Two-dimensional layered perovskites are attracting increasing attention as more robust analogues to the conventional three-dimensional metal-halide perovskites for both light harvesting and light emitting applications. However, the impact of the reduced dimensionality on the optoelectronic properties remains unclear, particularly regarding the spatial dynamics of the excitonic excited state within the two-dimensional plane. Here, we present direct measurements of exciton transport in single-crystalline layered perovskites. Using transient photoluminescence microscopy, we show that excitons undergo an initial fast diffusion through the crystalline plane, followed by a slower subdiffusive regime as excitons get trapped. Interestingly, the early intrinsic diffusivity depends sensitively on the choice of organic spacer. A clear correlation between lattice stiffness and diffusivity is found, suggesting exciton–phonon interactions to be dominant in the spatial dynamics of the excitons in perovskites, consistent with the formation of exciton–polarons. Our findings provide a clear design strategy to optimize exciton transport in these systems. The so-called two-dimensional (2D) layered perovskites possess distinct optoelectronic properties from their 3D counterparts due to their reduced dimensionality. Here Seitz et al. investigate the exciton transport dynamics in 2D perovskites and highlight the impact of the stiffness of the lattice. Currently, the main challenge in the applicability of perovskites is their poor environmental stability17. Reducing the dimensionality of perovskites has proven to be one of the most promising strategies to yield a more stable performance19. Perovskite solar cells with mixed two-dimensional (2D) and three-dimensional (3D) phases, for example, have been fabricated with efficiencies above 22%20 and stable performance for more than 10,000 h21, while phase pure 2D perovskite solar cells have been reported with efficiencies above 18%23. Likewise, significant stability improvements have been reported for phase pure 2D perovskites as the active layer in light emitting technologies29. The improved environmental stability in 2D perovskite phases is attributed to a better moisture resistance due to the hydrophobic organic spacers that passivate the inorganic perovskite sheets, as well as an increased formation energy of the material30.Metal-halide perovskites are a versatile material platform for light harvesting33. 2D perovskites are composed of inorganic metal-halide layers, which are separated by long organic spacer molecules. They are described by their general chemical formula L2[ABX3]n-1BX4, where A is a small cation , B is a divalent metal cation , X is a halide anion , L is a long organic spacer molecule, and n is the number of octahedra that make up the thickness of the inorganic layer. The separation into few-atom thick inorganic layers yields strong quantum and dielectric confinement effects34. As a result, the exciton binding energies in 2D perovskites can be as high as several hundreds of meVs, which is around an order of magnitude larger than those found in bulk perovskites37. The excitonic character of the excited state is accompanied by an effective widening of the bandgap, an increase in the oscillator strength, and a narrowing of the emission spectrum38. The strongest confinement effects are observed for n = 1, where the excited state is confined to a single B-X-octahedral layer.However, the reduced dimensionality of 2D perovskites dramatically affects the charge carrier dynamics in the material, requiring careful consideration in their application in optoelectronic devices39. This stands in contrast to bulk perovskites in which free charges are generated instantaneously thanks to the small exciton binding energy35. Particularly, with excitons being neutral quasi-particles, the charge extraction becomes significantly more challenging as they cannot be guided to the electrodes through an external electric field40. Excitons need to diffuse to an interface before the electron and hole can be efficiently separated into free charges41. On the other hand, for light emitting applications the spatial displacement is preferably inhibited, as a larger diffusion path increases the risk of encountering quenching sites which would reduce brightness. While charge transport in bulk perovskites has been studied in great detail, the mechanisms that dictate exciton transport in 2D perovskites remain elusive41. Moreover, it is unclear to what extent exciton transport is influenced by variations in the perovskite composition.Consequently, light harvesting using 2D perovskites relies on the efficient transport of excitons and their subsequent separation into free charges42. This technique allows us to follow the temporal evolution of a near-diffraction-limited exciton population with sub-nanosecond resolution and reveals the spatial and temporal exciton dynamics. We observe two different diffusion regimes. For early times, excitons follow normal diffusion, while for later times a subdiffusive regime emerges, which is attributed to the presence of trap states. Using the versatility of perovskite materials, we study the influence of the organic spacer on the diffusion dynamics of excitons in 2D perovskites. We find that between commonly used organic spacers , diffusivities and diffusion lengths can differ by one order of magnitude. We show that these changes are closely correlated with variations in the softness of the lattice, suggesting a dominant role for exciton–phonon coupling and exciton–polaron formation in the spatial dynamics of excitons in these materials. These insights provide a clear design strategy to further improve the performance of 2D perovskite solar cells and light emitting devices.Here, we report the direct visualization of exciton diffusion in 2D single-crystalline perovskites using transient photoluminescence-microscopyn = 1 phenethylammonium lead iodine (PEA)2PbI4 2D perovskite by drop-casting a saturated precursor solution onto a glass substrate44, as confirmed by XRD analysis and photoluminescence spectroscopy (see Methods section for details). Using mechanical exfoliation, we isolate single-crystalline flakes of the perovskite and transfer these to microscopy slides. The single-crystalline flakes have typical lateral sizes of tens to hundreds of micrometers and are optically thick. The use of thick flakes provides a form of self-passivation that prevents the typical fast degradation of the perovskite in ambient conditions.We prepare single crystals of λex = 405 nm) and an oil immersion objective (N.A. = 1.3). The image of the fluorescence emission of the exciton population is projected outside the microscope with high magnification (×330), as illustrated in Fig. 2PbI4. The fluorescence emission intensity I is normalized at each point in time to highlight the broadening of the emission spot over time. Each time-slice I is well described by a Voigt function45, from which we can extract the variance σ(t)2 of the exciton distribution at each point in time  = 171 nm to σ(t = 10 ns) = 448 nm, indicating fast exciton diffusion.To measure the temporal and spatial exciton dynamics, we create a near-diffraction-limited exciton population using a pulsed laser diode  = σ(t)2 − σ(0)2. Taking the one-dimensional diffusion equation as a simple approximation, it follows that MSD(t) = 2Dtα, which allows us to extract the diffusivity D and the diffusion exponent α from our measurement  a fast linear broadening occurs with α = 1.01 ± 0.01, indicative of normal diffusion, while for later times (t ≳ 1 ns) the broadening becomes progressively slower with α = 0.65 ± 0.01, suggesting a regime of trap state limited exciton transport 2PbI4. Our diffusivity of single crystalline (PEA)2PbI4 is around an order of magnitude higher than previously reported mobility values from conductivity measurements  of polycrystalline films46. This finding is reasonable as grain boundaries slow down the movement of excitons and conventional methods measure a time-averaged mobility that cannot separate intrinsic diffusion from trap state limited diffusion.To analyze the time-dependent broadening of the emission spot in more detail, we study the temporal evolution of the mean-square-displacement (MSD) of the exciton population, given by MSD background excitation of varying intensity  is obtained at a background illumination power of 60 mW cm−2. For comparison, this value corresponds roughly to a 2.5 Sun illumination. Additionally, we observe that the onset of the subdiffusive regime is delayed as more and more trap states are filled, as represented by the increasing tsplit parameter , which is of the same order of magnitude as previously reported values for bulk perovskites49. The inset in Fig. λ2 with increasing illumination intensity. We note that the exponential decay of Eq. D(t) by relating the subdiffusion directly to the trap density 1/λ2 rather than relying on the subdiffusive exponent α of a power law commonly used in literature42.To gain theoretical insights and quantitative predictions concerning the observed subdiffusive behavior of excitons and its relation with trap state densities, we performed numerical simulations based on Brownian dynamics of individual excitons diffusing in a homogeneously distributed and random trap field (see Supplementary Note α = 1) for all illumination intensities. This strongly suggests that the early diffusion dynamics is unaffected by energetic disorder, which would result in a sublinear behavior with α < 1, and any trap states, giving us direct access to the intrinsic exciton diffusivity of the material and allowing us to compare the intrinsic exciton diffusivity between perovskites of different compositions. To explore compositional variations, we substitute phenethylammonium (PEA) with butylammonium (BA)—another commonly used