The metal-semiconductor-metal transition in dense lithium is considered as an archetype of interplay between interstitial electron localization and delocalization induced by compression, which leads to exotic electride phases. In this work, the dynamic dielectric response and optical properties of the high-pressure electride phases of cI16, oC40 and oC24 in lithium spanning a wide pressure range from 40 to 200 GPa by first-principles calculations are reported. Both interband and intraband contribution to the dielectric function are deliberately treated with the linear response theory. One intraband and two interband plasmons in cI16 at 70 GPa induced by a structural distortion at 2.1, 4.1, and 7.7 eV are discovered, which make the reflectivity of this weak metallic phase abnormally lower than the insulating phase oC40 at the corresponding frequencies. More strikingly, oC24 as a reentrant metallic phase with higher conductivity becomes more transparent than oC40 in infrared and visible light range due to its unique electronic structure around Fermi surface. An intriguing reflectivity anisotropy in both oC40 and oC24 is predicted, with the former being strong enough for experimental detection within the spectrum up to 10 eV. The important role of interstitial localized electrons is highlighted, revealing diversity and rich physics in electrides. In fact, the bcc structure of lithium at ambient conditions will convert to an fcc structure at 7 GPa under compression, which then transforms to a low symmetry cubic polymorph phase cI16 through an intermediate rhombohedral structure hR1 at about 39 GPa, as discovered by synchrotron X-ray powder diffraction experiments5.Pressure alters the state of matter by squeezing electrons into smaller space and modifying the interaction between particles. It is not surprising that metals are usually anticipated to become more “metallic” at high pressures due to expected broadening of the bandwidth and ensued overlapping of electronic orbitals. However, the high-pressure behavior of matter sometimes is counterintuitive and convoluted. Lithium, as the simplest metallic element, is an archetype of how pressure turns a metal into insulator and induces intricate and fascinating phenomena. Since 1960 s, the unusual increase in the electric resistivity of lithium at a hydrostatic pressure of 30 GPa as observed in the Bridgman anvilp orbitals and making s-p hybridization, which then leads to an extended nesting structure, as well as the consequent phonon softening7. This gives rise to an anomalous melting in lithium compared to other ordinary metals, in which the melting temperature increases to a maximum and then decreases continuously under further compression starting from 520 K at ~10 GPa in the fcc phase9. A minimum of the melting curve is reached at 50 GPa in the semi-metallic cI16 phase, with a cold melting point as low as 190 K8, the lowest one among all known materials at this pressure range. The role of quantum ion dynamics in lithium is evident, considering which the accurate theoretical value of melting temperature was obtained as well10.With pressure increased, the Fermi surface of lithium is substantially distorted by excitation of electrons to 15. The physics behind these peculiar phenomena is that the Coulomb attraction of nucleus is reduced greatly for valence electrons at high pressure, as they suffer a strong repulsion from core electrons due to the overlap with the inner orbitals, as well as the required orbital orthogonality by quantum mechanics. Some valence electrons thus move from the neighborhood of ions to the interstitial positions of crystalline structure, and behave as anions, thus forming high-pressure electrides (HPE)19. These localized valence electrons are further enhanced by multi-centered chemical bondings that hybridize the valence and conduction bands, and then split them into bonding and anti-bonding states20. This exotic behavior was proposed for energy gap opening in dense lithium23, and was confirmed by a recent measurement in which a direct visual observation of the reflectance change and a distinct jump in the electric resistivity clearly reveals the existence of an insulating phase of oC4024. Under further compression, the multi-centered bonding/anti-bonding states overlap again and lithium returns into a semi-metallic electride phase of oC24 above 110 GPa27. Similar anomalous phenomena also present in other light alkali metals and dense hydrogen30. Especially the s to p electronic excitation in hydrogen has been predicted to result in a deep melting minimum32 and a mobile solid phase33. The interstitial electrons are also shown to be crucial to mix the unmixable lithium and sodium to form an interesting insulating electride alloy34.Along with this anomaly, the electron-phonon coupling in lithium becomes strong when above 25 GPa even in the fcc phase. This results in one of the highest superconducting temperature (~20 K) of elements34. They could provide important information on exotic collective and excitation behavior of valence electrons under an external electric field or interacting with light, which otherwise are not obvious from the bare band structure. These include the zone boundary collective state (as in the bcc phase) and the undamped interband low-energy plasmon that leads to a suppression of the reflectivity (as in the fcc phase) for lithium38. Such kind of pressure-induced interband plasmon, is not unique to lithium40, and cannot be described within the free-electron model41.The prominent variation of the atomic and electronic structure in dense lithium induced by pressure should also reflect in physical properties, especially in the vicinity of the boundaries of the metal-semiconductor-metal transition. Dynamic response and optical properties are indispensable for the purpose to acquire a thorough understanding of the high-pressure anomalies in dense lithium22. The structural details about these high-pressure phases will be covered in next section. We will then explore the optical properties of lithium from 40 to 200 GPa thoroughly, including the reflectivity, the electron energy-loss spectroscopy (EELS) and the optical conductivity. Both the intraband and interband contributions are carefully treated with the linear response theory. The property in the low energy regime of the spectra, especially within the visible light range, is highlighted for its importance and tight connection with experiments. Ground-state electronic structure and analyses are reported as well, which are prerequisite to interpret the optical spectra. Our investigated pressure range overlaps the transition point of the 2 s2 p excitation. Therefore, the influence of this excitation on the s-p hybridization, as well as the impact on the optical properties, are also discussed.So far, most theoretical calculation and analysis on dense lithium mainly focused on the static crystalline structure and ground-state band structure. The optical properties beyond fcc phase have not been systematically analyzed yet. Considering the drastic change in the crystalline and electronic structure through the metal-semiconductor-metal transition, it is imperative to explore how the optical properties of these HPE phases will evolve under compression. Especially, whether the localized interstitial electrons contribute significantly to the dielectric response? And how it will change at higher pressure beyond 100 GPa? In this work, we comprehensively investigate the dynamic dielectric response and optical properties of lithium at high pressure by first-principles calculations, mainly concentrating on the three intriguing HPE phases of cI16, oC40 and oC24. The intermediate phase oC88 is not considered here for its narrow pressure range of stability16c Wyckoff positions, so that one internal coordinates 5. cI16 is a HPE phase and the interstitial electrons locate at the vertices of an equilateral triangle perpendicular to the other diagonal of the bcc structure as shown in supplementary Fig. cI16 belonging to the space group et al.42. These peaks are mainly associated with the transitions near the 42  is not significant in physics, because the intraband contribution dominates in this frequency range.Five distinct peaks can be found in the imaginary part of interband dieletric function (DF) for cI16 at 40 GPa, as shown in Fig. The cI16 phase at 40 GPa is also taken as an example to demonstrate the contribution of intraband transitions and the crystalline local field effect (LFE). As Fig. et al.35, where the reflectivity has a value above 0.9 up to 5.8 eV and is then strongly suppressed by the interband plasmon at 6.0 eV. The reflectivity of cI16 by our calculations in the infrared region is about 0.3 smaller than fcc, namely, reduced by 40%, due to the LFE-induced small value in 43. The subtle details of the computed dynamic spectrum at 40 GPa, however, is a little bit different from the experiment at 36 GPa, as detailed in supplementary Fig. The reflectivity and the EELS of cI16 and fcc phase at 40 GPa were calculated and shown in Figs. 44The electronic structure of cI16 changes a lot under further compression, especially at In the visible light range 1.6–3.2 eV), the reflectivity of cI16 is strongly modified by pressure so that it is almost 0.3 smaller at 70 GPa than that at 40 GPa (or 43% reduction). This low-energy suppression originates in the shift of the intraband plasmon from 1.2 eV to 2.1 eV by compression see Fig. . At 40 GGPa Fig.  to 4.8 e.2 eV, thx, rather than the hydrostatic compression, according to two facts: (1) this x value in cI16 phase was analyzed and found significant to optical spectra by Alonso et al., but only in the range of 0.01–0.0542; (2) in our calculation, the value of x actually increases further under compression . To validate this assumption, we separate the structure change from 40 GPa to 70 GPa into two steps. In the first one, the lattice vectors of the structure are released to 70 GPa but with the internal coordinate x being fixed at the value of 40 GPa, i.e., x = 0.048. In the second step, the internal coordinate is also fully optimized. The reflectivity of this intermediate phase shows an overall agreement with that of 40 GPa, with just a slight shift of the spectrum towards higher frequency and reduction in the magnitude, as shown in Fig. p-contribution to the electronic states around ions, accompanying which, the x. A larger value of x indicates stronger deviation from the initially high symmetric phase. This process is more drastic in cI16 than in the other two electride phases, due to its evident localization change under compression. For example, the volume of interstitial space with electron localization function (ELF) greater than 0.9 increases five times from 40 to 70 GPa in cI16, compared to a mild increase of 18% in oC40 when compressed from 70 to 100 GPa (see Supplementary Information).We hypothesized the anomalous variation in the optical properties induced by compression to 70 GPa, especially the splitting of the plasmon at 6.0 eV and the emergence of the weak intraband plasmon at ~2.1 eV, might arise from the local distortion characterized by the internal coordinate 8. The same transition sequence was also predicted by ab initio calculations after including the zero-point energy22. The orthorhombic phase oC40 belongs to the space group of Aba2. It is the only phase that has an energy gap in all intrinsic phases of dense lithium, as shown in Fig. The phase transition sequence in lithium at ~60  GPa is From first-principles band structure calculations, we find an