To evaluate in a prospective study the possibility of visualization and diagnostic assessment of the recurrent motor branch (RMB) of the median nerve with high-resolution ultrasound (HRUS).HRUS with high-frequency probes (18–22 MhZ) was used to locate the RMB in eight fresh cadaveric hands. To verify correct identification, ink-marking and consecutive dissection were performed. Measurement of the RMB maximum transverse-diameter, an evaluation of the origin from the median nerve and its course in relation to the transverse carpal ligament, was performed in both hands of ten healthy volunteers (n = 20). Cases referred for HRUS examinations for suspected RMB lesions were also assessed.The RMB was clearly visible in all anatomical specimens and all volunteers. Dissection confirmed HRUS findings in all anatomical specimens. Mean RMB diameter in volunteers was 0.7 mm ± 0.1 . The RMB originated from the radial aspect in 11 (55%), central aspect in eight (40%) and ulnar aspect in one (5%) hand. Nineteen (95%) extraligamentous courses and one (5%) subligamentous course were detected. Three patients with visible RMB abnormalities on HRUS were identified.HRUS is able to reliably visualize the RMB, its variations and pathologies.Ultrasound allows visualization of the recurrent motor branch of the median nerve.• Ultrasound may help clinicians to assess patients with recurrent motor branch pathologies.• Patient management may become more appropriate and targeted therapy could be improved.• The online version of this article (doi:10.1007/s00330-016-4671-1) contains supplementary material, which is available to authorized users. The recurrent motor branch (RMB), sometimes also referred to as the muscular thenar branch of the median nerve, classically supplies innervation to the thenar musculature, including the abductor pollicis brevis, the opponens pollicis and the superficial head of the flexor pollicis brevis Fig.  1, 2]. , 2. 1, 2Damage to the RMB may lead to severely impaired function in patients, with loss of dexterity, pinch and grasp function. The main clinical relevance of the RMB is its susceptibility to iatrogenic injury, due to its variants, during decompression surgery for carpal tunnel syndrome (CTS) . This isIsolated RMB neuropathies are either rare or possibly underdiagnosed due to the lack of imaging modalities that can depict the nerve. Among these RMB neuropathies, anecdotal reports describe compression of the nerve due to schwannomas –15, gangTo date, evaluation and localization of the RMB has been restricted to electrophysiological assessment  and clinHigh-resolution ultrasound (HRUS), using linear, high-frequency probes, offers excellent tissue differentiation for the examination of superficial structures and may facilitate imaging of the RMB. As this has been described for the palmar cutaneous branch of the median nerve , which cTherefore, this study aimed to: (i) confirm the correct identification of the RMB by HRUS with ink-marking and consecutive dissection in anatomical specimens; (ii) provide the first measurements of RMB diameter, evaluating the origin, course and possible accessory branches in healthy volunteers; and (iii) present cases with RMB pathology found with HRUS.This prospective study was approved by the ethics committee of the Medical University of Vienna (EC-number 1529/2015) and was conducted between 1 February 2015 and 1 December 2015.HRUS examinations were performed using a GE LOGIQ e  ultrasound (US) platform with high-frequency probes . Two radiologists carried out all examinations. One had more than 20 years’ experience (G.B.) and one had 4 years’ experience (G.R.) in peripheral nerve imaging. Both raters were present during the collection of the subjects. G.B. performed all the interventions on all the anatomical specimens. G.R. collected all the images of healthy individuals, with G.R. watching the procedure.The examination followed a standardized assessment protocol that started with the transverse view of the median nerve or its digital cutaneous branches at the level of the metacarpal bodies III-IV. The probe was moved proximally until a tubular structure arising from the median nerve, in most cases curving radially and proximally and coursing toward the thenar musculature, was presumed to be the RMB. Subsequently, the origin was assessed by turning the probe until the longitudinal axis of the nerve was visible. In most cases, the RMB formed an approximately 45° angle with the median nerve. Subsequently, the nerve was followed until its entrance into the thenar musculature. To avoid confusion with the palmar cutaneous branch or the palmar digital branch of the median nerve, the RMB had to enter the thenar musculature in contrast to the other branches. To avoid confusion with vessels, colour Doppler was used. Probe positioning, probe track