spacer molecule for 2D perovskites51.Importantly, the early diffusion dynamics is unaffected by the trap density and shows normal diffusion 2PbI4, excitons in (BA)2PbI4 are remarkably less mobile, displaying a diffusivity of only 0.013 ± 0.002 cm2 s−1, which is over an order of magnitude smaller than that of (PEA)2PbI4 with 0.192 ± 0.013 cm2 s−1 2PbI4 2PbI4, we take a closer look at the structural differences between these two materials. Changing the organic spacer can have a significant influence on the structural and optoelectronic properties of 2D perovskites. Specifically, increasing the cross-sectional area of the organic spacer distorts the inorganic lattice and reduces the orbital overlap between neighboring octahedra, which in turn increases the effective mass of the exciton52. Comparing the octahedral tilt angles of (PEA)2PbI4 and (BA)2PbI4, a larger distortion for the bulkier (PEA)2PbI4 (152.8°) as compared to (BA)2PbI4 (155.1°) is found54. The larger exciton effective mass in (PEA)2PbI4 would, however, suggest slower diffusion, meaning a simple effective mass picture for free excitons cannot explain the observed trend in the diffusivity between (PEA)2PbI4 and (BA)2PbI4.To understand the large difference in diffusivity between (PEA)33. To investigate the possible role of exciton–phonon coupling on exciton diffusion, we first quantify the softness of the lattices of both (PEA)2PbI4 and (BA)2PbI4 by extracting the atomic displacement parameters from their respective single crystal X-ray data55. The atomic displacement of the different atoms of both systems are summarized in Fig. 2PbI4 as compared to (PEA)2PbI4 in both the organic and inorganic sublattice54. The increased lattice rigidity for (PEA)2PbI4 can be attributed to the formation of an extensive network of pi-hydrogen bonds and a more space-filling nature of the aromatic ring, both of which are absent in the aliphatic BA spacer molecule. Qualitatively, a stiffening of the lattice reduces the exciton–phonon coupling and would explain the observed higher diffusivity in (PEA)2PbI4 as compared to (BA)2PbI4. In addition to a softer lattice, we find that (BA)2PbI4 exhibits a larger exciton–phonon coupling strength than (PEA)2PbI4, as confirmed by analyzing the temperature-dependent broadening of the photoluminescence linewidth of the two materials . However, in the presence of dominant long-range lattice deformations, large exciton–polarons may form which extend across multiple lattice sites. The diffusion of large polarons decreases with increasing temperature (∂D/∂T < 0), resembling that of band-like free exciton motion, although with a strongly increased effective mass. In Fig. 2PbI4 and (BA)2PbI4. In both materials a clear negative scaling of the diffusivity with temperature is observed (∂D/∂T < 0), characteristic of band-like transport.Polaron formation can significantly modify the mechanism of transport, in some cases causing a transition from band-like to a hopping type transport60. Crucial in this respect will be further spectroscopic investigations of temperature-dependent optical properties of these materials, which should allow to better distinguish the influence of exciton–polaron formation from more traditional phonon-scattering mechanisms in these materials.The observed correlation between diffusivity and lattice softness in combination with band-like transport is in good qualitative agreement with the formation of large exciton–polarons. However, further studies will be needed to provide a more quantitative model that can explain the large differences in diffusivity between the various organic spacers. The correct theoretical description of exciton–phonon coupling and exciton–polarons in 2D perovskites is still the subject of ongoing debate, though the current consensus is that the polar anharmonic lattice of these materials requires a description beyond conventional Frohlich theoryn = 1 2D perovskites, we have observed consistent trends in the n = 2 analogues. Indeed, just like in n = 1, in n = 2 the use of the PEA cation yields higher diffusivities than for BA  cation, consistent with the trend in the atomic displacement parameters. It is important to note, though, that already for n = 2 perovskites a significant free carrier fraction may be present in the perovskites61, suggesting that transport in n > 1 perovskites cannot be assumed to be purely excitonic and needs to be evaluated more rigorously.Meanwhile, structural rigidity can be used as a design parameter in these systems for optimized exciton transport characteristics. Taking into account the close correlation between diffusivity and the atomic displacement, this parameter space can be readily explored using available X-ray crystal structure data for many 2D perovskite analogues. While the influence of the organic spacer is expected to be particularly strong in the class of 62. A trade-off therefore exists in choosing the optimal rigidity for bright emission. Meanwhile, for light harvesting applications, long diffusion lengths are essential for the successful extraction of excitons. While strongly excitonic 2D perovskites are generally to be avoided due to the penalty imposed by the exciton binding energy, improving the understanding of the spatial dynamics of the excitonic state may help mitigate this negative impact of the thinnest members of the 2D perovskites in solar harvesting.From a technological perspective, structural rigidity may play a particularly important role in light emitting devices. Long exciton diffusion lengths in light emitting applications can act detrimentally on device performance, as it increases the possibility of encountering a trapping site. From an exciton–polaron perspective, this suggests soft lattices are preferred. At the same time though, Gong et al. highlighted the role of structural rigidity in improving the luminescence quantum yield through a reduced coupling to non-radiative decay pathways2PbI4. We show that excitons undergo an initial fast diffusion through the crystalline plane, followed by a slower subdiffusive regime as excitons get trapped. Traps can be efficiently filled through a continuous wave background illumination, extending the initial regime where excitons undergo normal diffusion. By varying the organic spacer L we find that the intrinsic diffusivity depends sensitively on the stiffness of the lattice, revealing a clear correlation between the lattice rigidity and the diffusivity. Our results indicate that exciton–phonon interactions dominate the spatial dynamics of excitons in 2D perovskites. Moreover, the observations are consistent with the formation of large exciton–polarons.In summary, we have studied the spatial and temporal exciton dynamics in 2D metal-halide perovskites of the form L63 using transient-absorption microscopy to study excited-state transport in 2D perovskites, with a focus on the differences in the spatial dynamics as a function of layer thickness (n = 1 to 5).During the review process we became aware of a related manuscript by Deng et al.2PbI4 and (DA)2PbI4, were synthesized under ambient laboratory conditions following the over-saturation techniques44. In a nutshell, the precursor salts LI, PbI2, and AI were mixed in a stoichiometric ratio (2:1:0 for n = 1 and 2:2:1 for n  = 2) and dissolved in γ-butyrolactone. The solution was heated to 70 °C and more γ-butyrolactone was added (while stirring) until all the precursors were completely dissolved. The resulting solutions were heated to 70 °C and the solvent was left to evaporate. After 2–3 days, millimeter sized crystals formed in the solution, which was subsequently cooled down to room temperature. For this study, we drop cast some of the remaining supersaturated solution on a glass slide, heated it up to 50 °C with a hotplate and after the solvent was evaporated, crystals with crystal sizes of up to several hundred microns were formed. The saturated solution can be stored and re-used to produce freshly grown 2D perovskites within several minutes. We would like to note that drop cast n = 2 solutions form several crystals with different n values. However, n = 2 crystals can be easily isolated during the exfoliation (see next section) and the formation of n = 2 can be favored by preheating the substrate to 50 °C before drop casting.Chemicals were purchased from commercial suppliers and used as received (see Supplementary Methods). Layered perovskites, with the exception of (HA)2PbI4 and (DA)2PbI4 were synthesized by dissolving PbI2 (100 mg) in HI (800 µl) through heavy stirring and heating the solution to 90 °C. After PbI2 was completely dissolved a stoichiometric amount of the amine was added dropwise to the solution.