indirect band gap in oC40 about 1.03 eV  at 85 GPa. As a consequence, both the imaginary part of DF and the absorption spectrum show a feature starting from 1 eV. As shown in Fig. b axis, with each part including three different layers. Between these two parts is the largest interstitial space in oC40, where localized valence electrons locate, noted as M1 and M3. The other interstitial localization position is in the middle layer of each part, noted as M2  and the calculated band gap increases at a rate of ~0.013 eV/GPa when pressure rises from 70 to 100 GPa. The features in optical spectra correspondingly move to higher frequency and the reflectivity is slightly reduced under continuing compression. Unlike the local structural distortion observed in cI16, all pressure-induced variations in oC40 seem mild.Under compression, the energy of the ab initio method18. The metallic feature of this phase was experimentally confirmed by a rapid decline in electric resistivity at ~110 GPa25. By comparison to the semi-metal phase cI16 at low pressure, this high-pressure phase has an almost vanished electronic DOS at the Fermi level (EF). As shown in Fig. Further compression leads to band overlap again, and lithium transforms into a reentrant metallic electride phase oC24. The atomic structure of this phase, belonging to the space group see Fig. . The firIn view of the low frequency part of the calculated see Fig. , oC24 is24. The calculated reflectivity also strongly depends on the frequency of incident wave, and this dependence greatly differs from each other in these three electride phases, mainly due to their different plasmon frequencies. As demonstrated by EELS in Fig. On the whole, the reflectivity of lithium in the visible light range decreases as a function of pressure. It changes slowly within one stable phase and rapidly, even a jump at the transition pressure. Some specific values of reflectivity at given frequencies are provided in Table 25. For oC24, the calculated static conductivity is lower than cI16, but much higher than oC40  the regular effect of hydrostatic pressure, as shown in oC40 and oC24, as well as the intermediate step of cI16 discussed in Results section; (ii) the irregular effect that causes extra structural modification, such as the distortion in cI16 at 70 GPa. However, this irregular effect of compression of (ii) only emerges in some particular phases, according to above analysis. We infer that its working condition includes both the low symmetry crystalline structure and the metallic electronic structure with energy bands crossing the Fermi level. The first one is easy to understand since less constraint by spatial symmetry makes stronger modification on crystalline structure possible. Therefore, this effect does not show up in the bcc and fcc phase. The second one is closely associated with the electronic structure around the Fermi surface, because variation of this part impacts essentially on the electron localization in electrides and further modifies all physical properties, especially the optical properties in low frequency range. oC40 and oC24 phase do not have many such bands crossing the Fermi level, so that the role of compression in them belongs to the case (i).Except the evident variations of the electronic and optical properties at the phase boundaries, which are described by the s orbitals centered at ISQs rather than the p orbitals surrounding the ions. Consequently, the valence electrons from ISQs, when interacting with light, are inevitably excited to p orbitals that are usually surrounding the lithium ions. Because all electride phases are stabilized by the interstitial electrons, as well as the ensuing multicenter-bonding, this photon-induced electron transfer from ISQs to ions implies there might have a strong photon-phonon coupling, leading to polaritons or light-induced phase transformations. Our rudimentary calculation on oC40 with such photon-excited electronic configuration indeed suggests that dense lithium could be a prototype of elements with such kind of photon-induced transition, and should be the focus of future investigations.Furthermore, the optical electronic transitions occur only when the angular momenta satisfy the optical selection rule mentclass2pt{minimTo summarize, the dynamic dielectric response and optical properties of the three high-pressure electride phases of cI16, oC40, and oC24 in lithium were thoroughly and comprehensively investigated across a wide pressure range from 40 to 200 GPa by first-principles calculations. The reflectivity of dense lithium was predicted to decrease with increasing pressure in the visible and infrared range, changing from highly reflective metallic phases to semi-transparent electride phases. Two previously unknown interband plasmons were discovered in cI16 at 70 GPa. This explains the abnormal variation in the reflectivity, and reveals a strong dependence of the optical property on the subtle details of the crystalline structure. The atomic structure was found gradually distorted with the increasing localization of interstitial electrons under compression, even within the same cI16 phase. In contrast to the drastic jump of physical properties at the phase boundaries, this observed gradual evolution unveils how external pressure continuously modifies the microscopic structure, especially the irregular effect of compression, via localized interstitial electrons, and further impacts on the dielectric response. These interesting variations, however, are not obvious if viewed only from the band structure. The system accumulates these variations and finally reaches the condition of phase transitions. We also predicted the possible response of electrides when interacting with the light field, and discovered diverse and rich features in their optical properties. Two important characteristics are the strong anisotropy in oC40 phase and the possible photon-induced transformation. Even though both cI16 and oC24 are semi-metallic electrides, their optical properties, as well as the role of compression in their evolution, are very different, which is quite unexpected. Especially, oC24 becomes a semi-transparent metal, with a reflectivity and absorption coefficient even lower than the insulating oC40 phase, determined by its unique electronic structure. This work uncovers intriguing anomalous optical properties in the electride phases of dense lithium, and the underlying physics was elaborated. The results and findings could be the foundation for future experimental exploration of high-pressure electrides.q of the incident electromagnetic wave. For 45. In this work, the dynamic dielectric function at the long-wavelength limit of 46. The response to this electric field reveals a collective excitation of the Fermi sea, or the bulk plasmon behavior.The dielectric function (DF) 47The important optical properties, such as the reflectivity 48Here the EELS describes inelastic scattering of the incident electrons with narrow initial kinetic energy, including the inelastic interaction of inter- and intra-band transitions and plasmon excitations. The dynamic optical conductivity 49c and v refer to conduction and valence band states respectively, and k in the reciprocal space. The k-point weights 4544, and leads to an imaginary frequency shift after Fourier transform. This direct band summation method equation  only the direct transitions of electrons between valence and conduction bands are calculated, and the results include only the interband contribution, with the imaginary part of the DF having a direct connection with the electronic band structure; (ii) when deducing equation . The microscopic DF includes additional rapid oscillations of the total electric field on the scale of the primitive cell, whereas the macroscopic DF is homogenous on a coarse scaleThe rapid oscillations on the microscopic scale caused by slowly varying external electric field and local crystalline structure may have an influence on the macroscopic response of the material to this field, and this is called local field effects (LFE), which represent the inhomogeneity of the electron gas. These are reflected in the off-diagonal elements of DF and have been automatically included in equation  in the sab initio total-energy and molecular-dynamics program VASP53. The projector-augmented wave (PAW) pseudopotential of Blöchl is employed, which combines the all-electron level accuracy with the computational efficiency of the pseudopotential approach, as implemented in VASP with the frozen-core approximation54. The longitudinal expression of the frequency-dependent microscopic dielectric function is utilized in the frame of PAW. The results are proved to be more accurate than the transversal expressions and are largely independent of the applied potential49. The generalized-gradient approximation (GGA) of Perdew-Burke-Ernzerhof (PBE) is used for the exchange-correlation functional of the density functional theory (DFT)55. All electrons  mesh56 of 58, our results presented here are sufficiently accurate to give an quantitative optical spectra , which is enough for our purpose to understand the evolution of the optical properties of dense lithium with increasing pressure.The first step of the DF calculation as mentioned above is directly based on the static results of the ground state DFT calculation, so it is labelled as ‘interband part by DFT’ in Results section. The second step of the DF calculation is carried out by using the GW module as implemented in VASP, with an extra exchange-correlation kernel in the Dyson equation . In addiAll data generated or analyzed during this study are included in this published article (and its Supplementary Information files).Supplementary Material


Zika virus (ZIKV) is an emerging mosquito-borne flavivirus that can potentially threaten South China. A Chinese family of four returning from Venezuela to China was found to be positive for ZIKV when the youngest son's fever was first detected at an airport immigration inspection. They were isolated temporarily in a local hospital in Enping city, Guangdong province, where their clinical data were recorded and urine and saliva were collected to isolate ZIKV and to obtain viral sequences. All of them except the mother presented mild symptoms of rash and fever. Envelope gene sequences from the father, daughter and son were completely identical. Phylogenetic analysis demonstrated that this strain is similar to several imported strains reported in recent months, which are all clustered into a group isolated from 2015 ZIKA outbreaks in Brazil. Together with the climatic features in Venezuela, New York and Guangdong in February, it can be concluded that our subjects are imported cases from Venezuela. With the same viral sequence being shared between family members, neither direct human-to-human nor vector transmission can be ruled out in this study, but the former seems more likely. Although our subjects had mild illness, epidemiologists and public health officials should be aware of the risk of further expansion of ZIKV transmission by local competent vectors. ZIKV is a reemerging mosquito-borne flavivirus circulating in a wide range of regions including Africa, South America, and Asia.Aedes aegypti is considered the principal transmission vector of ZIKV,8 although Aedes albopictus, which caused several outbreaks of dengue fever in Guangdong Province of South China in the last two decades, may play a role in the spread of this virus because A. albopictus may be a competent vector.9 There are over 180 000 Chinese in