and measurement of the RMB diameter is presented in Fig. A second possibility to locate the RMB (considered by the authors to be more difficult) was to start with the transverse view of the median nerve 3 cm proximal to the pisiform bone. Following the median nerve, the probe was moved distally until its subdivision into terminal branches. At this level, the probe was moved proximally and distally to identify the RMB. After identification, the assessment of the nerve was performed in a manner similar to that described above.In four randomly selected fresh anatomical specimens in the legal custody of the Department of Systematic Anatomy, Medical University of Vienna, HRUS was performed as described above in both wrists (n = 8). After locating the RMB, a small amount of blue dye mixed with glue (0.1 ml) was injected into the nerve/adjacent to the nerve under HRUS guidance . Subsequent anatomical dissection was performed to confirm the exact location of the dye injection. A plastic surgeon (M.V.) and anatomist (P.B.) who performed the dissections determined the exact location of the dye injection. Correct dye injection was noted if at least some amount of the dye was injected into the nerve sheath.Ten healthy volunteers were recruited via notices at the Department of Biomedical Imaging and Image-guided Therapy and word-of-mouth acquisition. Written informed consent was obtained from all volunteers. Inclusion criteria were age over 18 years, and exclusion criteria were known polyneuropathy, known myopathy, chronic disease known to cause peripheral neuropathy, current or previous CTS and previous hand surgery.The RMB was assessed on both sides (n = 20). Measurements of the maximum transverse diameter were obtained immediately after the separation from the median nerve using the platform software of LOGIQ e. The origin of the RMB with respect to the median nerve was assessed according to Mackinnon and Dellon (4). The site of origin of the RMB from the median nerve was classified as either ulnar, central-volar or radial, since the intermediate type  described by Mackinnon and Dellon  is not cThe course of the RMB in relation to the transverse carpal ligament (TCL) was evaluated using the classification of Lanz : extraliBetween 1 February 2015 and 1 December 2015, we monitored patients who were referred to the Department of Biomedical Imaging and Image-guided Therapy, and in whom RMB pathologies were detected with HRUS. The referring diagnosis of all screened patients was clinically or electrophysiologically diagnosed carpal tunnel syndrome. All patients were referred to our department for preoperative evaluation prior to carpal tunnel surgery. HRUS examinations were performed using the same assessment protocol as described above.Descriptive statistics were performed using IBM SPSS Statistics for Windows Version 22.0.0.2 . Metric data (nerve diameter) are presented as mean ± standard deviation and range (minimum–maximum).The RMB was clearly visible in all anatomical specimens. Dissection confirmed the correct identification of the RMB (100%) on both sites in all subjects (n = 8). An example of a dissection finding is shown in Fig. Table The mean transverse diameter was 0.7 mm ± 0.1 . The maximum detectable intraindividual side difference was 0.03 mm.The RMB originated from the radial aspect in 11 hands (55%), the central aspect in eight hands (40%), and the ulnar aspect in one hand (5%).An extraligamentous course was seen in 19 hands (95%), and a subligamentous course in one hand (5%). No transligamentous course was observed. One accessory branch arising from the radial aspect of the median nerve, with a maximum transverse diameter of 0.6 mm, was detected Fig. .Fig. 4ExOf 189 patients with carpal tunnel syndrome, three patients (1.6%) with RMB pathologies were identified and are presented below. Figure 2 within/distal to the carpal tunnel . The clinically visible atrophy was also documented with US  of thumb abduction, thenar wasting and a mild hypoesthesia of the first digit. Motor conduction studies showed severe axonal damage of motor fibres on the left  and prolongation of distal motor latency, while sensory testing revealed a preserved sensory nerve action potential with a only slightly reduced amplitude of 12 μV and a decreased antidromic conduction velocity of 44 m/s. Sonographic assessment revealed a radially originating, extraligamentously coursing and severely thickened RMB (1.5 mm vs. 0.9 mm on the right). Further, the whole median nerve also exhibited an increased cross-sectional area of 0.23 cm2  of the median nerve. Further, atrophy of the thenar musculature was assessed  and mild paresis of thumb abduction (4/5). Atrophy of the lateral thenar and the opponens pollicis muscle was visible. Sonographic assessment revealed a radially originating, extraligamentously