(HA)43.The perovskite crystals of the thin film were mechanically exfoliated using the Scotch tape method (Nitto SPV 224). The exfoliation guarantees a freshly cleaved and atomically flat surface area for inspection, which is crucial to avoid emission from edge states and guarantee direct contact with the glass substrate. After several exfoliation steps, the crystals were transferred on a glass slide and were subsequently studied through the glass slide with a ×100 oil immersion objective . A big advantage of this technique is that the perovskites are encapsulated through the glass slide from one side and by the bulk of the crystal from the other side. It is important to use thick crystals to guarantee good self-encapsulation and prevent premature degradation of the perovskite flakes to affect the measurementλ = 1.5406 Å). The polycrystalline perovskite films were prepared by drop casting the saturated perovskite solutions on a silicon zero diffraction plate.X-ray diffraction (XRD) was performed with a PANanaltical X’Pert PRO operating at 45 kV and 40 mA using a copper radiation source (Perovskite flakes were excited with a 385 nm light emitting diode (Thorlabs) and the emission spectrum was measured using a spectrograph and an EMCCD camera coupled to a spectrograph . Temperature of the flakes was varied with a Peltier element , using a PID temperature controller  connected to a type K thermocouple  for feedback control and a fan for cooling.Perovskite flakes were excited with a 405 nm laser , which was focused down to a near-diffraction limited spot. The photoluminescence was collected with an APD . The laser and APD were synchronized using a timing board for time correlated single photon counting (Pico-Harp 300).45. In short, a near diffraction limited exciton population was created using a 405 nm laser  and a ×100 oil immersion objective . Fluorescence of the exciton population was then imaged with a total ×330 magnification onto an avalanche photodiode  with a detector size of 20 µm. The laser and APD were synchronized using a timing board for time correlated single photon counting (Pico-Harp 300). The APD was capturing an effective area of around 60 × 60 nm (= 20 µm/330). The APD was scanned through the middle of the exciton population in 60 or 120 nm steps, recording a time trace in every point. To minimize the degradation of the perovskites through laser irradiation, the perovskite flakes were scanned using an x-y-piezo stage (MCL Nano-BIOS 100), covering an area of 5 × 5 µm. Diffusion measurements were performed with a 40 MHz laser repetition rate and a laser fluence of 50 nJ cm−2 unless stated otherwise. The time binning of the measurement was set to 4 ps before software binning was applied. For the temperature-dependent measurements, the temperature was varied with a silicon heater mat , using a PID temperature controller  connected to a type K thermocouple  for feedback control. Here, a silicon heater mat was chosen over the Peltier element as a Peltier element expands during the heating process and causes mechanical vibrations that lead to drift.Exciton diffusion measurements were measured following the same procedure as Akselrod et al.r(t) is the position of the exciton in the plane at time t, its displacement Δr over a time Δt is given by,D is the free-diffusion coefficient and dW is taken from a Wiener process, such that 〈dWdW〉 = Δt. Traps were scattered throughout the plane following a uniform random distribution. The exciton is considered to be trapped as soon its location gets closer than Rtrap = 1.2 nm to the trap center. The value was taken from estimations of the exciton Bohr radius and corresponds to a trap area of 1.44 nm2 37. In any case, in the dilute regime, the diffusion is not sensitive to the trap size Rtrap, because the trap radius is much smaller than the average separation between traps, Rtrap ≪ λ. To numerically integrate the equation of motion, we used a simple second-order-accurate modification of the well-known Euler Maruyama algorithm: the BAOAB-Limit method64. Trajectories were computed for many independent excitons and the data was averaged to determine the MSD as a function of time. While the simulation of the MSD was done in two dimensions, we used the MSD in one dimension to match the experimental conditions: We have performed Brownian dynamics simulations of a single exciton diffusing in a field of traps, representing ideal (non-interacting) excitons in the dilute limit carried out in experiments. In these simulations, an exciton diffuses freely until it finds a trap, where it just stops. Free diffusion is modelled using the standard stochastic differential equation for Brownian motion in the Itô interpretation. If Supplementary InformationPeer Review File


H-MX2 . We show that while NbSe2 exhibits a strongly enhanced CDW order in two dimensions, TaSe2 and TaS2 behave oppositely, with CDW being absent in NbS2 entirely. Such a disparity is demonstrated to arise from a competition of ionic charge transfer, electron-phonon coupling, and electron correlation. Despite its simplicity, our approach can, in principle, explain dimensional dependence of CDW in any material, thereby shedding new light on this intriguing quantum phenomenon and its underlying mechanisms.Charge density wave (CDW) is a startling quantum phenomenon, distorting a metallic lattice into an insulating state with a periodically modulated charge distribution. Astonishingly, such modulations appear in various patterns even within the same family of materials. Moreover, this phenomenon features a puzzling diversity in its dimensional evolution. Here, we propose a general framework, unifying distinct trends of CDW ordering in an isoelectronic group of materials, 2 H-MX2 and provide a unified understanding involving several microscopic factors.The dimensional dependence of charge density wave (CDW) in two-dimensional dichalcogenides remains puzzled. Here, Lin et al. study trends of CDW ordering in an isoelectronic group of materials 2 The appearance of the latter in the 2D limit has, in particular, attracted a great deal of attention. By definition, CDW is the ground state of a one-dimensional metallic chain, leading to a simultaneous periodic modulation of its charge density and lattice dimerization9. In the 2D limit and beyond, CDW does not follow a unique pattern even within the same family of materials with identical electronic properties. While it is believed that phonon vibrations at low temperatures play a major role in the emergence of CDW ordering, its underlying mechanism has remained an unsettled question for decades11 and its relation with superconductivity is still a subject of debates13. The advent of layered 2D materials14 ushers in a new era of exploring CDW in confined dimensions16. Mechanical exfoliation19, molecular-beam epitaxy (MBE)22, and chemical vapour deposition24 have produced a plethora of 2D materials exhibiting CDWs. Dimensionality reduction has been shown to enhance the CDW order in some of them24 while suppress it in others25. Even for the same compound21, results from different studies are scattered. For better understanding and control of the CDW states, it is thus imperative to determine the key factors governing the intrinsic effects of dimensionality on CDWs.Two-dimensional (2D) materials have become a fertile playground for the exploration and manipulation of novel collective electronic states. Recent experiments have unveiled a variety of robust 2D orders in highly crystalline materials ranging from magnetismH-MX2  are among the most studied CDW systems11. They all share similar crystal structures at high temperatures with isoelectronic chemical properties. At low temperatures, they behave differently, manifesting as CDW patterns unique to each compound. More interestingly, 2H-NbS2 stands out as CDWs are known to be absent in its bulk limit26. In a broad picture, two constraints facilitate the emergence of CDW instability and its diversity in this group of materials. The first is the fact that TMDs, in general, possess a quasi-2D electronic structure regardless of their thickness. This is due to the weak van der Waals stacking of MX2 layers, allowing resonance of phonon vibrations along certain crystalline directions. Second, the metallic TMDs, as will be discussed later, exhibit a strong tendency to form additional covalent bonding between their chalcogens to compensate for the deficient charge transfer from the existing transition metals. When combined, these two can modulate the otherwise uniform charge distribution of metallic bands, such that the distorted state is chemically more robust.The group-V transition metal dichalcogenides (TMDs) 2H-MX2  family. We show disparate dimensional dependence of the CDW properties in these materials, when the samples are reduced from their bulk limit to atomically thin layers. Based on these experimental observations, we establish a general framework that unifies all possible driving forces behind CDW ordering in 2D materials. We show that in principle, three major factors orthogonally govern the fate of CDW: ionic charge transfer, electron–phonon coupling, and the spreading extension of the electronic wave functions. We further explain how these factors compete with each other and manifest in CDW as such systems approach their bulk limit. The simplicity of our approach, combined with its success in accounting for such sophisticated features, paves a new way to study CDW and exotic quantum phenomena emerging from it in low dimensions.In this work, we report systematic Raman scattering spectroscopy of the 2Ha structure10  and the neighbouring chalcogens (each in 2−  state). As such, they all show a metallic behaviour with a mixed ionic-covalent character. Figure QI and determined by their electronegativity difference, see Supplementary Fig. M–X bonds; (2) intralayer axial and planar σ-electron hoppings between the chalcogen p orbitals, both forming covalent X–X bonds; (3) interlayer σ-hoppings across the van der Waals gap.The prototypes chosen here all crystalize in the so-called 2e10 Fig. . The tra2 is nearly 140% of that for monolayer NbSe2  as well as the superconducting transitions at a lower temperature are consistent with those established in the literature27. Atomically thin samples were mechanically exfoliated from the bulk crystals, with special care taken to minimize sample degradation (see Methods).High-quality single crystals were chosen for our experiments. Their temperature-dependent resistance data in Fig. 28. Figure Ag1 and Eg2 symmetry while the latter only couples to Eg2 (ref. 8). Four common features are noted in the data: (1) a rigid layer mode with Eg2 symmetry (labelled as shear mode), which corresponds to interlayer shearing vibration and distinguishes the 2H from 1T and 3R polytypes; (2) an Ag1 phonon mode, which only involves the chalcogen atoms vibrating against each other along the c-axis; (3) an Eg2 phonon mode, which involves the transition metal and chalcogen atoms vibrating along opposite directions within the layer plane; (4) a broad two-phonon