Venezuela, which is one of the regions most heavily affected by ZIKV infection in South America.10 With frequent people shuttling between South America and Guangdong, there is a potential risk of spreading ZIKV to South China, where A. albopictus are active in densely populated communities.By far, 13 it was also necessary to explore this possibility in this family.In this study, a family of four flying from Venezuela to Guangzhou of Guangdong Province was found to be ZIKV positive in their peripheral blood. To gain a better understanding of transmission among communities, the phylogenetic relationship between the isolates from this family and others from diverse regions of the world was analyzed. Because this virus may be transmitted directly by body fluids,Four hospitalized individuals from a family  were diagnosed with ZIKV infection at Enping People's Hospital. This family had lived in Venezuela for more than two months before 20 February 2016; then they flew to New York on that day and stayed there for ~4 days. Finally, they flew from New York to Guangzhou, China on 24 February 2016 . These fBefore RNA extraction, urine samples were concentrated with Amicon Ultra-0.5 Centrifugal Filter Units with Ultracel-10 membrane . The QIAamp Viral RNA Mini Kit  was used to extract viral RNA from urine and saliva according to the manufacturer's instructions. RNA was eluted in 50 μl of AVE buffer and stored at −80 °C until use.http://www.oligo.net/.Samples positive for ZIKV were selected to amplify genes encoding Envelope protein (E) and nonstructural protein 1 (NS1). Complementary DNA (cDNA) was synthesized from viral RNA by using the RevertAid First Strand cDNA Synthesis Kit  according to the manufacturer's instructions. Four pairs of primers were designed to generate overlapping DNA fragments covering the E and NS1 gene regions by using the software Oligo7.0 . The reference sequences selected were those with highly similar BLAST  scores. Phylogenetic trees were drawn using the maximum likelihood method in the Tamura-Nei model with gamma-distributed evolutionary rates in MEGA 7.0 (www.megasoftware.net).14 An initial tree was made automatically with the nearest-neighbor-interchange method. The gaps/missing data treatment was set as complete deletions. Bootstrap analyses with 1000 replications were utilized to determine confidence values for groupings within the phylogenetic trees. Other parameters were set to default style.Before phylogenetic analysis, the E and NS1 coding sequences of four individuals in our study were aligned with other reference sequences using Clustal 2.1 software , patient 3 (daughter) and patient 4 (son) were found to be positive for ZIKV. Only patient 4 was positive for ZIKV in a saliva sample . The urine and saliva samples from patient 2 (mother) were all negative for ZIKV detection.et al, Guangdong Provincial Center for Disease Control and Prevention), the viral sequence from the same patient. We found that all our sequences were similar to those of strains circulating in South America, especially Brazil  is another cluster located a long distance from the cluster of current imported Venezuelan strains in our study. Our strains from the family we are studying, as well as other strains isolated from imported cases in China during the same period of time, form a cluster near the Brazilian strains isolated in 2015. As there is a high bootstrap value at the node of this branch, together with enough evolutionary distance, HQ234499.1-P6-740 (isolated from A. aegypti) is an ideal outgroup strain in the Asian lineage.PCR products were successfully obtained only from patient 1, patient 3 and patient 4. After sequencing and fragment assembly, the E gene sequences of the three patients were found to be exactly the same. The E gene sequence of patient 1 is identical to Z16019  are milder than those of Dengue virus (DENV) infection; the latter often include high-grade fever with myalgia, headache, arthralgia and nausea. Leukopenia (<4000/mm3) has been detected in ~30% of Dengue fever patients.21 In our study, one adult and two children had the symptoms of fever, rash and conjunctival congestion, and no one complained of any pain. Unexpectedly, the three patients with fever all had normal leukocyte counts (>4000/mm3). Whether the milder symptoms of ZIKV infection (milder than DENV infection) mean a higher leukocyte count is unclear because of the lack of data obtained in past reports on ZIKV outbreaks. Hence, if the mother without any symptoms in our study is considered only a carrier of ZIKV (not a patient), this proportion of patients in our study is almost consistent with previous reports, in which rashes were presented by almost all patients.20 Information about pruritus, the second most common clinical symptom in the confirmed cases in previous study,22 was not obtained from the family while inquiring them regarding their case history. Perhaps this symptom is transient and unnoticeable at the time of disease onset.Rash and low-grade fever seem to be the main symptoms for most ZIKV-infected individuals. Rash was reported to be the most frequent symptom (presented in 95.7% cases), followed by fever and arthralgia.24 because enzyme-linked immunosorbent assays (ELISAs) for IgM antibody against ZIKV would cross-react with other flaviviruses,26 and the results must be validated by a plaque reduction neutralization test, a labor-intensive and costly method.27 Moreover, compared with blood drawing, urine and saliva collection are more acceptable options for suspected individuals, and the latter have an advantage for viral detection and isolation because virus can be detected at higher titers and for a longer period in urine than in serum.28 A previous study showed that a patient had prolonged shedding of viral RNA in saliva and urine for up to 29 days after symptom onset.29 Nevertheless, positive TaqMAN RT-PCR detection is not usually a guarantee of successful sequencing or isolation of target viruses. Urine and saliva usually need to be concentrated before RNA extraction or viral inoculation, which was performed in our study.RT-PCR based on TaqMAN probes may be the most convenient tool to detect ZIKV infection in suspected patients19 based on experience from molecular study of dengue virus.30Like other flaviviruses, the structure of the single polyprotein encoded by ZIKV genomic RNA is 5′-C-prM-E-NS1-NS2A-NS2BNS3-NS4A-NS4B-NS5-3′, in which the E (envelope) protein is the main antigen recognized by the host immune system, and NS1 is the principal component of the replication complex of the virus. In previous research, E gene sequences of ZIKV isolates were usually utilized to construct phylogenetic trees31 meaning that the phylogenetic tree based on the E gene is strong enough to distinguish all clusters of ZIKV isolates from all over the world. Because complete genome sequencing of an RNA virus is a time-consuming procedure, constructing a phylogenetic tree using E protein gene sequences is a simpler method for molecular epidemiologic study, especially in an outbreak. In our phylogenetic tree constructed from E protein gene sequences, ZIKV can be divided into two lineages, African and Asian.33 Although there is a very short evolutionary distance between our strains and the Venezuela imported strain KU744693.1-VE Ganxian, the diversity of the strains is apparent, and the latter is nearer to KJ776791.1-H/PF/2013 French Polynesian strains.34 Currently, all cases reported in China seem to be imported from South America and Pacific islands, which also indicates there was a great diversity of strains circulating during early 2016 in Venezuela. Our strains are more similar to the strains circulating in South America in late 2015, where there were more strains isolated from patients. A phylogenetic tree based on NS1 gene sequences cannot efficiently distinguish different subgroups of ZIKV from various sources of isolates (Our phylogenetic tree constructed based on the E protein coding gene is topologically similar to that based on the complete ZIKV genomic sequence ,31 meaniisolates  because isolates .Aedes mosquitoes circulating in the African continent according to the phylogenetic tree, probably because ZIKV did not draw much attention before massive microcephaly cases were reported in Brazil. ZIKV is spread primarily by A. aegypti, a dominant vector for several mosquito-borne viruses in the Western Hemisphere.35 ZIKV was detected in A. albopictus from Gabon, a country in Central Africa.9 No direct evidence has demonstrated that this virus could be spread by A. albopictus, a local vector for Dengue virus in South China, yet the threat is quite real. The fact that ZIKV can propagate in the C6/36 cell line derived from A. albopictus suggests that this mosquito may be a competent vector and have the potential for spreading the virus,5 although there have not been any reported isolates from mosquitoes in South China. A previous experiment conducted in laboratory indicated that ZIKV could be detected in the midgut and salivary glands of A. albopictus ten days after oral infection.36 Outbreaks of DENV have proved that South China may be a permissible environment for transmission of emerging viral diseases such as ZIKV, and imported cases may be sources of subsequent autochthonous cases.38It is worth noting that strains of the African lineage were almost solely isolated from A key difficulty in preventing the spread of infection is that a high proportion of infected individuals have no symptoms. Similar to the mother in our study, asymptomatic individuals with viremia may be a source of infection when they are exposed to competent vectors. If the son's fever had not been found at the airport entrance, our subjects would not have been suspected of being infected with ZIKV, and thus their viremia would not have been confirmed. It is difficult to convince ZIKV carriers to be examined even if they are returning from an affected region. Furthermore, in addition to the mosquito-borne route, other complex modes of transmission such as body fluid transmission will make controlling ZIKV infection even more difficult than controlling Dengue fever. The priorities for control include strengthening the education of travelers from infected areas to non-epidemic regions, identifying virus carriers, preventing local transmission caused by imported cases, and an effective vector management program.In our study, four subjects in one family had lived and traveled together for the past few months, so they had similar opportunities for exposure to mosquito bites. However, because they are a family, the probability of human-to-human transmission by close contact between family members should not be ignored. If their ZIKV infections were all caused by mosquito-biting, from a time axis showing when symptoms occurred, the incubation periods of three of the patients were all more than five days , which i12 Because an in vitro experiment demonstrated that human skin cells are permissive to ZIKV,6 it might be possible that ZIKV can be transmitted by other bodily fluids such as saliva and urine. RT-PCR positivity for ZIKV RNA together with successful viral isolation from the three patients' urine samples proved that there were live viruses present in these samples. RT-PCR also indicated the existence of viral RNA in the patients' saliva. However, currently there is no reliable evidence demonstrating ZIKV transmission via these types of bodily fluids.Regarding direct transmission, sexual contact may be a route, for a relative case had been reported previously but with no direct evidence.


