.The humoral response elicited by the vaccination of gilts was evaluated biweekly over the course of 6 weeks. At 2 weeks post-vaccination, the agglutination response was highest among all groups regardless of the formulation or the vaccine strain (Table S2). There was no statistical difference between the different vaccine candidates. Starting at 4 weeks post-vaccination, the agglutination response started to decrease in all groups and varied from mild to weak agglutination, with only 2 animals from the unencapsulated S19 group still remaining as strong positives. At 6 weeks post-vaccination, regardless of the treatment, all gilts became serologically negative. One gilt vaccinated with unencapsulated S19 Δ3.5.2Brucella-specific IgM and IgG antibodies by ELISA. Immunization with the S19 or S19ΔvjbR vaccine candidates elicited an anti-Brucella specific IgM and IgG response that was clearly detectable at 2 weeks post vaccination for either encapsulated or unencapsulated vaccine. Anti-Brucella IgM and IgG levels were similar at 2 weeks post-vaccination, however, by 4 weeks, the IgM levels were significantly reduced compared to IgG levels which persisted for 4 weeks and then started to decrease . In the cases of stillbirths , there was no evidence of an inflammatory or an infectious process and these were considered to be unrelated to the vaccination. Two of the stillborn observed were from the control.Complete gross and histopathological evaluations were conducted in all piglets (181). Tissue sections from all organs were unremarkable with no differences observed between the treatment groups and controls . Fetal d4B. suis in the feral swine population and the imminent zoonotic threat, only a handful of studies have been performed throughout the years to develop and evaluate potential vaccine candidates for swine B. suis infection, obvious alternatives include the potential use of the commercially available vaccines for cattle (S19 and RB51) in swine. Vaccination of pregnant sows with S19 has only been described in one study conducted in a brucellosis endemic farm in 1948, in which abortions were reported to occur in pregnant swine that were vaccinated with S19 B. suis challenge Brucella vaccine for swine that provides protection, not only against abortion but most importantly that it will prevent the organism from shedding into the environment. Ideally, this vaccine should be safe regardless of the reproductive status of the animal and should provide differentiation of vaccinated from naturally exposed animals.Despite the endemic nature of vjbR in which the vjbR gene (BABII 0118) is absent vjbR mutants have revealed a significantly attenuated phenotype  while conferring high levels of protective immunity vjbR vaccine, part of our strategy involves the use of a delivery system designated alginate microencapsulation Over the past years, our laboratories have been interested in the development of improved vaccines, with a strong emphasis on designing new LAV. Among these, we have developed and tested the LAV candidate designated S19ΔAs the first step towards the development of a vaccine for swine, we sought to concentrate our efforts in evaluating the safety profile by (1) assessing the potential risk of inducing abortion when inoculated into pregnant animals, (2) determining the presence of any adverse clinical symptoms associated with vaccination, (3) determining organism excretion into the environment via different routes including placenta, vaginal secretions as well as vertical transmission to the fetuses and (4) determining if there is a persistent infection in sows following delivery.Brucella spp induces abortions in livestock including swine. Under experimental conditions, B. suis can induce fetal death when animals are infected after day 40–45 of pregnancy with abortion occurring usually at mid to late gestation vjbR did not induce abortion and the vaccine was incapable of colonizing either placental or fetal tissues which further supports its attenuated phenotype. It is well known that S19 vaccination induces abortions in multiple animal species. Interestingly, abortion is not significantly reduced when a lower dose of 3.0 × 108 organisms is used, clearly indicating the residual virulence of S19 in cattle 8 CFU of Brucella abortus S19 B. suis to establish a marked or persistent infection in pigs. Studies conducted by Stuart et al Brucella abortus wild-type Strain 544 can only establish a short-lived infection for up to 99 days post-inoculation. This is different than what is usually observed in cattle where vaccination of adult pregnant cattle with S19 induces abortions at an approximate rate of 22% when animals are inoculated with the standard dose of 5.8 × 109 CFU/ animal It is well-known that infection with The reproductive status and stage of gestation when a susceptible animal becomes infected (either by vaccination or infection) will have a different clinical manifestation. For example, if females become infected very early during pregnancy, sows will develop placentitis that can vary in their degree of magnitude and, if severe enough, can impair early fetal growth resulting in early embryonic death approximately 3–4 weeks post-infection Brucella