scattering peak commonly assigned as due to the CDW soft phonons30.We used Raman scattering to obtain both CDW signatures and phonon informationAg1 and Eg2 modes suggest that the ionic bonds dominate their eigenfrequencies. As shown in Fig. Ag1 and Eg2 modes are found to have much higher frequencies in S-based 2H-MX2 compounds than in Se-based ones. Similar results are found for the monolayer samples  compounds. In a glance, one can notice a systematic difference between the S- and Se-based compounds. In the former, the optical modes appear at much higher frequencies, and the gap between the Ag1 and Eg2 modes at Γ point ) is much larger. Such a hardening of phonon modes is clear evidence that the chemical bonding in S-based compounds is more ionic (and thus stronger) than that in Se-based compounds. To be more precise, the higher electronegativity of sulfur enables it to gain more charge from the M site, thereby making the resulting M–S bond harder than its M–Se counterpart in 1H–MSe2. This is consistent with the trend of ionic charge transfer shown in Fig. σ = 0.03 Ry) for treating the distribution of electrons at the Fermi level31 (see Methods). Nevertheless, the calculated values for the Ag1 and Eg2 mode frequencies at Γ point agree reasonably well with our experimental data  as well as more accurate pseudo-potentials.The vibration patterns for the les Fig. . To confata Fig. , even co2. These are the amplitude modes or zone-folded modes unique to the CDW phase33. The former features characteristic softening and broadening upon approaching TCDW from below, while the latter shows only a minute change of the frequency. Fig. 2, TaSe2, and TaS2, respectively. We focus on the amplitude modes, which are the most pronounced features in the spectra below 100 cm−1. NbSe2 exhibits a single broad peak that shifts from below 50 cm−1 in the bulk to 73 cm−1 in the monolayer, while the intensity is less affected. In contrast, both TaSe2 and TaS2 exhibit multiple sharper amplitude modes below 100 cm−1. Each peak shows a weak layer number dependence for its frequency, while the intensity dramatically diminishes in atomic layers. The blueshift of the mode frequency (suppression of the mode intensity) correlates well with enhanced (reduced) TCDW in atomically thin NbSe2 (TaSe2 and TaS2), as will be detailed below. The completely different behaviours of the amplitude modes in the Nb- and Ta-based compounds call for further investigations.Figure 2 down to the monolayer  when approaching the monolayer limit. The trends are further confirmed by analysing the amplitude mode frequency and the zone-folded mode intensity , we have calculated ΔQσ for all four compounds in the bilayer and bulk configurations , TaS2, and TaSe2 (ref. 35).TaSe2 Figs. , suggestQI we discussed earlier, implying a trade-off between ΔQI and λ. In addition to these two parameters, the spatial extension of the wave functions (represented here as 1/〈r2〉) plays an important role. The more localized are the wave functions, the less effective become both ΔQI and λ. This is because it enhances the electron–electron correlation, demobilizing the carriers and so preventing the formation of any superlattice charge ordering. As a result, if λ is not large enough, CDW remains forbidden. This then solves the last piece of the puzzle, i.e. why NbS2 is so robust against CDW instability. We note that van Loon et al.36 have already given a detailed account for the competing nature of electron–phonon coupling versus the short- and long-range electron Coulomb interaction, and its prominent role in the absence of CDW instability in NbS2. Interestingly, they suggest that the interplay between these parameters strongly enhances both charge and spin susceptibilities in NbS2, meaning that it is at the verge of collapsing to a CDW or even a spin density wave phase, if sufficient perturbations are introduced 36. As such, NbS2 appears to be an ideal candidate for exploring thermal and quantum fluctuations.In a broader context, we can again attribute this tendency to the ionic charge transfer ΔQI and λ and high 1/〈r2〉. Above the surface of the resulting terrain is a parameter-space within which the CDW is forbidden and below it is where CDW is allowed to emerge. We also have shown the schematic location of each of the 2H-MX2 compounds, studied here. Obviously, NbS2 is the only member of this family appearing in the region where CDW is prohibited.Altogether, the three parameters (1) ionic charge transfer, (2) electron–phonon coupling, and (3) the spatial extension of the electronic wave functions are the key components defining the fate of CDW ordering and its thickness dependence in this and potentially other layered materials. Thus, they can be used to create a unified phase diagram describing such instabilities in low-dimensional limits. We have schematically illustrated such a phase diagram in Fig. 2, TaSe2, and TaS2. For NbSe2, a strongly enhanced TCDW from  ~33 K in the bulk to  ~145 K in the monolayer was previously reported in mechanically exfoliated samples on sapphire substrates8 and confirmed here. This is in stark contrast to the almost unchanged TCDW in MBE-grown monolayer NbSe2 on bilayer graphene7. For TaSe2, we found a rather weak suppression of TCDW in the exfoliated monolayer, while MBE-grown monolayer TaSe2 on bilayer graphene shows slightly enhanced TCDW with respect to its bulk value22. The exfoliated samples transferred on sapphire substrates are expected to exhibit intrinsic CDW properties. The MBE-grown samples may be affected by charge transfer from the underlying graphene substrate. For monolayer NbSe2, such charge transfer can increase the intralayer covalency and suppress the strongly enhanced TCDW. For monolayer TaSe2, due to its extended charge distribution, charge transfer tends to accumulate between TaSe2 and graphene, so that the intralayer covalency is reduced; CDW is therefore enhanced.Lastly, we address some controversies in the studies of 2D CDWs in NbSe2 on Au(111) substrate37, but persist in MBE-grown monolayer TaS2 on graphene/Ir(111)38 as well as in the exfoliated monolayer studied here. These findings provide further evidence that the CDW formation in such atomically thin TMDs is, in general, highly susceptible to the surrounding environment, which could be a neighboring layer or even a substrate. As we discussed earlier, the deficient charge transfer in metallic TMDs makes them active in finding new pathways for electron hopping so that the chalcogen p orbitals can gain all electrons they need to form a closed shell system. Such pathways could, in principle, extend to a substrate. As an example, with Au(111) surface as a substrate, one can expect electron charge transfer from spatially extended 4s electrons of Au to the chalcogens above them. Meanwhile, the trigonal symmetry of Au(111) substrate enforces the original symmetry of pristine monolayer TaS2, thereby avoiding any lattice deformation. Of course, hybridization between monolayer TaS2 and the Au(111) substrate could significantly affect its electronic band structure40, and correspondingly, its intrinsic properties. Alternatively, when the substrate is made of chemically inactive orbitals (such as pπ states in graphene), the CDW is still inevitable.Interestingly, CDWs were found to be absent in epitaxial monolayer TaSTCDW, which is essential for developing CDW-based devices for applications. It may also be applied to explain the distinct dimensionality effects in the growing family 2D CDW materials41. Future work should address the connection of the current framework with the existing theories for CDWs, for instance, Fermi-surface nesting and momentum-dependent electron–phonon coupling42.In conclusion, our chemical-bonding framework provides an intuitive guide for boosting Bulk single crystals were synthesized by the chemical vapour transport method. Atomically thin flakes were mechanically exfoliated from the bulk crystals on silicone elastomer polydimethylsiloxane stamps and transferred on sapphire substrates for Raman study. To minimize sample degradation, we prepared them in a glove box filled with nitrogen gas, followed by encapsulation with thin h-BN. The flake thickness was determined by the shear mode frequency in the Raman data  was sampled by a 24 × 24 × 1 k-mesh. The ionic charge transfer and hopping parameters were obtained by constructing a 22-band tight-binding model by downfolding the DFT Hamiltonian using maximally localized Wannier functions49. The M-d and X-p atomic orbitals were taken as projection centres.The electronic structure of 1−5 Ry Bohr−1 and the total energy was converged below 10−10 Ry. To avoid the negative phonon modes, resulting in an overestimation of λ, the Methfessel–Paxton smearing scheme with a relatively large broadening parameter σ = 0.03 Ry was used, as suggested in ref. 31. The dynamical matrix was then calculated based on density-functional perturbation theory employing an 8 × 8 × 1 q-mesh. Finally, we computed λ using the interpolation scheme implemented in Quantum Espresso46. In this scheme50, λ is obtained by summing up the electron–phonon coupling arising from each individual phonon mode ν at all available phonon wave vectors q, i.e. λ =  ∑qνλqν. λqν is expressed as follows:ωqν is the phonon frequency for mode ν at the wave vector q; εnk is the eigenvalue of the nth Kohn–Sham eigenfunction ψnk at wave vector k in the BZ; εF is the Fermi level; N(εF) corresponds to the density of states per spin at εF; and ΩBZ is the volume of the BZ. Above, qνV being the first derivative of the self-consistent potential associated with phonon ωqν.For the calculation of phonon modes and the corresponding electron–phonon coupling parameters, we first fully optimized both the lattice parameters and atomic positions until the magnitude of the force on each ionic site was less than 10Supplementary InformationPeer Review File