Astrocytes are the most populous glial subtype and are critical for brain function. Despite this, historically there have been few studies into the role that they may have in neurodegenerative diseases, such as Parkinson’s disease (PD). Recently, however, several studies have determined that genes known to have a causative role in the development of PD are expressed in astrocytes and have important roles in astrocyte function. Here, we review these recent developments and discuss their impact on our understanding of the pathophysiology of PD, and the implications that this might have for its treatment. PD is characterised by the degeneration of dopaminergic neurons in the substantia nigra pars compacta (SNc).Neuroinflammation in the SNc is a consistent feature of PD.Recent studies of eight proteins implicated in PD demonstrate that they have functional roles in astrocyte biology.These studies provide evidence that PD may develop, at least in part, due to astrocyte dysfunction. They provide structural and metabolic support, and regulate synaptic transmission, water transport, and blood flow within the brain Evidence is emerging to suggest that disruption of astrocyte biology is involved in dopaminergic neuron degeneration in PD. Although most PD cases are idiopathic, monogenic mutations in 17 genes have been identified and implicated in the development of the disease PARK7 gene. Expression of PARK7 has been shown to be higher in astrocytes than in neurons in postmortem human brain samples, and to be upregulated in reactive astrocytes in patients with PD Park7 were shown to result in the increased degradation of the lipid raft proteins flotillin-1 and caveolin-1, and to lead to disrupted lipid raft assembly Park7 knockout (KO) and mutant astrocytes were found to exhibit impaired glutamate uptake The most evidence for a PD-related gene having a role in astrocyte biology has been found for DJ-1, which is encoded by the Park7-KO astrocytes display alterations in inflammatory cytokine production Park7-KO astrocytes with IFN-γ Park7-KO brain slices compared with wild-type (WT).Lipid rafts also have an important role in astrocyte immune response to lipopolysaccharide (LPS). It has been shown that the disruption of lipid raft assembly that occurs in the absence of DJ-1 results in impaired TLR3/4-mediated endocytosis Park7 in astrocytes also results in a reduction in their ability to protect neurons against neurotoxicity in rotenone and 6-hydroxydopamine neurotoxin models of PD Park7 increases the neuroprotective capacity of astrocytes in the rotenone model Park7 reduced astrocytic mitochondrial motility in the same manner as rotenone treatment. Furthermore, a decrease in astrocyte mitochondrial membrane potential caused by rotenone treatment was exacerbated by Park7 knockdown. Mitochondrial fission was also shown to be reduced in the presence of Park7 knockdown and rotenone treatment, although no changes were detected in mitochondrial fusion or respiration.Knockdown or KO of SNCA gene, and mutations as well as duplication or triplication of the gene have been shown to lead to development of the disease. Expression of SNCA in astrocytes is low, whereas it is abundantly expressed in neurons Snca-KO astrocytes showed that the incorporation and distribution of the fatty acids arachidonic acid (AA) and palmitic acid was disrupted One of the pathological hallmarks of PD is accumulation of α-synuclein-positive cytoplasmic inclusions in neurons. α-synuclein is encoded by the SNCA was overexpressed under an astrocyte-specific promoter ventral tegmental area (VTA) (26.1%), mirroring the relative susceptibilities of the different regions, as seen in PD.High concentrations of extracellular α-synuclein have been shown to induce a TLR4-dependent inflammatory response in primary astrocyte cultures PLA2G6 encodes group VI Ca2+-independent phospholipase A2 (iPLA2), an enzyme that catalyses the release of fatty acids from phospholipids. Mutations in iPLA2 cause infantile neuroaxonal dystrophy and neurodegeneration with brain iron accumulation and have recently been shown to cause PD 2 has been shown to inhibit the release of the fatty acid AA from phospholipids in astrocytes Pla2g6 knockdown and inactive mutants of Pla2g6 have both been shown to result in a reduction in calcium responses  in primary astrocyte cultures 2, suggesting a link with TLR4 signalling 2 mutations.ATP13A2 encodes the lysosomal type 5 ATPase protein (ATP13A2), mutations of which cause an early-onset form of PD Atp13a2-KO astrocytes were shown to exhibit increased expression of proinflammatory cytokines, and decreased expression of anti-inflammatory cytokines and neurotrophic factors, such as GDNF, compared with WT cells. This was demonstrated to be mediated through the upregulation of the lysosomal protease Cathepsin B, which was subsequently released into the cytosol via damaged lysosomal membranes, resulting in the activation of the NLRP3 inflammasome. Conditioned medium from Atp13a2-KO astrocytes was found to be less neurotrophic than medium from WT astrocytes, and was less able to protect dopaminergic neurons from MPP+ toxicity. Interestingly, the authors also found that, in response to MPP+ treatment of WT astrocyte cultures, ATP13A2 expression was reduced, suggesting that MPP+ toxicity proceeds through a similar pathway.LRRK2 gene LRRK2 are the most common genetic cause of PD, and result in a patient phenotype similar to the idiopathic disease Leucine-rich repeat kinase 2 (LRRK2) is a large protein with dual kinase and GTPase activity that is encoded by the GBA gene encodes β-glucocerebrosidase (GCase), a lysosomal enzyme that is involved in glycolipid metabolism. Heterozygous mutations are a risk factor for developing PD Gba-KO mice that lack Gba only in neurons and macroglia  have been found to display an increase in expression of cathepsin lysosomal proteases in both astrocytes and neurons Gba-KO astrocytes from another study demonstrated a reduced number of LC3-positive puncta, indicating further deficits in the autophagy pathway Gba-KO neurons and astrocytes exhibited decreased mitochondrial resting membrane potential and increased mitochondrial fragmentation, and showed that the fragmentation of mitochondria was more severe in astrocytes than in neurons.The PINK1 encodes PTEN-induced putative kinase 1 (PINK1), a protein widely shown to be involved in mitophagy, a process that selectively degrades damaged mitochondria Pink1 expression in the brain has been shown to increase as embryonic development progresses, and has an important role in the development of astrocytes Pink1-KO mouse brains exhibit a reduction in the number of astrocytes compared with WT mice Pink1, proliferation was markedly reduced PINK1 are loss-of-function mutations PARK2 gene. Postmortem studies of brains from patients with PD and PARK2 mutations have shown the accumulation of α-synuclein inclusions in astrocytes PINK1, PARK2 mutations tend to confer a loss of function, and Parkin has been shown to have a role in astrocyte proliferation, as demonstrated by the decreased proliferation of Park2-KO astrocytes Park2-KO astrocytes have also been found to have decreased neurotrophic capacity, mediated by a reduction in glutathione secretion Park2-KO astrocytes had increased levels of damaged mitochondria Parkin is another protein that has been widely implicated in mitophagy The roles of each of the eight PD genes described above that have been studied to date converge on four main cellular functions: inflammatory response, lipid handling, mitochondrial health, and lysosomal function . Perturb2As discussed above, multiple genes that have been implicated in PD have been demonstrated to have a role in the initiation and regulation of astrocyte activation in response to inflammatory stimuli. Not only have high levels of exogenous α-synuclein been shown to initiate a TLR4 signalling cascade Pla2g6-KO astrocytes display a diminished ability to release AA from phospholipids Snca-KO astrocytes, it is the uptake and distribution of AA that are disrupted As well as responding to inflammatory cues in the brain, astrocytes have a role in the metabolism of lipids and the release of fatty acids. This process has been shown to be disrupted in PD gene-KO models, especially in the case of the fatty acid, AA. Further to the roles that PD-related genes have in astrocyte inflammatory response and fatty acid metabolism, we have discussed their role in global cellular pathways, such as mitochondrial function and lysosome biology, in the context of astrocytes. Dysfunction of both of these cellular processes been implicated in both familial and idiopathic PD PINK1 and PARK2With regards to lysosome biology, astrocyte uptake of α-synuclein from the extracellular space via endocytosis results in its degradation by lysosomes 2, ATP13A2, PINK1, and Parkin, there is now clear evidence that they are involved in astrocyte-specific functions, including inflammatory responses, glutamate transport, and neurotrophic capacity. In the cases of LRRK2 and GCase, no studies have yet investigated their role in astrocyte-specific functions, but they have been shown to have a role in the general physiology of astrocytes. Clearly, a wealth of research points to a role for many of the PD genes in neurons, although there is now also substantial evidence to suggest that non-cell-autonomous processes contribute to development of disease pathology. We believe that further investigation of the role of astrocytes in PD will be paramount to furthering our understanding of the disease  or overexpression (SNCA) was conducted to dissect the contribution of individual cell types to the pathogenesis of disease. The overexpression of mutant SNCA under an astrocyte-specific promoter resulted in the degeneration of dopaminergic neurons in the SNc and VTA as well as the onset of a movement disorder. It will be valuable to develop similar astrocyte-specific overexpression and KO models for other genes that have been shown to cause PD, to determine whether they also develop such phenotypes.In this article, we have discussed data produced from various models, including studies conducted in whole animals and in primary culture. In two animal models, cell type-specific KO (induced pluripotent stem cell (iPSC)-derived astrocytes may go some way to enable the research community to answer this question Likely due to the lack of availability of living human brain tissue for the derivation of primary cultures, the only human data available to cover in this review refer to postmortem pathology. All mechanistic insights into the role of PD-causative genes have been generated using animal models. However, it has been found that there is some variability in the expression levels of PD-related genes between human and mouse astrocytes and neurons  is one such treatment that can be used to successfully alleviate the motor symptoms of PD, and it has been demonstrated that its mechanism of action may include the activation of astrocytes Previous studies have shown some benefit of transplanting human foetal midbrain tissue into the brains of patients There are promising data that show a reduced risk of developing PD in humans who have taken nonsteroidal anti-inflammatory drugs Alt-text: Box 2Although the number of studies that have been conducted to investigate the contribution of astrocytes to the pathogenesis of PD is few compared with those investigating neuronal function, a picture of astrocyte involvement in the disease is beginning to emerge. Here, we have reviewed the literature supporting the concept that genes known to be causative in PD have important roles in astrocyte biology. We have drawn on this research to describe how the implicated pathways intersect in the involvement of astrocytes in PD. In the cases of DJ-1, α-synuclein, iPLA disease .Outstand