coursing, moderately thickened RMB (1.3 mm) and a normal cross-sectional area of 0.10 cm and mildA 45-year-old female presented with a severe paresis of the thenar musculature on her right side for the 6 months prior to presentation. No paraesthesias or pain were reported. Clinical examination revealed plegia of thumb abduction (0/5). A clear thenar atrophy was visible. Sonographic assessment revealed a radially originating, extraligamentously coursing and moderately thickened RMB (1.3 mm). Moreover, the radial-sided motor fascicles within the median nerve were clearly swollen. Further, atrophy of the thenar muscles was assessed . Subsequently, a broad variation in point of origin and course was observed.Ultrasound measurements of the transverse diameter of the RMB in healthy volunteers revealed clearly lower values than previously described in anatomical studies. Wang and colleagues  reportedIn addition to the depiction of the RMB, HRUS provides further information about the course, origin and branching of this nerve. The course of the RMB in relation to the TCL has gained extensive attention in various studies, due to the fact that the ‘anomalous’ trans- and subligamentous variants, in particular, are at risk during both open and endoscopic surgery , 8, 32. The origin from the radial aspect of the median nerve was described as a possible site of entrapment because of separate obliquely arranged fibres from the TCL encircling the RMB . As the Iatrogenic RMB injury during CT release seems to be a rare complication, with approximately 0.5% in the reported literature , 32. NevIn 1982, Bennet and Crouch  reportedin vivo were uncontrolled. However, accuracy in anatomical specimens was 100% and RMB in volunteers could be followed into the abductor muscle. A further limitation is the fact that pathological findings in this manuscript are case reports and do not provide reliable information about the future role of HRUS in RMB pathology detection. For this reason, further comparative studies between normal and pathological conditions are needed.This study has several strengths and limitations. Its strengths include the first-time use of HRUS for specific assessment of the RMB and confirmation of the findings by the gold standard of anatomical dissection. Its limitations include the fact that findings In conclusion, this study confirms the reliable ability to visualize the RMB and its variations with HRUS, in anatomical specimens and in healthy volunteers. We therefore encourage the use of HRUS, especially for preoperative evaluation for carpal tunnel release or if thenar muscle weakening is present. Further studies are needed to assess the value of HRUS in diagnosing RMB pathologies.MovieAnatomical variant. Video sequence of sonographic findings in a volunteer with a recurrent motor branch originating from the ulnar aspect of the median nerve, crossing the anterior aspect of the median and coursing beneath the TCL (subligamentous) toward the thenar musculature (MP4 40229 kb)Below is the link to the electronic supplementary material.


The type I clathrate framework can be described as being composed of two types of polyhedral cages made up of tetrahedrally coordinated Si: pentagonal dodecahedra with 20 atoms and tetrakaidecahedra with 24 atoms in the ratio of 2:6. The cation sites, A, are found in the center of each polyhedral cage. This review focuses on the newest discoveries in the group 13-silicon type I clathrate family: A8E8Si38  and their properties. Possible approaches to new phases based on earth abundant elements and their potential applications will be discussed.Earth abundant element clathrate phases are of interest for a number of applications ranging from photovoltaics to thermoelectrics. Silicon-containing type I clathrate is a framework structure with the stoichiometry A Inorganic clathrate phases which can be described as all-inorganic frameworks with guest atoms have been of interest for a wide variety of applications, including thermoelectrics, Li-ion batteries, hydrogen storage, and photovoltaics . In part8-xSi46 is the first observed silicon-based compound discovered to crystalize in the type I clathrate structure. The structure can be described as having a covalently bonded silicon (Si) framework with sodium (Na) as the guest atom centered within the polyhedra [c, 16i, and 24k. The eight guest atoms in the unit cell are located at the Wyckoff positions 2a and 6d, which centers the two types of polyhedra found in type I clathrate: 20-vertex pentagonal dodecahedra and 24-vertex tetrakaidecahedra. Following the discovery of Na8-xSi46, the alkali metals series  of type I clathrate were synthesized from the elements by annealing, with the Cs clathrate being a high pressure phase [Naolyhedra . The strre phase . The alk8E8Si38  are considered to be electron-balanced