Understanding how these interactions vary through space and time allows for the management of these resources to be improved, and for isotope data to be useful in other disciplines. The seasonal variation of δ2H and δ18O in tap water within South Africa was assessed to identify municipalities that are supplied by seasonally invariant sources that have long residence periods, such as groundwater, and those supplied by sources that vary seasonally in a manner consistent with evapoconcentration, such as surface water—the proposed two tap water “worlds”. Doing so allows for the cost-effective spatial interpolation of δ2H and δ18O values that likely reflect that of groundwater, removing the residual error introduced by other sources that are dependent on discrete, isolated factors that cannot be spatially generalised. Applying the proposed disaggregation may also allow for the efficient identification of municipalities that are dependent on highly variable or depleted surface water resources, which are more likely to be vulnerable to climate and demographic changes.Stable isotope ratios of hydrogen and oxygen (δ In many contexts, the management of water resources is expected to become increasingly challenging given the predicted impacts of climate change, urbanization, economic development, and increasingly complex water resource supply systems and infrastructure3.Understanding the variation of stable hydrogen (H) and oxygen (O) isotopes in tap water has global application for several diverse fields5. A theoretical understanding of the seasonal variability of stable isotopes in precipitation, surface water and groundwater  post office branches, with 233 (69%), 203 (60%) and 158 (46%) branches returning viable sample kits in May, November and both campaigns, respectively  following the methods of West et al.8. Prior to analysis, each sample was filtered through a 0.2 µm syringe filter. Six injections of 1.9 µl were analysed for each sample, with the results of the first three injections discarded as conditioners. All samples were assessed for additional components that could affect the analysis following the methods of West et al.8. Isotopic ratios are expressed in ‰ as:2H/H or 18O/16O). The d-excess is defined as16:Stable water isotopes were analysed at the University of Cape Town by wavelength-scanned cavity ring-down spectroscopy using a Picarro L2120-i  where analysed after at most six consecutive samples were run. Tap water samples were calibrated to VSMOW using two in-house standards. The accuracy and precision of the calibration were assessed with a third quality control standard not used in the calibration. The mean (± one standard deviation) accuracy and precision of the isotope analyses across all batches were 0.4‰ ± 0.2‰ and 0.5‰ ± 0.2‰ for δ2H and 0.10‰ ± 0.05‰ and 0.09‰ ± 0.05‰ for δ18O, respectively.For drift correction and quality control, three in-house standards of known δ2H, δ18O and d-excess values in R version 3.6.3 (https://cran.r-project.org/)17 using the dismo18 and spdep19 packages. Universal kriging models were applied to interpolate the observed δ2H, δ18O and d-excess values and construct prediction isoscapes for each. The additional explanatory variables that were considered include the mean annual precipitation (MAP) and temperature (MAT), elevation (elev) and A-pan equivalent potential evaporation (PE), obtained from Schulze et al.20. The shortest Euclidean distance to the coast (tocoast) was calculated in R17 using the rgdal21 and sf22 packages. Lastly, the predicted mean annual δ2H (MAH), δ18O (MAO) and d-excess (MAD) in precipitation26 were also considered.A Monte Carlo simulation permutation test for Moran’s I statistic was calculated using 5,000 random permutations for the observed δ2H in tap water is being predicted, MAH is considered as an explanatory variable but not MAO or MAD). The explanatory variables for the final model were selected by first assessing the stepwise effect on the AIC (Akaike information criterion) value and then the statistical significance of the variable coefficients. The kriging models were run in R17 using the automap27, gstat28, sp29, and raster30 packages, the and plotted using the tmap31 package. Where the model uncertainty was greater than one standard deviation of the underlying training data, hatching was overlaid on the isoscapes to represent a lower confidence in the model predictions.A linear model was constructed for each isotope ratio from the November and May tap water sampling campaigns using all five climatological and topographical variables , the interaction between elev and tocoast (elev*tocoast) and between MAP and apan (MAP*apan), and the corresponding isotope ratio in precipitation  between May and November , those that varied in a manner consistent with evaporation  and vice versa for sample locations categorized as having a negative direction of variation .The seasonal variability in δ2H, δ18O and d-excess November 2017 tap water  samples made up 43% of all repeated sample locations, with categories B, C and D making up 40%, 8% and 9% respectively Fig. . The disles Fig. . Despitegns Fig. . The varThe distribution of the four categories of seasonal isotope variation varied spatially across the region Fig. . Sample 2H, δ18O and d-excess values for category A and B sample locations were produced by universal kriging. The density and distribution of category C and D sample locations are too limited to allow for representative kriging of national scale isoscapes.Isoscapes for the May Fig.  and Nove2H and δ18O values for category A were more negative in the interior than on the coast; the eastern coast in KwaZulu-Natal province, in particular, had more positive δ2H values than the rest of the study area.The spatial variability of category A samples broadly reflected that of the predicted precipitation isotopes Fig. . The δ2H2H and δ18O values and atypically negative d-excess values  are consistent with the expectations for groundwater8. Sampling locations that exhibited seasonal variability that approximate local evaporation . The extrapolation of δ2H and δ18O values beyond the location of sampling is unreliable for this category  have several possible interpretations. The source of tap water supply could entirely or partly include surface water reservoirs that experience a greater degree of evapoconcentration in the dry than the wet season, or the source could entirely or partly include non-local water that has stable isotope ratios distinct from that of local water. Non-local water could be distant in space  or distant in time . The gradient of change in water stable isotopes The sample locations that demonstrated medium to high seasonal variability  schemes12.The seasonal variation from local evapoconcentration can only result in isotope ratio changes along the local evaporation line (LEL) in the positive direction4.Figure 4. Two expectations are provided, based on the principles outlined above. The climatic and topographical dynamics between the Gauteng and Lesotho catchments suggest that the scenarios illustrated in Fig. 2H and δ18O values between May and November (Fig. The water requirements of the urban municipalities and industrial sectors in Gauteng are met by a large IBWT scheme sourced from Katse Dam in Lesothober Fig. .Figure 112. The use of other geochemical tracers may further distinguish between modern and fossil groundwater aquifers39.Future studies can improve the interpretation of seasonally variable tap water isotopes by quantifying the spatial and seasonal variability in the LEL gradient. Additionally, further comparisons between the national-scale groundwater isotope ratio predictions based on tap water samples and catchment-scale groundwater isotope data would be valuable to determine the accuracy at scales relevant to local water resource management. Comparisons between the predicted groundwater isotope ratios and that of local modern precipitation would also contribute to determining whether or not the groundwater being extracted was likely recharged recently or is non-renewable2H, δ18O and d-excess values in tap water aid in to their application to urban hydrology. In particular, it has allowed for the identification of the two tap water “worlds”—municipalities that likely rely on groundwater and surface water dependent tap water sources. This has several implications for fields such as water resource management, ecology and forensics1. This study has demonstrated that stable H and O isotopes in tap water have the potential to complement traditional water resource management practices. The degree of observed temporal variability suggests that future studies of stable isotopes in tap water need, at a minimum, to incorporate seasonal sampling in order to understand the variable source and transport dynamics of a given location.The seasonal variability of δSupplementary Information.Supplementary Tables.


It should be “A Hydrogel Drink With High Fructose Content Generates Higher Exogenous Carbohydrate Oxidation and a Reduced Drop in Dental Biofilm pH Compared to Two Other, Commercially Available, Carbohydrate Sports Drinks”.In the original article, the title 13CO2 measurements. The authors wish to add the second sentence below (in black) to the first paragraph in the Results, Exogenous and Endogenous Carbohydrate Oxidation section:In the original article, there was an error. The authors forgot to results on the Reproducibility of δEXO) for each participant (Supplementary Table 2) was calculated from changes in δ13CO2 of breath samples taken during the 3.5 h cycling exercise . Reproducibility of δ13CO2 measurements was at an average 0.06‰ (range 0.03–0.09‰).”“Exogenous carbohydrate oxidation (CHOThe authors apologize for this error and state that this does not change the scientific conclusions of the article in any way. The original article has been updated.