FOXI2, FANK1 and GLRX3 remain candidates for further investigation. This, the largest genetic study of VUR to date, highlights the 10q26 region as a major genetic contributor to VUR in European populations.Vesicoureteric reflux (VUR) is the commonest urological anomaly in children. Despite treatment improvements, associated renal lesions – congenital dysplasia, acquired scarring or both – are a common cause of childhood hypertension and renal failure. Primary VUR is familial, with transmission rate and sibling risk both approaching 50%, and appears highly genetically heterogeneous. It is often associated with other developmental anomalies of the urinary tract, emphasising its etiology as a disorder of urogenital tract development. We conducted a genome-wide linkage and association study in three European populations to search for loci predisposing to VUR. Family-based association analysis of 1098 parent-affected-child trios and case/control association analysis of 1147 cases and 3789 controls did not reveal any compelling associations, but parametric linkage analysis of 460 families  under a dominant model identified a single region, on 10q26, that showed strong linkage  to VUR. The ~9Mb region contains 69 genes, including some good biological candidates. Resequencing this region in selected individuals did not clearly implicate any gene but  Other congenital anomalies of the kidney and urinary tract (CAKUT) commonly occur along with VUR. The oft-quoted prevalence of VUR is 1–2% but the true prevalence may well be higher4. The disease has been suggested to be twice as common in females as males5 but this most likely reflects an ascertainment bias, and other studies have detected only a slight excess of incidence in females compared to males7. The prevalence of VUR tends to decrease with age5, and serial studies of individual patients show VUR can spontaneously regress during childhood in a subset of initially affected individuals8. Screening studies of first-degree relatives of individuals with VUR identifies VUR in one third to one half of siblings10 and 65% of offspring11. This observation, coupled with the high concordance of primary VUR in identical twins12 and the identification of families with multiple generations affected by primary VUR and RN14, suggests that there may be a substantial genetic component to VUR. However, large-scale genetic studies of VUR carried out to date have been somewhat disappointing and generally rather inconclusive. Although there have been some compelling findings in individual large families, overall, little concordance is seen between the results from different studies23, supporting the notion that the condition is genetically heterogeneous. Here we combined data from the two largest genetic studies of VUR conducted to date22, comprising three separate cohorts , to investigate whether the increased power obtained from use of a larger sample size could help identify genetic contributors operating across multiple affected families/individuals from these three European populations.Primary vesicoureteric reflux (VUR), the retrograde flow of urine from the bladder through the vesicoureteric junction into the upper urinary tract, is the most common renal tract malformation. VUR is usually a benign condition but chronic kidney damage triggered by ascending pyelonephritis and also congenital kidney hypo/dysplasia  can occur and lead to end stage renal disease24 produced no compelling association signals 22 and the UK (2938 Wellcome Trust Case Control Consortium controls)25 similarly produced no compelling association signals. We note that the relatively sparse SNP set available for association analysis (see Methods) provides incomplete genome coverage with levels that are probably, at best, close to the 31% coverage provided by the Affymetrix 111 K array26. Therefore, our results do not preclude the possibility that common variants associated with VUR exist, but we would need to genotype our UK/Slovenian samples  with a much denser genotyping array in order to answer this question definitively.Family-based association analysis carried out using the transmission/disequilibrium test (TDT)27 with the Haplotype Reference Consortium (HRC) reference panel28, treating the Irish and UK/Slovenian cohorts separately (since they had very different numbers of QCed genotyped SNPs available to inform imputation). We then repeated the TDT association analysis at all SNPs passing post-imputation QC  when analysis was restricted to the UK/Slovenian cohort, however this association was not replicated in the Irish or combined cohorts ) than is seen at the lead SNP, suggesting an inconsistency between results that should in reality be highly concordant. A single SNP on chromosome 6 showed genome-wide levels of significance  in analysis of the combined cohorts  of linked families of α ≈ 30%. This region had shown some evidence of linkage in the previous separate analyses of these cohorts22 and, indeed, had been highlighted as one of the top findings in the Irish cohort by Darlow et al.22, although the significance achieved in that previous analysis  was not sufficient to definitively conclude the presence of a disease locus in the region. In previous analysis of the UK/Slovenian cohorts19 this region had achieved an HLOD of 2.44 and a ZLRLOD of 1.55 in an expanded set of families where affection status was coded as positive for either VUR or RN. Cordell et al.19 had further noted that the signal of linkage increased to ZLRLOD = 2.32 when analysis was restricted to families where affection status was coded as positive for VUR (regardless of RN status), as in the current analysis.Parametric linkage analysis under a recessive model gave no strong evidence for linkage at any genomic location (data not shown). However parametric linkage analysis under a dominant model, as well as non-parametric linkage analysis, identified a single genomic location (focused around rs7907300 on chromosome 10q26) showing strong evidence  for linkage to an underlying disease locus , resulted in a slightly increased maximum HLOD of 5.07 at the same genomic location (rs7907300).We investigated the sensitivity of our parametric linkage analysis results to slight changes in the assumed parameters of the underlying dominant model, but found this did not have any substantial effect. In particular, changing the assumed disease allele frequency from 0.001 to 0.01 19 produces a reasonably compelling linkage signal. Neither the individual cohorts nor the UK/Slovenian combined cohort achieve the HLOD of 3.3 that would most probably be required to conclude ‘significant’ linkage31  but the results are certainly consistent with the much stronger (and highly significant) effect seen here when all three cohorts are combined.To clarify the relative contributions of the individual cohorts to the current results, we repeated the parametric (dominant HLOD) analysis within each cohort separately and within the UK/Slovenian cohorts combined Fig. . The str29 plot of the equivalent p-values 32 for those SNPs lying within the main peak of significance bounded by red dashed lines in Fig. 33, with genome-wide significance in affected-sib-pair linkage studies corresponding to a p-value of around 2.2 × 10−5). The 4 genes omitted from the LocusZoom plot in Fig. C10orf88, ACADSB, HMX2 and BUB3) all lie to the left of  GPR26 and thus would probably not be considered good candidates for harboring causal mutations, given the decrease in linkage signal observed within this section of the plot, but any of the genes to the right of GPR26 might be considered good positional candidates.As is often the case in linkage studies, the plausible region within which the underlying disease lies is very large, containing 44 genes in the central ~7 Mb region or 69 genes in the wider ~9 Mb region (Supplementary Table −6) is seen at around 129.5 Mb (Build 37). This is not replicated in the UK/Slovenian cohorts  within the 12 Mb region spanning the linkage signal , 127.6 Mb , 128.4 Mb  and 131.5 Mb . However these signals are not replicated in the Irish cohort .In the UK/Slovenian cohorts  that was used to generate the linkage signal in the first place. A far more likely scenario is that the linkage signal is caused by heterogeneous mutations operating within different families in a dominant or dominant-like fashion. Given that the TDT can be considered as a test of linkage in the presence of association  we conclude that the apparent TDT association signals seen most likely reflect linkage between alleles at the relevant SNPs and mutations at the underlying disease locus, coupled with perhaps low levels of allelic association (linkage disequilibrium) between alleles at these loci in the founders.40, especially when applied to multiple affected individuals originating from the same family. A caveat in relation to our own study, however, is that we expect that only around 30% of our families will actually harbor mutations at the disease-causing genetic element in the 10q26 region . Prioritizing affected individuals from families where affected sibs shared alleles IBD, we whole-exome sequenced 29 individuals from the 8 families  showing the strongest evidence of linkage in the 10q26 region. There were 37,930 non-synonymous variants called across the whole exome in the 29 individuals. Of these, 139 occurred within the 10q26 region of interest and 37 of these were relatively rare  in the 1000 Genomes and Exome Sequencing Project data sets. Of these relatively rare variants, 23 were found within the same gene in two or more individuals (for 7 different genes) and 6 were predicted to be deleterious by at least one predictor out of SIFT/PolyPhen/MutationTaster43 . Only 7 of the 189 1000 Genomes Project sequences had no rare variants at all, suggesting that the detection of rare variants in FANK1 in our own VUR cases is not, in fact, a rare event, and so may not necessarily be related to their phenotype. Moreover, given a VUR population prevalence of up to 3%, we note that the ‘rare’ FANK1 variants listed in Supplementary Table To interrogate in more detail the genes in the implicated 10q26 region, we performed whole-exome sequencing in a subset of our families. Next-generation sequencing has been extremely successful for the detection of disease-causing mutations in Mendelian, monogenic disordersFANK1, while 11 occurred within the promoter of genes other than FANK1 , a plausible explanation is that such variants may be found within non-coding, regulatory regions. To investigate this possibility, we performed targeted genomic resequencing of the implicated 9 Mb region of 10q26 (from 123 Mb–132 Mb) in 32 unrelated affected individuals from those families in our data set that contributed the most to the linkage signal (the 8 families that had been whole-exome sequenced and 24 others). Of the 34,412 single nucleotide variants (SNVs) passing standard quality control steps, we identified 9170 that were either absent or present at less than 1% in the 1000 Genomes European data, 3625 of which were also shared between affected individuals . We consider this large number of SNVs identified as showing frequency differences between our samples and the 1000 Genomes European samples as most likely to be artefacts arising from the systemic differences in read depth, alignment and variant-calling pipelines between cases and controls. Concentrating on rare SNVs found within promoters in our 9 Mb target region, we found that 69 of 80 promoter-region SNVs occurred within the promoter of FANK1, further interrogation of the .bam files showed that the FANK1 region appears to be hypervariable and many of the called SNVs were clearly artefacts  mapping to chromosome 10. This suggests that a number of the reads identified by our pipeline as mapping to 10q26 may in fact really come from chromosome 22, offering a possible explanation for the unusually high level of variability observed , as well as to the desired 10q26 region. This observation led us to re-align our targeted sequences to the GRCh38 assembly. 