suis S2 vjbR or S19 vaccines to colonize the fetuses, multiple tissues were examined for the presence of residual vaccine strains. None of the 181 fetuses had any recoverable bacteria in either the lungs, stomach, gastric contents and umbilicus which are all preferential sites for isolating Brucella in fetuses vjbR in pregnant sows cannot cross the placental barrier when animals are vaccinated at mid-gestation.The ability of the vaccine to colonize the placenta with subsequent vertical transmission to the fetus was also investigated. In swine, the transplacental transmission has been described in both naturally infected sows as well as in animals vaccinated with B. abortus Strain 544 demonstrated the presence of bacteria in vaginal swabs when animals were inoculated with a dose ranging from 5 × 108 to 5 × 1011 CFU via the subcutaneous or intraconjunctival route B. abortus wild-type.Brucellosis in swine, like any other animal species, is characterized by the excretion of the bacteria into the environment through different secretions such as milk, urine or vaginal discharges Brucella infection in humans Brucella abortus S19 vjbR developed fever. Whether fever can be used as a predictor of abortion or wild type infection in swine is yet to be studied, since none of the sows in this study aborted. However, vaccination did not induce fever in any of the gilts, further supporting the vaccine safety profile of both the encapsulated and non-encapsulated vaccines.Despite the fact that undulant fever is one of the major symptoms of B. abortus in swine has demonstrated that pigs are susceptible to infection, develop bacteremia and excrete bacteria into the environment up to 99 days post-inoculation Brucella in swine, suggesting that handling of vaccinated carcasses after 7 weeks post-vaccination is safe. Based on the bacteriology, histopathology and Immune response data, vaccine clearance occurs between 6 and 7 weeks.Although brucellosis is usually associated with reproductive failure, this is not pathognomonic nor the only clinical sign associated with the disease in swine. Brucellosis in swine has been considered to be somewhat different from the disease in ruminants with the development of a non-specific inflammatory process characterized by the formation of granulomas with extensive areas of necrosis that can be present in different organs such as lymph nodes, joints, bones and liver among others Brucella specific IgG and IgM responses peaked at two weeks post-vaccination and dropped significantly by six weeks except for the IgG response of encapsulated S19ΔvjbR group. These findings correlate with the unpublished results referenced in a study from the 40′s in which they indicate that swine vaccinated with S19 had a positive agglutination response that dropped rapidly by 4 weeks following vaccination vjbR in swine is a transient, short-lived response compared to what it is typically observed in mice, cattle or red deer inoculated with S19 or S19ΔvjbRB. suis. Experimental infection of swine with 2 × 107 CFU of Brucella suis biovar 2 through the conjunctival route results in a Brucella-specific IgG response that lasts approximately 21 weeks following inoculation B. abortus wild-type in swine induces a shorter response compared to B. suis in swine that persists for 10 weeks following inoculation Brucella vaccines has been primarily studied in the mouse model and to a lesser degree in other species including cattle vjbR vaccination elicited a transient humoral response, this could potentially be used to our advantage to distinguish between vaccinated and naturally exposed animals. Naturally, infected swine will have a sustained response for at least 21 weeks (or 10 weeks if infected with B. abortus) as opposed to swine vaccinated with S19 or S19 ΔvjbR which will become negative after 6 weeks via RBT [43]. Practically, a screening test within 2 week and 4-week post-vaccination interval can be introduced in the field and serve as a DIVA  strategy while vaccinating swine with either S19 or S19 ΔvjbR.As the first step towards the characterization of the immune response of the vaccine candidates in sows, we investigated the humoral response associated with vaccination using RBT and iELISA. As a confirmatory test, iELISA is commonly employed following a positive RBT. During these studies, 5Overall, the vaccine candidates regardless of the formulation proved to be safe in pregnant swine and did not induce any undesirable side effect. Since no adverse pregnancy outcomes, tissue pathology or organism excretion was observed in these studies, efficacy studies in pregnant swine are the next logical step towards the development of a vaccine for swine. Future efficacy studies will also support or refute the use of alginate microencapsulation as a delivery system to improve vaccination efficacy.The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Allison Rice-Ficht, managing partner of NanoRelease Technologies (NRT), LLC Inc., has a 95% equity interest in NRT, a company involved in vaccine delivery platforms. The terms of this arrangement have been reviewed and approved by TXAgriLife Research and Texas A&M University in accordance with their conflict of interest policies.