phases as the compounds contain an equivalent amount of group 13 elements to accept the donated electrons from guest atoms and were predicted to show semiconducting behavior [8E8Si38  were calculated using density functional theory [c framework site with a small amount on the 24k site in the ratio of approximately 5:3, when A = K, Rb, Cs. This is consistent with experimental observations for these alkali metal clathrates [The band structures of the series Al theory ,26. The l theory . Site spl theory . Figure athrates ,11,13.8Al8Si38 showing a quasi-direct bandgap of ~ 1 eV with relatively high mobilities determined [8Ga8Si38 [Theoretical calculations suggest that these phases should have applications in photovoltaics with Ktermined . Additio8Ga8Si38 . Overall8Ga8Si38 .7.62(1)Si46 and Rb6.15(2)Si46 to examine their crystal structure [8Si46 [c site [Powder X-ray diffraction was performed on the binary type I clathrate phases Ke [8Si46 . Fully o [c site ,31,32,338E8Si38, A = Na, K, R, Cs and E = Al and Ga have been prepared to date and their lattice parameters are provided in c, 16i, 24k) can affect the band structure and transport properties significantly. In the A8E8Si38 framework, Ga occupancy can be distinguished by single-crystal X-ray diffraction as Ga and Si atoms have electron densities that provide significantly different scattering factors. For A = K, Rb, Cs compounds, the Ga occupancy ratio at the 6c site is in the range of 61%~46% and 17%~20% at the 24k site. At the 16i site, the Ga occupancy ratio is low, only 1%~2%, which indicates Ga preference for the 6c site  is difficult, so neutron powder diffraction was performed to study the Al/Si mixed occupancy ratios [s.o.f.) is shown in s.o.f. The group 13 atoms’ preference to occupy 6c site is attributed to the fact that there is no direct bonding between 6c sites  of a mixture of NaSi [8Al8Si38 . Rietveld and single-crystal refinements indicate that Al preferentially resides at the 6c site in the ratio Al:Si 0.79:0.21. The 24k site to a significantly lesser extent with the ratio Al:Si 0.13:0.87 and no evidence for Al on the 16i site. This result is similar to what has been determined for A8Al8Si38.Na of NaSi  and NaAl of NaSi . This ph7B7Si39, where B is a much smaller atom than Si, B is found to substitute 16i sites by single-crystal X-ray diffraction refinement, while only Si is found at 6c and 24k sites [8E8Si38 . In c and 24k sites, has bond angles deviating from an ideal tetrahedron and therefore creates chemical stress if substituted by the small B atom. Therefore, B atoms have a preference for the 16i site [In the case of K4k sites . This is16i site .a inside the 20-vertex pentagonal dodecahedral cages and 6d inside the 24-vertex tetrakaidecahedral cages. Large anisotropic atomic displacement parameters (ADPs) have been reported for the 6d sites guest atoms, and therefore they are considered as rattler and contribute to phonon scattering. For compounds K7.62(1)Si46 and Rb6.15(2)Si46, when guest atom vacancy is present, occupancy at 6d site is preferred and 2a site has lower s.o.f, 88.5(4)% at K7.62(1)Si46 2a site and 21.8(5)% at Rb6.15(2)Si46 2a site [7.8(1)Si46, s.o.f of 2a site is 0.881(4) while 6d site is close to fully occupied [In the type I clathrate crystal structure, the guest atom Wyckoff nomenclature is 28AlxSi46-x, since the amount of Al can vary from x ~ 8–15. Neutron powder diffraction at 35 K of Ba8AlxSi46-x showed that when Al composition increases from 8 to 15, the anisotropic ADPs behave differently as presented in 11 and U33 are the two thermal displacement parameters parallel to the six-membered ring in the E24 cage, and U22 is direction perpendicular to six-membered ring. As the Al composition increases and the cage volume increases as well, U22 changed from a value larger than U11, U33 to a value smaller than those two. As shown in d site in the case of A8Ga8Si38 and A8Ga8Si38 : the alkali metals at 6d sites behave similarly with those of Ba8Al15Si31 instead of Ba8Al8Si38, with the larger U elongated in the directions parallel to the six-membered ring  [Although this review is concerned with the alkali metal guest group 13 Si clathrate type I phases, it is informative to review the guest ADPs for Ba11, U33) .8AlxSi46-x, the composition for A8Al8Si38 appears to be fixed [8-xBaxAl8+xSi38-x was investigated using metal hydrides for the alkali and alkaline earth metals and Al/Si arc melted mixtures heated together. The nominal compositions of x = 1.0, 1.5, and 2.0 were investigated. The structures were investigated for each composition with synchrotron radiation at 295 and 100 K. The 2a site with the smaller volume polyhedron was mostly substituted by Ba2+ rather than K+ guest atoms, consistent with the smaller ionic radius of Ba2+ [d site, the guest atom in the 6d site shows a larger