9 of the 11 previously identified SNVs in the FANK1 and its promoter, which therefore represent artefactual findings unrelated to the VUR phenotype. More sophisticated experimental methods  will therefore be required in order to robustly interrogate this 42.4 kb region, without danger of contamination from other genomic regions.Further investigation revealed that the 42.4 kb region of chromosome 10 from 125,885,884 to 125,928,375 contains segmental duplications from at least 4 other genomic locations  whereas our own sequences have up to 1000 fold coverage in the target region. Analysis of sequence data where there are systemic differences in coverage between cases and controls typically leads to inflated type I errors46, but discarding those samples with insufficient read depth can result in a loss of power . We therefore used the software package TASER47, which effectively re-calls variants in both cases and controls, allowing for systematic differences in read depth (and other factors), at the same time as constructing a test statistic.The analyses described above compared observed allele frequencies in our VUR cases to those in publicly available summary data provided by the 1000 Genomes Project (and similar resources), in order to identify putative causal variants. To achieve a better comparison between our sequenced VUR cases and phenotypically normal controls, we obtained the .bam files from 1106 ALSPAC controls48λ = 0.76). With the MAF filtering threshold set to 0.05 in the base population, we obtained a few signals that were mildly suggestive of association but nothing that reached experiment-wide significance. The output of the current implementation of TASER does not indicate the position of the variants contributing to the test statistic within each window, but by inspecting the input and .bam files we were able to identify the probable variant driving the signal and discount any signals caused by sequencing error . The top findings , a SNP approximately 3 kb upstream of FAM175B (rs533072490) and a further three SNPs in the intergenic region between LINC01163 and MGMT: rs146303503, rs182100352 and an unknown G/A substitution at position 128770839. Given the relatively low frequencies of all these variants seen within the VUR cases, and the relative lack of statistical significance obtained , we do not consider any of these results particularly compelling.In an adaptation of the original implementation of TASER, which splits the entire genome into small windows of interest , we considered our entire target region to be ‘of interest’. For each of the 1106 ALSPAC control sequences and 32 VUR case sequences, we split the target DNA sequence into consecutive 120 bp windows with 30 windows included within a processing ‘block’. This resulted in 2490 blocks covering the entire sequence from 121,240,479 bp to 130,201,779 bp (GRCh38) on chromosome 10. Only bases called with a quality score >30 were added to the read count at each position. We found that only counting alleles that are seen at least twice in the low depth control sequences and at least 5 times in the high depth VUR sequences (in order to reduce the expected number of sequencing artefacts) improved the overall distribution of test statistics . Thus this region, containing the key candidate gene FANK1, remains effectively untargeted by our current sequencing endeavours, due to our inability to reliably map the sequence reads in this region.In addition to the findings listed in Supplementary Table in vitro-propagated cultures of Normal Human Urothelial (NHU) cells in different differentiation states were obtained from Fishwick et al.49. Results  argue against these being strong positional candidates for explaining the linkage signal. OAT, the only one of the three highest expressed genes in normal urothelium to be moderately near to the centre of the linkage region, encodes the mitochondrial enzyme ornithine aminotransferase, so is an unlikely candidate on grounds of function.To help further prioritize genes in the implicated chromosome 10q26 region, data on gene expression by GLRX3 remains a key region for further investigation. Moreover, given our discovery that the 42.4 kb region of chromosome 10 from 125,885,884 to 125,928,375 contains segmental duplications from other genomic locations, this region (which contains the key candidate gene FANK1) remains effectively untargeted by our current sequencing endeavours. More sophisticated methods  will be required in order to robustly interrogate this region, without danger of contamination with sequence from other genomic regions.Here we have identified, through genome-wide parametric and non-parametric linkage analysis, a region on 10q26 that shows strong evidence of harboring genetic variants contributing to primary nonsyndromic VUR. As is often the case in linkage studies, the plausible region in which the underlying disease locus must lie is very large, and, despite our attempts via association analysis and targeted sequencing (in a subset of samples), we have, as yet, been unable to find variants within the region that plausibly account for the linkage signal observed, although the promotor of 22, four of which lie close to the 10q26 region implicated here, in addition to another, as yet unidentified, gene near the 10q telomere50. However, these previously-implicated genes lie mostly outside our main peak of linkage. According to the gene expression atlas GUDMAP (GenitoUrinary Development Molecular Anatomy Project)52, mouse homologues of most of the human genes in the critical Chr10 locus are normally expressed in at least one site in the developing murine kidney and urinary tract. It is, however, of particular note that: Cpxm2 is expressed in the mature ureter and bladder; Fam53 is expressed in the developing and mature ureter; Dhx32 is expressed in the developing and adult ureter and bladder; Adam12 is expressed in the developing ureter and bladder; and Ptpre is expressed in the developing and adult ureter. Expression during in vitro differentiation of normal human urothelial cells might be considered a more relevant factor than mouse gene expression data obtained from GUDMAP, and indeed there are proven human VUR genes that encode tenascin XB and uroplakins which are expressed in the urothelium54. Cross-referencing the genes in our implicated 10q26 region with expression data from normal human urothelial cells49 highlighted several genes of interest including BUB3, OAT and GLRX3. Of these, only OAT would seem a strong positional candidate, but is a highly unlikely candidate on grounds of function. Our other key candidate, FANK1, was also expressed, albeit at a low level. However, this analysis of human urothelial cells comes with the caveat that epithelium is just one type of tissue found in the urinary tract, and we do not currently have similar data for other components such as nerve and muscle/mesenchyme.There are five genes on chromosome 10 that are already known to be involved in urinary tract developmentFGFR2, which has been implicated in induction abnormalities of the ureteric bud and development of VUR in mice58. However, the localisation of FGFR2 outside our main peak of linkage argues against it being a strong candidate for harboring the mutations that contribute to the linkage signal detected in our families. It is worth mentioning that FGFR2 is involved not only in the embryonic development of the metanephric urinary tract but also in that of the skeleton and skin, and mutations in the gene in humans can cause Crouzon syndrome, Pfeiffer syndrome, Apert syndrome, Jackson-Weiss syndrome, Beare-Stevenson cutis gyrata syndrome, or Saethre-Chotzen syndrome. Demonstration of VUR in mice58 was achieved by conditional knock-out of Fgfr2 very specifically in the peri-Wolffian duct stromal cells only, and therefore any mutation causing non-syndromic VUR or other CAKUT in humans that affects FGFR2 is likely to be in a non-coding regulatory element that affects expression of the gene in the developing urinary tract but has no effect on its expression in other tissues. Since most, if not all, genes already known to be involved in urinary tract development are also known to be involved in the development of other structures, including the eye, the ear, the branchial clefts  the skeleton and the gut, it is quite possible that most mutations that cause VUR will be regulatory mutations and in non-coding DNA, whatever gene they affect.Arguably the best candidate gene in the region is FOXI2. GUDMAP shows that Foxi2 is very highly expressed in the mouse metanephric mesenchyme at the appropriate time to stimulate the growth of the ureteric bud. However, our investigation of expression in human urothelial cells failed to detect FOXI2 expression. In 2009 the Irish group used Sanger sequencing to explore this gene, including all the untranslated sequence, 1 kb of promoter sequence upstream, and several highly conserved non-coding regions in the gene desert at distances up to 1 Mb away, in 232 VUR index cases. Eleven of the variants found in the conserved intergenic elements had ‘Restricted Substitution’ (GERP) scores >4, of which four have still not appeared in dbSNP, and, of these, one is immediately adjacent to a predicted POU3F1 binding-site and another is within a predicted PRRX2 binding-site. Pou3f1 and Prrx2 are both expressed in mouse metanephric mesenchyme, the latter very highly. However, functional experiments would need to be carried out to determine whether any of the variants actually affect expression and, if so, which gene is affected. FOXI2 itself was sequenced at that time in case there might be a mutation in the gene which would help to confirm its candidacy. Only two novel variants with GERP scores >2 were found in the gene, neither of which were likely pathogenic, and there was not an opportunity to pursue investigation of the intergenic variants at that time. Sequencing right across the whole linkage region with a larger number of patients (and controls) should hopefully enable identification of the correct regulatory element(s), but it is still very likely that functional testing will be required to determine which gene is