To determine the regional- and district-level newborn prevalence of sickle cell trait and disease, and the prevalence of haemoglobin variants and genetic modifiers of sickle cell disease, in the nine regions of north-western United Republic of Tanzania.We repurposed dried blood spot samples from children (aged 0–24 months) born to mothers living with human immunodeficiency virus (HIV), collected as part of the HIV Early Infant Diagnosis programme, for sickle cell diagnosis. We performed isoelectric focusing to determine whether samples had normal haemoglobin, sickle cell trait, sickle cell disease or a rare haemoglobin variant. We shipped samples diagnosed as disease or variant to Cincinnati Children’s Hospital in the United States of America for deoxyribonucleic-acid-based analyses to determine the prevalence of α-thalassaemia, glucose-6-phosphate dehydrogenase (G6PD) deficiency or fetal haemoglobin genetic modifiers.– deficiency in 19.2% (14/73) of males.We analysed a total of 17 200 specimens during February 2017–May 2018. We observed a prevalence of sickle cell trait and disease of 20.3% (3492/17 200) and 1.2% (210/17 200), respectively. District-level trait varied from 8.6% (5/58) to 28.1% (77/274). Among confirmed sickle cell disease specimens, we noted 42.7% (61/143) had 1-gene deletion and 14.7% (21/143) had 2-gene deletion α-thalassaemia trait. We documented G6PD AOur calculated prevalence is twice as high as previously reported and reinforces the need for enhanced sickle cell diagnostic services. Our district-level data will inform public health policy, allowing screening and disease-modifying hydroxyurea therapy to be focused on high-prevalence areas, until universal newborn screening is available. Sickle cell disease is an inherited disorder of haemoglobin, caused by a mutation in the β-globin subunit of adult haemoglobin. In classic autosomal recessive fashion, inheritance of one abnormal and one normal allele confers sickle cell trait, a carrier state without clinical symptoms. Inheritance of two mutated alleles causes sickle cell disease, characterized by varying amounts of chronic haemolytic anaemia, recurrent debilitating pain and an array of clinical sequelae, including increased risk of infection, stroke, lung disease, splenic dysfunction and bone infarction.–,Sickle cell disease imposes a significant global burden of disease that remains underrecognized,,,,,The World Health Organization acknowledged the global importance of addressing sickle cell disease almost 15 years ago,–,The United Republic of Tanzania does not yet have a national newborn screening programme. In the absence of recent reports, Tanzanian estimates of sickle cell disease are based on sparse data from studies performed over the past 65 years in only seven of the 30 regions of the country.In 2006, the International Center for AIDS Care and Treatment Programs, the Tanzanian Ministry of Health and Social Welfare, Bugando Medical Centre and the United States Centers for Disease Control and Prevention implemented the HIV Early Infant Diagnosis programme with the aim of preventing mother-to-child transmission of HIV.An infant born to a mother living with HIV is brought to a Reproductive and Child Health clinic at 4–6 weeks of age for the first preventive health visit. A dried blood spot is collected from the infant, and transported to a reference laboratory for detection of HIV using a polymerase chain reaction (PCR). The dried blood spots are labelled with key demographic information including date of birth, sex, referring health facility, date of collection, date of dispatch and date of receipt at the laboratory. HIV results are communicated to the referring health facility for prompt initiation of antiretroviral medication. Dried blood spots are then stored at room temperature and made available for additional testing for sickle cell trait and disease. We analysed all repurposed dried blood spots collected as part of the HIV Early Infant Diagnosis programme during February 2017–May 2018 by using the isoelectric focusing technique.All dried blood spots were analysed with a standard control specimen containing adult, fetal and sickling haemoglobin  and haemoglobin C. Isoelectric focusing results were scored independently by two Bugando Medical Centre staff for the presence and abundance of each type of haemoglobin. The results were interpreted as: normal if haemoglobin A (± haemoglobin F) was present; sickle cell disease if haemoglobin S (± haemoglobin F) was present; sickle cell trait if both haemoglobin A and S (± haemoglobin F) were present; variant if a band was present at any location other than haemoglobin A, S or C (± haemoglobin F); and uninterpretable if poor quality precluded interpretation. Dried blood spot analyses interpreted as sickle cell disease, variant or uninterpretable were repeated for confirmation and frozen for later deoxyribonucleic acid (DNA) studies. Regular teleconferences were convened with collaborators based in the Cincinnati Children’s Hospital, USA, to provide ongoing feedback on the quality of laboratory techniques and interpretation of gels; however, the Tanzanian team was responsible for all final interpretations. HBA1:p.Asp75Asn) and haemoglobin Stanleyville II (HBA1:p.Asn79Lys), and the fusion variants haemoglobin P-Nilotic (β-globin gene (HBB)–δ-globin gene (HBD) fusion: β31-δ50) and haemoglobin Kenya (γ-globin gene (HBG1)–HBB fusion: Aγ81-β86).Any dried blood spot diagnosed as sickle cell disease or variant was stored at −20 °C then shipped to the USA for future DNA testing. Upon arrival, we stored dried blood spots at −80 °C until testing could be performed. We extracted DNA from dried blood spots using an adapted protocol from Instagene .– variant using DNA-based techniques,–BCL11A polymorphisms  and HBS1L-MYB intergenic polymorphisms (HMIP) (rs28384513 and rs9399137) using commercially available real-time PCR assays . To identify the XmnI single-nucleotide polymorphism at −158 basepairs to G-γ globin (rs7482144), we amplified the G-γ gene (HBG2) using PCR with G-γ-specific forward and reverse primers to ensure that the G-γ rather than an A-γ product was isolated. We then further amplified this product by performing PCR with Classic 1 forward and reverse primers. We genotyped the final product using a custom-made TaqMan PCR probe set .We detected α-thalassaemia trait resulting from the 3.7-kilobase α-globin gene deletion and glucose-6-phosphate dehydrogenase (G6PD) A2χ test.We entered data into an Excel database , treating age as a continuous variable (summarized using median and interquartile range) and treating haemoglobin type, HIV status, sex, region of origin, district of origin, G6PD status and α-thalassaemia status as categorical variables (summarized using frequencies). We calculated the prevalence of sickle cell trait and disease by dividing the number of specimens with sickle cell trait and disease by the total number of non-missing specimens with interpretable results. We determined allelic frequency by dividing the number of times that an allele was observed  by the total number of all alleles . We compared the proportions of participants living with HIV with and without sickle cell disease using a Our study protocol was approved with a waiver for informed consent by the joint Catholic University of Health and Allied Sciences–Bugando Medical Centre Research and Ethics Committee, as well as the Tanzanian National Institute for Medical Research, to perform disease surveillance on de-identified archived dried blood spots previously collected by the HIV Early Infant Diagnosis programme. The study was also approved by the Cincinnati Children’s Hospital Medical Center Institutional Review Board. A formal material transfer agreement was obtained so that specimens could be shipped to the USA for genetic analysis.Our local Bugando Medical Centre staff completed a total of 232 isoelectric focusing gels during February 2017–May 2018. After samples from children older than 24 months were excluded to obtain a more accurate prevalence for infants, the median age of infants included in the sampling population was 52 days . Staff scored a total of 17 274 unique dried blood spot specimens from the catchment area. The quality of laboratory testing was extremely high with only 20 specimens scored as uninterpretable and 54 with missing results, meaning that we performed our primary analysis on 17 200 specimens.We observed an overall prevalence of sickle cell trait and disease in our cohort of 20.3% (3492/17 200) and 1.2% (210/17 200), respectively, with a 0.1% (17/17 200) prevalence of atypical or uncommon haemoglobin variants. Our data yielded an allelic frequency of 0.114 ([3492+(2 × 210)]/(2 × 17 200)) for the sickle gene (haemoglobin S) mutation, and demonstrate perfect Hardy–Weinberg equilibrium, as expected in a stable population.Using our regional prevalence estimates of sickle cell trait and disease, and regional census data from the 2012 Population and Housing Census for the United Republic of Tanzania,For the 16 479 samples for which HIV results were available, we analysed the co-morbidity of HIV and sickle cell disease to compare the potential effect of HIV status on mortality, as previously performed in Uganda.HBA1:p.Asp75Asn), two haemoglobin Kenya (HBG1–HBB fusion Aγ81-β86) and a haemoglobin P-Nilotic (HBB–HBD fusion β31-δ50). We identified 1-gene deletion α-thalassaemia trait in 42.7% (61/143) and 2-gene deletion α-thalassaemia trait in 14.7% (21/143). We detected G6PD A– deficiency in 19.2% (14/73) of males, and 25.7% (18/70) of females were heterozygous carriers  and included four haemoglobin G-Pest  to 0.325 (rs4671393). Our frequencies were slightly higher than those observed within the African subgroup of the 1000 Genomes Project,HBG2 was not detected, as expected in an East African cohort.We provide the minor allelic frequencies for genetic modifiers that affect haemoglobin F production for the 143 samples available for genetic testing in We estimate that the annual number of live births with sickle cell disease is at least twice that previously thought to occur in the United Republic of Tanzania; we project that over 10 000 births among just 40% of the Tanzanian population are affected annually, compared with previous estimates of 8655–Our data reinforce the urgent need to enhance sickle cell diagnostic services, the obligatory first step in the cascade of care for this neglected patient population. The sickle cell clinic at Bugando Medical Centre has approximately 600 patients currently enrolled in care, some of whom travel from neighbouring regions. However, we estimate that 1730 infants are born with sickle cell disease in Mwanza region alone each year, which indicates that many affected children in sub-Saharan Africa have not even been diagnosed with the condition. The nine regions included in this study have the highest childhood mortality in the country, ranging from 30 to 38 deaths per 1000 live births,We also investigated the extent to which additional erythrocyte disorders are commonly co-inherited with sickle cell disease; cross-sectional analyses in the United Republic of Tanzania have reported on the impact of such disorders on sickle cell patients.,An important strength of our study is that we developed a sickle cell disease diagnostic service using existing infrastructure used for HIV infant diagnosis, demonstrating a potential of using this approach across sub-Saharan Africa. For example, in Uganda decision-makers have successfully expanded the use of their HIV infant diagnosis screening platform for sickle cell disease diagnosis, and have launched focused screening in 18 high-prevalence areas.