displacement than the direction perpendicular to the six-membered ring of the 24-vertex cage. This is also observed for the Zn-containing phases [Unlike Babe fixed ,37, althbe fixed ,38. Therg phases .8Ga8Si38 were first calculated using density-functional theory [8E8Si38 for E = Al, Ga, In was also calculated and showed a slight increase in bandgap with decreasing mass of E. Overall, the gaps were calculated to be 0.45 eV for Na8Ga8Si38 to 0.89 for Cs8Ga8Si38 while Eg value of Si with the diamond structure is calculated to be 0.65 eV. Since the experimental value of Eg of Si is 1.12 eV, these calculated values are adjusted according, corresponding to 0.92 and 1.36 eV for Na8Ga8Si38 and Cs8Ga8Si38, respectively. The experimental determination was for K8Ga8Si38 and the optical bandgap was estimated to be indirect at 1.2 eV [8Al8Si38 was found to be larger than 1 eV [8Ga8Si38 follow the predicted trend, but are larger than the original calculations [8Al8Si38 and A8Ga8Si38  were determined from surface photovoltage spectroscopy [8Al8Si38 indicated an optical bandgap of 0.64 eV [The band structures of Al theory ,26 and dt 1.2 eV . With a han 1 eV . The expulations . The valtroscopy ,42 indictroscopy . Na8Al8S8Ga8Si38 is predicted to provide a zT of 0.75 at 900 K. The figure of merit is a dimensionless parameter that provides a way to distinguish a favorable material for high performance as it combines the following thermoelectric parameters: Seebeck coefficient, resistivity, and thermal conductivity for a specific absolute temperature. 8Al8Si38, it is apparent from the reported results that the precise stoichiometry greatly affects the electrical resistivity, as can be seen in 8Al7Si39 [There has been significant interest and recent progress in the measurement of thermoelectric properties of these clathrates. Theoretical calculations indicated that that the dimensionless figure of merit, zT, for optimally doped compounds can reach 0.5 at 600 K. Hole-doped Cs8Al8Si38 phase showed the most promise with ease of synthesis, further optimization of that phase was considered. As indicated above, one method to optimize the transport properties is to focus on the Al-Si ratio and K8Al7Si39 shows the promise of this approach. Another method for decreasing the resistivity of the stoichiometric A8E8Si38 samples is to attempt to control the electron donation to the framework with mixed cations. The mixed alkali metal-alkaline earth Al-Si clathrate phases, K8-xBaxAl8+xSi38-x  were prepared and their thermoelectric properties measured. The K8Al8Si38 used in this study [8Al8Si38 phase was stable up to 900 K , high temperature spark plasma sintering of larger pellets provided samples with lower densities thereby making it difficult to obtain reliable experimental measurements of thermal conductivity. Pressed pellets of K8-xBaxAl8+xSi38-x  were prepared and the Seebeck coefficient and electrical resistivity measured  and kinetically controlled thermal decomposition (KCTD) reactions with NaAlSi and NaSi mixture [8AlxSi46-x compositions were investigated, but while the diffraction peaks broadened, there was no direct evidence for a composition other than x ~ 8. −6 torr) at 953 K. During this reaction, the Na is removed in a chemically controlled manner from the NaAlSi/NaSi mixture via interaction with a graphite flake which is spatially separated from the mixture via NaCl. The NaCl stops a direct reaction of the Na with the graphite, so it is presumed to be a vapor-phase interaction of Na with the graphite flake. This procedure has been described in detail for the synthesis of Na-Si clathrate phases [Na mixture . This roe phases . The pro8AlxSi46-x has also been investigated employing a mixture of NaSi, Al and Si as reagents, but only x~0.5 was prepared at 5.5 GPa and 1570 K [8Al8Si38 was reported to be synthesized from KH and arc-melted Al/Si, sealed in a Ta tube jacketed in fused silica at 973 K. Ball milling KH and Al/Si together subsequent to heating enhanced the sample purity [8-xBaxAl8+xSi38-x could be prepared from ball milling BaH2 with KH and arc-melted Al/Si in the same manner as described above [8Ga8Si38  can be prepared from the pure elements reaction in a Ta tube jacketed in a fused silica ampoule at 973 K [8Cd4Sn42, phase-pure samples could be prepared by simple heating and annealing in a sealed metal tube [8Al8Si38  can also be prepared from Al and the respective alkali-metal halide salt flux, as reported by Baran et al. Similarly, Cs8-xGa8-ySi38+y was synthesized from a stoichiometric reaction of the elements in an alkali metal halide flux, while K8Zn3.5Si42.5 and Rb7.9Zn3.6Si42.4 were synthesized using a combined alkali metal halide/zinc flux [High pressure synthesis of Nad 1570 K . This loe purity . K8-xBaxed above . While tat 973 K . Excess tal tube . It is pinc flux ,17. Exceinc flux  