affected.A stronger positional candidate, given the linkage evidence, is In conclusion, this, the largest genetic study of VUR to date, provides strong evidence for the 10q26 region as the major genetic contributor to primary nonsyndromic vesicoureteric reflux in European populations. Definitive identification of the causal variants contributing to this observation will be required in order to identify the mechanism involved. Removal of families with pathogenic mutations in this region (once identified) from the data set should make it easier to identify, by reanalysis, the next most important of the many loci expected to be responsible for VUR in the 70% or more of families that are not linked to 10q.22. Full details of sample collection, phenotype definition and genotyping can be found in the primary publications from these studies22. In brief, one study22 consisted of 235 Caucasian families  collected from two hospitals in Dublin, Ireland. Families with two or more affected members with primary VUR of any grade were collected; the majority of the families were nuclear (affected-sib-pair) families containing two siblings with VUR, with genotype data available for siblings and both parents. Six family-members (three parents and three siblings) of VUR patients who had CAKUT (without detected VUR) were also counted as affected. The other study19 included two separate cohorts, one from the UK and one from Slovenia. The UK cohort comprised 165 Caucasian families (303 affected offspring) collected from multiple centres across the UK; similar to the Irish cohort the majority of the families were nuclear (affected-sib-pair) families containing two siblings with VUR and with genotype data available for siblings and both parents. The Slovenian cohort comprised 148 Caucasian families  collected from the University Medical Centre, Ljubljana; again the majority of the families were nuclear (affected-sib-pair) families containing two siblings with VUR, with genotype data available for siblings and both parents.We combined data from the two largest genetic studies of VUR conducted to date59 involves classifying the disease into (increasingly severe) grades from I-V according to the site and severity of reflux based on the degree of filling and dilation of the ureter and upper urinary tract, as assessed by cystography. In the Irish cohort, VUR was diagnosed via micturating cystourethrograms allowing VUR grade to be determined in 96% of patients. In the UK/Slovenian cohorts, however, we elected at the start not to include this concept in the analyses. This was because a. the classic grading system was not in fact always used in the micturating cystogram reports and b. the same grading system is not generally used for radioisotope cystograms, a techique increasingly used to diagnose VUR. Thus, for the analyses described here, we considered VUR to be radiologically proven, but did not consider its grade.The classical grading system for VUR60 and 2.04 in the Slovanian cohort. We chose not to reduce our sample size by stratifying the analysis by gender, which for genetic studies would in any case only be likely to have any substantial effect with regards to variants on the X chromosome. We also chose not to formally account for age of diagnosis in the analysis, taking the view that for genetic studies the most relevant measure is simply the fact that an individual is affected, and whether this can be related to their genotype or shared genotype with other affected relatives. However, given that the prevalence of VUR decreases with age (on account of spontaneous resolution), we counted siblings of affected children who were not investigated until later ages as unknown for VUR affection status, if VUR was not found.Because of sample drop-out, affection status and family structure, not all samples/families constributed to all analyses. We estimated that 199 Irish families , 121 UK families  and 140 Slovenian families  contributed to the linkage analyses presented here. Slightly larger numbers of families/individuals contributed to association analysis (family-based and case/control), as association analysis could potentially make use of family structures that were uninformative for linkage  procedures). The female:male gender ratio for patients used in the genetic analyses was 1.49 in the Irish cohort, 1.29 in the UK cohort (slightly higher than the 1.21 ratio seen in the full UK cohort)19, 134,350 SNPs in the UK and Slovenian cohorts remained available for analysis. The Irish cohort was genotyped on the on the Affymetrix Genome-Wide Human SNP Array 6.0, containing 834,482 genome-wide SNPs. Following QC checks22, a final set of 643,691 SNPs in the Irish cohort remained available for analysis. The density of the final set of SNPs available is more than sufficient for genomewide linkage analysis  in all three cohorts; however, for genomewide association analysis, the UK and Slovenian cohorts are most likely underpowered with respect to providing full genome coverage, as illustrated by the fact that in imputation analysis (see below) only 3,600,157 SNPs were successfully imputed for the UK/Slovenian cohorts, in comparison to 6,277,126 SNPs for the Irish cohort.The UK/Slovenian study, intitially conceived as a linkage study, made use of a relatively sparse genotyping array, the Affymetrix NspI array, containing 262,264 genome-wide SNPs. Following ‘medium-level’ QC checks24, implemented in the program PLINK61 and applied to all affected sibs , was carried out at the subset of 119,548 genotyped autosomal and X-chromosomal SNPs passing quality control checks in all three cohorts.  associations or any significant asociations that replicated across different cohorts).Family-based association analysis using the transmission/disequilibrium test (TDT)22 and the UK (2938 Wellcome Trust Case Control Consortium controls)25 at 108,134 autosomal SNPs passing QC in all case and control cohorts. This analysis was carried out using a linear mixed modelling approach implemented in the software FaST-LMM62 in order to adjust for both relatedness between cases and population differences between cases and controls66.We also performed case/control analysis of our VUR cases together with population-based controls from Ireland (851 Trinity College Dublin/Irish Blood Transfusion Service BioBank controls)67. To construct a suitable SNP set for multipoint linkage analysis, we first pruned our data set to include only those SNPs with minor allele frequencies greater than 0.3 and showing low levels of inter-marker linkage disequilibrium (using the PLINK command “–indep 50 5”), and then thinned the data to use only the two SNPs with the highest heterozygosity in each 1 cM window, using the program MapThin (http://www.staff.ncl.ac.uk/richard.howey/mapthin/). Information content plots from MERLIN and MINX indicated that the resulting SNP set  achieved high levels of information content (≈95%) across the entire genome (data not shown).We carried out multipoint parametric and non-parametric linkage analysis on the combined Irish/UK/Slovenian cohorts using the software packages MERLIN and MINX68, which we denote here as “ZLRLOD”.Parametric linkage analysis allowing for heterogeneity  was carried out assuming a disease allele frequency of 0.001 and either a recessive  or dominant  model. . We chose to assume a slightly rarer disease allele frequency (0.001) than the 0.01 that had been used in previous (separate) analyses of these data sets, in order to better match our expectation that the disease is highly genetically heterogeneous and so the population frequency of any particular causal genetic variant is likely to be small. Non-parametric linkage analysis was carried out using the equivalent LOD score to the Kong and Cox exponential model likelihood-ratio allele-sharing test27 with the Haplotype Reference Consortium (HRC) reference panel28 and pre-phasing performed via Eagle269. ‘Best-guess’ imputed genotypes were used, subject to the posterior probability of the genotype call exceeding 0.9. To ensure that only high-quality imputed SNPs were included in the final set, imputed SNPs were filtered to include only those with minor allele frequency >0.02 and imputation Rsq >0.8.To improve genome coverage, allowing interrogation of a denser set of SNPs, we carried out SNP imputation followed by TDT association, treating the Irish and UK/Slovenian cohorts separately (since they had very different numbers of QCed genotyped SNPs available to inform imputation). We used the Michigan imputation server70 and variant calling was performed with the GATK HaplotypeCaller algorithm following best practice recommendations73. Sequencing reads were examined via visualisation of the .bam files in IGV74.We whole-exome sequenced 29 individuals from the 8 families  showing the strongest evidence of linkage in the 10q26 region. Sequencing was carried out by AROS using Illumina’s Nextera Rapid Capture Exome Kit followed by 100 bp paired end sequencing (6 samples per flow cell lane) on the Illumina HiSeq 2000/2500 platform. Sequences were aligned to the Human GRCh37 assembly using BWA74.We performed targeted genomic resequencing of the implicated 9 Mb region of 10q26 (from 123 Mb-132 Mb) in 32 unrelated affected individuals from the families in our data set that contributed most to the linkage signal. Sequencing was carried out by Oxford Gene Technology (OGT) (whose next generation sequencing services have now been transferred to Source BioScience) using SureSelectXT Custom 9 Mb design and capture followed by 100 bp paired end sequencing (11 samples per flow cell lane) on the Illumina HiSeq 2000 platform. Sequences were again aligned to GRCh37 with BWA, variants were called with GATK HaplotypeCaller following best practice recommendations, and reads were visualised via IGV44 sequenced as part of the UK10K study45. We used the software package TASER47, which effectively re-calls variants in both cases and controls, allowing for systematic differences in read depth (and other factors), at the same time as constructing a test statistic. TASER uses the total number of reads mapped to a variant, and the number carrying the minor allele, to calculate a score statistic at each position in a gene (or window) of interest, thus providing an assessment of the effect of each individual variant on the disease phenotype. A burden statistic is then calculated for each window as the sum of the score statistics for each of the variants within that window, allowing identification of windows that have a higher or lower accumulation of rare variants in the cases than might be expected, compared to controls. This procedure therefore effectively looks for windows containing clusters of rare variants seen in different affected individuals (and not seen - or at least not at comparable frequencies - in controls).We analysed the targeted resequencing data from the 32 unrelated affected individuals together with whole-genome sequence data from 1106 ALSPAC controlsin vitro-propagated cultures of Normal Human Urothelial (NHU) cells were obtained from Fishwick et al.49 for the genes in the implicated chromosome 10q26 region (Supplementary Table μM troglitazone (TZ) as PPAR γ ligand with concurrent 1 μM PD153035 to block epidermal growth factor receptor activation. Change in expression of MK167 was used as marker of cell cycle activity.Data on gene expression by The data sets analysed and full sets of results obtained during the current study are available from the corresponding author on reasonable request.Supplementary Information