ZSM-5 can be an excellent substitute to sulfuric acid which caused corrosion and equipments damage.A modified hydrothermal method for ZSM-5 synthesis was described. The crystals gave a typical pattern ), which indicated that the subnanocrystals could have the primary structure of MFI-type zeolites. the FT-IR spectra of subnanocrystals which have the primary structure of MFI zeolites. Oleic acid methyl ester (OAME) was prepared via a rapid derivatization procedure. the acidic strength is determined by the zeolite crystal structure and the higher esterification rate of ZSM-5 can be attributed to its stronger acidity compared to H•Zeolites are popular industrial catalysts comprised of crystalline microporous materials.•A modified hydrothermal method was used for ZSM-5 synthesis.•Synthesized ZSM-5 has a typical subnanocrystals structure corresponds to MFI- type Zeolite.•ZSM-5 can be an excellent substitute to sulfuric acid for the catalysis of esterification reactions. Zeolites are popular industrial catalysts comprised of crystalline microporous materials of different molecular sizes and shapes. They are widely used in oil refining, petrochemical production and organic synthesis of important chemicals. The intricate channel structure of zeolites provides selectivity based upon shape and polarity, and is particularly effective for reactant molecules with a kinetic diameter below 10 A° . Biodies2O3 (0.0018 mol), 0.075 mol of NaOH, Aqueous amount of 0.030 mol H2SO4 (96%), 4 g of  tetraacetic acid (EDTA) and 25 ml of SiO2 30 wt.% (0.15 mol) were mixed and stirred for 30 min, pH should be in the 11–12 range until formation of zeolite gel. The filtered gel was placed in an oven at ∼95 °C for a week. After a week of heating, the product was gravity filtered and rinsed with water until the pH is about 8 and dried in air. To eliminate organic compounds and zeolite formation, the powder was calcined under 800 °C for 2 h at a rate of 10 °C/min. X-ray powder diffraction spectrum was obtained using D8 Advance Bruker X-ray diffractometer by scanning from 10 to 50°. FT IR spectra were obtained using a SHIMADZU 8400 s spectrometer in the range of 400–4000 cm−1.A modified hydrothermal method for ZSM-5 synthesis can be described as the following: Aqueous amount of Alg for 2 min and 1 μl of hexane phase was injected for GC analysis. Results are obtained from GC data and the conversion of OAME was calculated by the formula [OAME was prepared via a rapid derivatization procedure. An aliquot (35.15 ml) of OFFA, 20 ml of methanol and 1.77 ml sulfuric acid solution (10 Wt%) were added into the Pyrex screw-cap tube (16 × 125 mm). The tube was vortexed for 10 s before being stirred to become homogeneous under 85 °C water bath for 15 min for acidic esterification. Afterwards, catalysis tests were carried out in the reactions and performed in a round-bottom glass flask with two-neck vol–250 ml equipped with a reflux condenser in a crystallizer. An excess of methanol was added to the flask containing the oleic acid in order to shift the equilibrium towards ester formation with a molar ratio of 1:4.5 (oleic acid:methanol). 4.5 (oleic acid:methanol). ZSM-5 catalyst  was added to catalyze the esterification without it depends on the concentration of the reaction substrate. The crystallizer is placed on a hot plate about 70 °C with magnetic stirring about 450 rpm, maintaining the stability of the degree of the temperature through the thermocouple installed. Connecting the condenser in a small submersible pump placed in a water bath containing an amount of ice. This system condenses the vapor from the reaction to maintain the amount of the reactants. The esterification reaction is started and stay for three hours, several samples are taken at different times  by Pasteur pipette, the samples are then separated by centrifuge. In order to regularize biodiesel acidity, OAME was placed in a separator funnel 60 ml, washed with distilled water and then with dilute solution of sodium bicarbonate, until having a value (PH = 7). The tube containing the OAME extract was centrifuged at 1500Crystallization temperature of calcination is one parameter that plays an important role on the properties of the catalyst. The good crystallinity of the ZSM-5 catalyst proves that the choice of the calcination temperature of 800 °C for 2 h is just .−1 and 540 cm−1, all spectra of the samples show a typical ZSM-5 structure [−1 can be attributed to both structure sensitive external tetrahedron and structure insensitive internal tetrahedron symmetric stretching vibrations. Meanwhile, the characteristic band of the double five rings structure of MFI-type zeolites could be observed at 620 cm−1 from all the samples, which can be used to estimate the crystalline degree of the samples. Although the band of the subnanocrystals at 620 cm−1 is weak, it may be proofed that the subnanocrystals could have the primary structure of MFI-type zeolites [FT-IR spectra of zeolite lattice vibration modes are depicted in tructure , are cauzeolites .Fig. 2FTThe conversion of oleic free fatty acid into Oleic acid methyl ester was determined by measuring the acid value at the end of the reactions. To compare the catalytic behavior of the two catalysts in the reaction using the same molar amount of sulfuric acid  and synthetized ZSM-5  was performed .Fig. 3OA2SO4 catalyst, excellent yields of OAME, 60%, 71% and 72.5% were obtained at 10, 20 and 30 min respectively. However, in the same conditions the OAMEs were obtained with lower yields (60–70%) using ZSM-5 catalysts. The higher esterification rate of ZSM-5 can be attributed to its stronger acidity compared to H2SO4, especially after 50 min of reaction. At 60 min, using the same catalyst… the conversion ratio was 74% while, under the same conditions, reached 50% in previous work [Using Hous work .The esterification rate is believed to be determined by the acidity strength and pore size of the catalyst . GeneralThe synthesis of ZSM-5 can be realized with a simple hydrothermal method with modifications result a subnanocrystals which can catalysis an esterification of a free fatty acid with short chain to produce biodiesel with a performance better to this observed in commercialize Zeolite. The catalyst was shown to be efficient and promising for the esterification of oleic acid with methanol. ZSM-5 can be than an excellent alternative to sulfuric acid which caused corrosion and equipments damage.This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.No conflict of interests including financial, personal or other relationships with other people or organizations.