–The testing of infants was another strength of our study. By excluding samples from children older than 24 months from our analyses, we avoided the errors of previous reportsOur study had two limitations. First, we analysed dried blood spots only after they had been processed by the HIV Early Infant Diagnosis laboratory. In some cases, several months had elapsed before the dried blood spots became available for sickle cell disease testing. During that time, dried blood spots were stored at suboptimal conditions for haemoglobin preservation ; however, our isoelectric focusing testing was robust and able to score 99.6% (17 200/17 274) of all dried blood spot specimens. Second, our study population was restricted to children born to mothers living with HIV; however, it has been reported that HIV status is unlikely to affect the genetic inheritance of the sickle allele.Since universal newborn screening is not immediately feasible in most countries, we recommend initially focusing screening efforts on high-prevalence districts to invest in those communities most affected by sickle cell disease, using existing public health infrastructure with minimal start-up cost and training. The cost–effectiveness of implementing a screening programme in conjunction with treatment for children who are subsequently diagnosed with sickle cell disease varies between regions and countries, especially regarding salary for personnel, but a detailed analysis in Angola provides some helpful comparisons.Inaccurate national and subnational estimates obscure the true burden of sickle cell disease, which is a common and important cause of death in young children in low-income countries. It is imperative that health ministries and international groups such as the Global Burden of Disease project


In OSA patients, TGF-β1 levels correlated with mitotic index, Breslow index and melanoma growth rate, and were increased in presence of ulceration or higher Clark levels. In CM patients, OSA was associated with higher TGF-β1 levels and greater melanoma aggressiveness only in non-obese subjects. An in vitro model showed that IH-induced increases of TGF-β1 expression in melanoma cells is attenuated in the presence of high leptin levels. In conclusion, TGF-β1 levels are associated with melanoma aggressiveness in CM patients and increased in moderate-severe OSA. Moreover, in non-obese patients with OSA, TGF-β1 levels correlate with OSA severity and leptin levels, whereas only associate with leptin levels in obese OSA patients.Active transforming growth factor-β1 (TGF-β1), a cytokine partially regulated by hypoxia and obesity, has been related with poor prognosis in several tumors. We determine whether obstructive sleep apnea (OSA) increases serum levels of active TGF-β1 in patients with cutaneous melanoma (CM), assess their relationship with melanoma aggressiveness and analyze the factors related to TGF-β1 levels in obese and non-obese OSA patients. In a multicenter observational study, 290 patients with CM were underwent sleep studies. TGF-β1 was increased in moderate-severe OSA patients  OSA effects on tumor development and progression have been mainly attributed to the impact of intermittent hypoxia on the local tumor microenvironment, promoting melanoma cell proliferation, the release of pro-angiogenic factors, and alterations of the immune surveillance system5. However, the impact of OSA on the intrinsic properties of tumor cells is less well understood.The putative association between obstructive sleep apnea (OSA) and cutaneous melanoma (CM) aggressiveness7. TGF-β is a multi-factorial peptide growth factor that is synthesized by macrophages, lymphocytes, fibroblasts, platelets, epithelial and cancer cells, and plays a key role in the regulation of several physiological processes, including cell proliferation, differentiation, migration, adhesion, and tissue repair8. TGF-β synthesized forms a dimer of with a latency-associated peptide, remaining inactive and anchored in the extracellular matrix by the TGF-ß binding proteins. TGF-ß activation requires its release form the extracellular matrix by proteolytic cleavage9. To adequately evaluate serum TGF-ß, it is important to consider that most of it corresponds to the latent complex form, while only a small fraction is biologically active10.There is increasing evidence that the aggressiveness of CM is largely due to the intrinsic plasticity of melanoma cells, which is regulated by a variety of mechanisms that prominently include the transforming growth factor-β (TGF-β) pathway7. Among the several TGF-β isoforms, TGF-β1 has been shown to be expressed by melanoma cells and to promote invasiveness in cell cultures11, whereas attenuation of TGF-β signaling by treatment with a small molecule inhibitor reduced bone metastasis formation12. Although a previous study did not identify a relationship between serum TGF-ß1 concentration and melanoma stage13, probably because circulating bioactive TGF-β1 levels were not specifically measured, it has been demonstrated that overall canonical TGF-β/SMAD signaling is a potent promoter of melanoma progression, even at early stages of the disease14, and that soluble active TGF-β1 is directly related to the risk of melanoma metastasis16.In melanoma cells, extracellular active TGF-β regulates gene expression by receptor-mediated activation of SMAD  transcription factors that regulate the transcriptional output of active genes and can also open repressive chromatin19. Along the same line, it has been described that patients with severe OSA have higher circulating levels of active TGF-β1 than control subjects, which decreases after six months of CPAP treatment20. Interestingly, it has also been reported that leptin synthesized and secreted by adipose tissues, after binding to its specific receptor on tumor cells, activates an intracellular signaling pathway that recruits transcription factors to the promoter of the TGF-β1 gene, increasing synthesis and secretion of TGF-β1 by tumor cells21.The involvement of the TGF-β/SMAD pathway in melanoma progression could be potentially important in OSA patients, since previous studies have shown that increased hypoxia-induced factor (HIF) secondary to hypoxia induces the activation of the TGF-β1/SMAD signaling pathway23, and such observations lead to the question whether OSA may have any effect on the activation of the TGF-β1/SMAD pathway in patients with melanoma, and if so, whether there is any synergistic effect with obesity. Therefore, our study objectives were to compare serum levels of active TGF-β1 in patients with CM according to the presence and severity of OSA, assess the correlation between active TGF-β1 levels and melanoma aggressiveness indices, and analyze their relationship with nocturnal hypoxia and serum levels of leptin in both obese and non-obese patients. Moreover, we explored the leptin effect on TGF-β1 released by melanoma cells under intermittent hypoxia in an in vitro model of OSA.Circulating TGF-β1 levels have been shown to exhibit dependency on both intermittent hypoxia and obesity (mediated through serum leptin levels)−2. Local melanoma extension was detected in 250 patients (87%), and evidence of ulceration was found in 49 (17%). Median (IQR) Breslow and mitotic indices were 0.84 (0.50–1.93) mm and 1 (0–2) cells mm−2, respectively; while the growth rate was 0.15 (0.05–0.45) mm month−1. The overall prevalence rates of mild and moderate-to-severe OSA were 31.7 and 33.8%, respectively. The comparison between groups illustrates that, as the severity of OSA increases, patients were increasingly more likely to be men, older and more obese.The general characteristics of the study participants are shown in Table Active TGF-β1 serum levels showed significant differences among the three study sub-groups (p < 0.001). When adjusted for sex, age, BMI and neck circumference, TGF-β1 serum levels were higher in moderate-severe OSA patients versus mild OSA or non-apneic patients . TGF-β1 levels were also higher in OSA patients with ulceration of melanoma than in those without ulceration . Finally, differences in TGF-β1 levels according to Clark levels were also detected (p < 0.001) , Breslow index  and melanoma growth rate  Fig. . OSA pat.07 4.03–1.67 vs. No significant differences in leptin levels were found among the melanoma patients without OSA and those with mild or moderate-severe OSA Fig. B. In the−1, p = 0.001), whereas active TGF-β1 levels did not differ between subgroups of OSA patients  showed similar results.Table ls Table , Fig. 3.When all obese patients with CM were analyzed together, the presence of OSA was not associated with higher levels of TGF-β1 or with greater tumor aggressiveness. In contrast, non-obese patients with OSA exhibited higher levels of TGF-β1 and melanoma aggressiveness indices Table . The corIH exposures in vitro increased bioactive TGF-β1 levels in the culture supernatant of melanoma cells subjected to the lowest leptin concentration, while such effect disappeared with the higher leptin concentration Fig. . TGF-β1 In patients with cutaneous melanoma, moderate-severe OSA is associated with increased circulating levels of active TGF-β1 which correlate with tumor aggressiveness. In addition, while TGF-β1 levels are independently related to serum leptin and AHI in non-obese subjects, they associate with leptin levels only in obese subjects, suggesting that the potential contribution of OSA to increase active TGF-β1 may be limited to non-obese patients. Moreover, the in vitro melanoma cell culture model showed that the IH-induced increase of active TGF-β1 expression is attenuated by the presence of higher leptin levels.24. The roles of TGF-β1 have been more extensively investigated relative to other isoforms of TGF-β. When bound to a specific receptor on the target cell membrane, TGF-β1 activates TGF-β receptor type I kinase, resulting in the phosphorylation of SMAD2 and SMAD3, which subsequently form oligomeric complexes with SMAD4, and translocate to the nucleus to regulate the expression of a large repertoire of genes25. In light of the biological roles of TGF-β1 in cancer, the strong relationships between serum levels of active