The Vanderbilt Holistic Face Processing Test (VHPT-F) is a new measure of holistic face processing with better psychometric properties relative to prior measures developed for group studies . In fiel People extract a wealth of socially relevant information from a single face, such as identity, emotional expression . The VHPT-F produces very large average effect size for holistic processing  than the composite task, and measures a stable trait , the composite task with bottom judgments (completed by 181 participants), the VET-bird , and the VHPT-F (completed by 174 participants).All participants were recruited in accordance with approval of Vanderbilt University Institutional Review Board. Out of a pool of 1000 participants who performed the VET-car on Amazon Mechanical Turk see , we sentIn total 166 subjects completed all tasks. We discarded data from 30 participants: 20 participants failed to follow instructions on the VHPT-F and did not view the study face for the entire 2 s on more than 25 trials; 8 participants failed 2/3 easy VHPT-F practice trials; 1 participant was an extreme univariate outlier on the VHPT-F (7 SD above the mean); finally, we screened for multivariate outliers in the correlation between holistic processing for top and bottom judgments in the composite task, and 1 participant had an externally studentized residual greater than 3. Therefore, data from 136 participants  are included in the analyses.The Vanderbilt Expertise Test was created as a battery of domain-specific tests to measure object recognition ability in different domains . We usedFigure 1A), a study composite face was shown for 2 s followed by a test display with three composite faces. Participants were instructed to select the composite face containing the target part with the same identity (but different image) as the target part in the study composite, while ignoring the rest of the face. The target part was outlined in red at study and test. The correct target part was paired with either the same distractor parts  or different distractor parts  relative to study . There were nine blocks of 20 trials, each with a different target part , for a total of 180 trials2. On 116 trials, face composites were created from grayscale male and female faces obtained from the internet. The faces used to create composites on a given trial were either all male or all female. On the remaining 64 trials, face composites were created from grayscale computer-generated faces. Both real and computer-generated trials were used to increase variability in trial types, which benefits reliability. Holistic processing scores are calculated using accuracy for congruent trials minus that for incongruent trials.On each trial (see 3 (° rotation4). The five face tops and bottoms were randomly combined to create composites (169 × 175 pixels). A white line four pixels thick separated face halves so it was unambiguous where the top half ended and the bottom half began.Because previous work found that the composite task was most reliable when a small set of stimuli (five tops and five bottoms) were used to create composites , composi3  presenteFigure 1B). The study and test composite faces were shown for 200 ms in the first 40 trials, and 100 ms in the second 40 trials. Presentation time was reduced in the second half of trials to add variability in trial difficulty, which is beneficial for test reliability. Otherwise, the trials were identical, with a mask for 500 ms (and a test image for 200 ms). Participants indicated if the target half (top or bottom) of the test composite was the same or different by clicking on the appropriate response option displayed on the screen. Each block of 40 trials included 10 trials for each combination of response (same/different) and congruency (congruent/incongruent).Each composite task (top and bottom judgments) included 80 trials  as a factor revealed significant holistic processing  across top and bottom composite task trials was r134 = 0.24. This value approaches the reliability of each congruency effect individually (see Table 1), which suggests a considerable amount of shared variance when the correlation is disattenuated . The correlation between holistic processing in the composite task and VHPT-F was r134 = 0.27 . These correlations were similar when only Caucasian subjects (n = 107) were included . Holistic processing in the VHPT-F was equally correlated with holistic processing in top and bottom composite task trials 5. There was no evidence for any relationship between car expertise measured by the VET-car and holistic processing in any task .Reliability for all tasks is shown in The composite task that is frequently used to measure holistic processing in group studies often has low internal consistency ∼0.2; , which lReliability is not a property of tasks but of measurements , and theDespite the differences in format, holistic processing measured in the VHPT-F was significantly correlated with holistic processing measured in the composite task, with the disattenuated correlation suggesting approximately 40% shared variance. Observing a correlation between the composite task and VHPT-F may seem obvious in retrospect, because there are many similarities between the tasks . However, the tasks differ in many important ways, such that if, we had not found a correlation, we could have pointed to several factors as explanation. For example, differences in task format  could have led to very different task strategies. Moreover congruency effects do not consistently correlate across various versions of the Stroop task  – if the6. This suggests that holistic processing measured in the VHPT-F and composite task is correlated to the extent that holistic processing correlates within the composite task itself, across top and bottom judgments. While some have proposed that holistic processing should only be measured with top part judgments to maximize the size of the effect , suggesting that they are not in fact measuring the same thing. However, a more optimal comparison would be to test the correlation between measures of holistic processing across participants. Thus, in Experiment 2, we included misaligned trials in the composite task so, we could assess the correlation between two different holistic processing measures in the composite task that have been widely used in group studies.In addition, the VHPT-F is based on the congruency measure of holistic processing. However, a large literature has used an alternative measure of holistic processing in the composite task, the alignment effect  are included in the analyses.The VHPT-F 2.1 was used in Experiment 2. This version is identical to version 2.0 used in Experiment 1, except trials that were programmed incorrectly were fixed. In addition, based on item analyses of data from 525 subjects, 16 trials that were not correlated with overall condition scores  were replaced or modified.The composite task was identical to Experiment 1, with the following exceptions. Stimuli were the top halves of five female faces and the bottom halves of five different male faces from the Max Planck Institute database that were converted to grayscale.On misaligned trials, the study face was aligned, and the top and bottom face halves of the test face were offset such that the edge of one half fell in the center of the other half. The target part was always the top face half. Because the alignment effect measure of holistic processing is calculated based on data from only a quarter of the total trials, we included more trials in Experiment 2 to increase the reliability of this measure. There were 40 trials for each combination congruent/incongruent, aligned/misaligned, and same/different for a total of 320 trials.Figure 3. A repeated measures ANOVA on VHPT-F accuracy with congruency (congruent/incongruent) as a factor revealed significant holistic processing . Surprisingly, however, neither composite task measure of holistic processing was significantly correlated with the VHPT-F .Reliability for all measures is shown in Given that one of the goals of Experiment 2 was to recruit a more diverse sample than Experiment 1, we tested whether differences in demographic variables between samples could explain the failure to replicate correlations between the composite task and VHPT-F measures of holistic processing.n = 47), we found significant correlations between holistic processing in the VHPT-F and both composite task measures that were comparable in magnitude to the correlations in Experiment 1 . These correlations were not significant for female participants . However, differences between correlations in male and female participants were not significant . The congruency effect and alignment effect in the composite task were significantly correlated in both male  and female  participants.In Experiment 1, we only tested male participants. When, we restricted our analyses in Experiment 2 to only male participants , with more older participants in Experiment 2. As can be seen in Table 3, age was only significantly correlated with the VHPT-F holistic processing measure. Notably, this effect was not present in Experiment 1 , and is larger than in a previous study  suggest a severe restriction in range for congruent trials in participants older than 45. When, we restricted our analyses to participants 45 years old or younger (n = 87), we found a significant correlation between the congruency effect measure of holistic processing in the composite task and the VHPT-F that is comparable in magnitude to that observed in Experiment 1 . In this restricted sample, the correlation between the congruency effect and alignment effect measures was r85 = 0.48 , and the correlation between the alignment effect measure and the VHPT-F was not significant . Mean-level effects for all subsamples  were qualitatively the same and quantitatively very similar as for the full sample (see Table 4).We also found significant differences in the age distribution between Experiment 1 and Experiment 2 . The samples differed in age and sex distributions, and when we restricted our sample to be more similar to Experiment 1, the results replicated. This suggests that there may be subject properties that mediate performance in these measures, particularly the VHPT-F. In a prior report, the VHPT-F showed a particularly large holistic processing effect in women older than 45 [relative to younger women and to men regardless of age  and the difference between them is not significant (s of age ]. Neithen = 101) that also measured the alignment effect  in the context of the composite design used to measure congruency effects (complete design) with different face stimuli and a larger set of face parts . This replicates findings in a study  it produces measurements that are more reliable than with large sets of face parts . For theC-CW and JR contributed to the design and study concept. All authors contributed to collect the data, the data analysis, and interpretation. JR drafted the early manuscript, and C-CW provided the revised version. DR and IG provided critical comments. IG replied to the reviewers.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.