TGF-β1 and all indices of melanoma aggressiveness in OSA patients are therefore not unexpected. The increased TGF-β1 levels identified in patients with moderate-severe OSA indicate that this disorder has an incremental effect on the expression and release of active TGF-β1 even in patients who have already developed a melanoma. Currently available evidence suggests that this effect is likely due to the OSA-related intermittent hypoxia. In fact, TGF-β1 has been shown to be increased in a variety of cells cultured in hypoxia and also in hypoxic ischemic tissues26. Several reports show that hypoxia activates latent TGF-β1 via HIF-1α in hepatocytes, placental fibroblasts, primary human lung fibroblasts, smooth muscle cells and cancer cells26. Using in vitro models, it has been recently confirmed that HIF-1α induction by hypoxia promotes the expression of TGF-β1, activates its receptors, and increases the phosphorylation of the SMAD2/3 proteins, indicating an activation of the TGF-β/SMAD signaling pathway, probably through the TLR/MyD88/NF-κB pathway23.The TGF-β pathway plays a major role in the evolution of cancer, and has been implicated with tumor progression by driving epithelial mesenchymal transition, tumor cell migration, invasion, metastatic spread and angiogenesis27.Although our results do not establish causal inferential mechanisms, the correlation found between the different nocturnal hypoxemia indices and the levels of active TGF-β1 among patients with OSA and melanoma concurs with the potential relevance of intermittent hypoxia in the enhanced induction of TGF-β1. The selection of AHI instead of the nocturnal hypoxemia indices by the multiple regression model could be due solely to the limited number of patients with more severe OSA (cutaneous melanoma was the driving selection criterion rather than OSA). However, it is also not possible to exclude that other OSA-related perturbations contribute to TGF-β1 production. Indeed, in a murine model exposed to intense sleep fragmentation, increases in TGF-β1 expression in the hypothalamus and hippocampus were reported28. High serum levels of leptin, which are strongly correlated with adipose tissue mass, are recognized as a factor driving cancer development and progression, as leptin exhibits mitogenic, proinflammatory, anti-apoptotic, and proangiogenic properties31. The finding of a significant relationship between leptin and TGF-β1 levels in our patients is consistent with previous studies that showed that leptin binds to leptin receptors on the surface of cancer cells, activates JAK/STAT3 signaling, and consequently increases TGF-β expression21. Moreover, it has been demonstrated that leptin can be a co-factor for TGF-β in cancer using an in vitro model of kidney fibroblasts whereby leptin treatment enhances SMAD2/3 phosphorylation by TGF-β33. Interestingly, the relationship between leptin and TGFβ could be bidirectional, since TGFβ/SMAD3 signaling appears to be crucial for the development of obesity, as demonstrated in mice in which TGFβ signaling was blocked or that were deficient in SMAD3 and consequently were protected from developing obesity35. Additionally, other members of the TGF-β family are involved in regulation of adipogenesis and energy metabolism through SMAD and P38 signaling pathways36.Leptin is primarily produced by fat cells and regulates food intake, energy homeostasis and cell proliferation37. It has been estimated that roughly 20% of all cancers are caused by excess weight gain39 and, although the mechanisms are unclear, some studies have suggested that obesity enhances metastases in tumors such as melanoma, colon, lung and breast cancer41. In fact, there is greater risk of recurrence among cancer patients with higher BMI42, and cancer patients with high BMI have higher mortality rates compared to those with average BMI38. Although in our subgroup of patients with OSA and melanoma the differences in Breslow index between obese and non-obese subjects did not reach statistical significance . Although no changes in patient weight or any other potential confounding factors were identified, we cannot rule out that such factors were void of any influence on our results. Third, as in most studies recruiting patients who had not been referred to sleep units for suspicion of OSA, daytime sleepiness in the OSA groups is low, which may be a limitation to extrapolate our findings to much more symptomatic patients. Fourth, we did not have a group of patients with distant metastatic melanoma to evaluate their serum TGF-β1 and leptin levels. Fifth, our study does not provide any information on the effect of OSA treatment on TGF-β1 levels or its effect on melanoma aggressiveness, and therefore no therapeutic recommendations beyond those in place for OSA can be formulated.In addition to its multicenter character and the relatively large cohort size, as well as the homogenization and rigorous control of sleep studies performed by the Spanish Sleep Network, we believe that the main strength of this study was the measurement of free active TGF-β1 in serum, which more reliably reflects effector activity of its several biological functions, such as activating epithelial mesenchymal transition, proliferation and the spread of metastasisIn summary, our study shows that melanoma patients with moderate-severe OSA have higher serum levels of active TGF-β1, which are directly related to melanoma aggressiveness. Furthermore, in non-obese patients with OSA TGF-β1 level variance is dependent on both AHI and serum leptin levels, while in obese patients with OSA only leptin levels retain their independent association with TGF-β1 levels.1. Exclusion criteria included extra-cutaneous location of the melanoma, daytime respiratory or heart failure, and current or previous use of home oxygen therapy, continuous positive airway pressure (CPAP) or noninvasive mechanical ventilation. Out of 443 recruited patients, serum samples were obtained in 360 subjects and after their clinical use, were available in 290 patients. The study was approved by the Institutional Ethics Committee of the Hospital Universitario y Politécnico La Fe, Valencia, Spain (2016/0223), and all subjects gave their written informed consent. All the experiment protocol for involving humans was in accordance to Declaration of Helsinki.Adult patients with new diagnoses of cutaneous melanoma were consecutively selected from a multicenter, observational study involving 29 teaching hospitals in Spain46, patients were evaluated by a dermatologist at each hospital, and data for tumor location and clinical stage at diagnosis  were registered. All tumors were surgically removed, and the Breslow tumor thickness, ulceration, tumor mitotic rate, and Clark levels were calculated47. Melanoma growth rate was calculated as the Breslow index divided by the difference between the date of the tumor excision and the date on which patients noticed changes suggestive of malignant transformation in a stable, pre-existing lesion or the time when they noticed the appearance of a new and changing lesion46. All dermatological and pathological evaluations were blinded to the sleep study and biochemical analysis findings.As previously described−2. Simultaneously, all patients underwent overnight respiratory polygraphy within a maximum of 4 months after CM diagnosis. All scoring and readings were conducted manually by experienced and trained personnel. Apnea was defined as an interruption of oronasal flow of > 10 s. Hypopnea was defined as a 30–90% reduction in the oronasal airflow for > 10 s associated with an oxygen desaturation ≥ 3%. The apnea–hypopnea index (AHI) was defined as the number of apneas plus hypopneas per hour of recording, while tSat90 was defined as the percentage of recording time with SaO2 < 90%. In addition, mean saturation, minimum saturation and oxygen desaturation index (ODI) were measured. According to the AHI, patients were divided into three groups: non-OSA (AHI 0–5 h−1), mild OSA (AHI 5–15 h−1) and moderate-severe OSA (AHI > 15 h−1)48.Anthropometric characteristics were measured and, according to WHO criteria, obesity was defined as a body mass index (BMI) ≥ 30 kg m−1 and 2.3 pg mL−1, respectively. The intra-assay and inter-assay variations were below 10% in all cases.Fasting venous blood samples were drawn between 8 and 9 am. The blood samples were centrifuged to separate the serum, and all specimens were immediately aliquoted, frozen and stored at − 80 °C. Free active TGF-β1 and leptin in serum were assayed using human free active TGF-β1 and leptin enzyme linked immunosorbent assay  according to the manufacturers’ instructions. The assays were done in duplicates in all samples. The detection limits of the assays were 63 pg mL49 was cultured under normoxia or intermittent hypoxia (IH) conditions over night, as previously described50. The culture medium was supplemented with human leptin  at either a concentration similar to that detected in patients or two-fold that level . Supernatant concentrations of active TGF-β1 were measured by ELISA as in serum.C81-61 human cutaneous melanoma cell line20. The products were amplified using primers for TGF-β1 5′-GGCCAGATCCTGTCCAAGC-3′ (forward) and 5′- GTGGGTTTCCACCATTAGCAC-3′ (reverse).Total RNA was purified using the High Pure RNA Isolation Kit . cDNA was obtained using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems). Gene expression levels were analyzed by real-time quantitative PCR using the LightCycler System ; and quantitative PCR was performed using a QuantiMix Easy SYG Kit . The results were normalized to the expression of 18S, and the cDNA copy number of each gene of interest was determined using a 7-point standard curve. The products were amplified using specific primers as described previously51. Analyses were performed using the Statistical Package for the Social Sciences .According the type and distribution of the variables, data are expressed as mean ± SD, median (interquartile range [IQR]) or absolute numbers and percentages. Normality was explored using the Shapiro–Wilk and skewness-kurtosis tests. Differences between groups were analyzed using the Chi-squared or Fisher’s exact tests  and Kruskal–Wallis or Mann–Whitney U tests . For between-group comparisons, TGF-β1 and leptin levels were adjusted for gender, age, body mass index and neck circumference using general linear models. Correlations between parameters were evaluated using Spearman’s correlation. Those variables exhibiting statistically significant findings were then introduced into a multiple linear regression analysis to identify independent determinants of TGF-β1 levels. Stepwise methods were used to include or remove individual independent variables at each step, based on the probability of F . Other aspects explored included residual standard deviation, changes in the distribution of the residuals and the homogeneity of the variance over the predictorsSupplementary file1