or Ge clinc flux ,17. TherzT, suggesting that further improvements are possible. Controlling the guest atoms by means of solid solutions of K and Ba have been shown to be a productive way to systematically change the n-type carrier concentration. Further investigations of Si-Ge, Si-Sn, and more complex solid solutions  of these phases should be investigated with guidance from theory. The solid solutions of Zn(Cd)-E  might provide a way to access hole-doped (p-type) clathrates. This is a rich area of chemistry, and progress towards other clathrate structures along with both guest and framework substitutions, provides a rich playground for new materials with technologically relevant properties.New synthetic processes to prepare single-phase clathrates of all earth abundant and non-toxic elements have provided new clathrate type I phases that have potential for applications in energy storage and energy conversion. Metal hydrides, salt fluxes, and kinetically controlled decomposition of ternary phases provide methods for precise control over composition. All of the compositions discussed above show reasonable bandgaps for photovoltaics and low thermal conductivity, important to thermoelectrics. Controlling the carrier concentration by alkali metal-alkaline earth metal solid solutions resulted in a reasonable


We describe the development of a CAM4712, a novel CK2α inhibitor which does not interact with the ATP binding site and shows improved properties over the first-generation inhibitor CAM4066. CAM4066. Whilst CAM4066 bound to this new pocket it was also interacting with the ATP site: herein, we describe an example of a CK2α inhibitor that binds completely outside the active site. This second generation αD-site binding inhibitor, compound CAM4712 , has numerous advantages over the previously reported CAM4066, including a reduction in the number of rotatable bonds, the absence of amide groups susceptible to the action of proteases and improved cellular permeability. Unlike with CAM4066, there was no need to facilitate cellular uptake by making a prodrug. Moreover, CAM4712 displayed no drop off between its ability to inhibit the kinase in vitro (IC50) and the ability to inhibit cell proliferation (GI50).CK2 is a critical cell cycle regulator that also promotes various anti-apoptotic mechanisms. Development of ATP-non-competitive inhibitors of CK2 is a very attractive strategy considering that the ATP binding site is highly conserved among other kinases. We have previously utilised a pocket outside the active site to develop a novel CK2 inhibitor,  In vivo it exists mainly as a holoenzyme composed of two catalytic (α and/or α′) and a dimer of regulatory (β) subunits, but it can also be found as the isolated subunits.CK2 is a serine/threonine kinase that is a key regulator of many cellular processes and is involved in cellular proliferation and anti-apoptotic mechanisms.1CX4945 (known as silmitasertib), the first in class CK2 inhibitor currently in phase II clinical trials.50 of CX4945 against CK2 is 1 nM but it also inhibits 12 other kinases with nanomolar affinity and it is more potent against Clk2 than against CK2.Like the majority of kinase inhibitors, most of the known CK2 inhibitors target the ATP binding site, presenting the issue of poor selectivity over other kinases.CAM4066 , shown in Previously we have reported our preliminary studies on the exploration of the αD pocket, based on the development of primary hits from the crystallographic screen, which led to the identification of compound 1 ; the 2-methyl derivative 2 was promising with a Kd of 41 μM. The introduction of an ethyl group in the same position (3) resulted in the highest affinity (Kd = 17 μM) and highest ligand efficiency (LE = 0.39) of the series and the crystal structure  showed how the lateral chain grows towards the water channel, displacing the first water molecule on the left (Kd = 205 μM) nor the LE of the isopropyl derivative 4. The 2-MeO and 2-F derivatives, 5 and 6 respectively, had a comparable Kd to 1 while the 2-OH group (compound 7) was detrimental to binding even though it formed a hydrogen bond to the conserved water . Substitution in the 3 position of ring A (compounds 8 to 10) appeared not to be tolerated as only the fluoro-derivative 9 yielded a co-crystal structures. The disubstituted 2-Me-4-F derivative 11 was found to have a Kd of 105 μM, marginally higher than the 2-methyl compound 2, but with a lower ligand efficiency (LE = 0.33). Expanding ring A to a naphthyl group 12 improved the affinity only slightly, while the indolyl derivative 13 did not show any significant binding and we were unable obtain structures of these in complex with CK2α. Therefore, compound 3 represented the fragment with the best LE and it was chosen as the best optimized fragment as far as the ring A is concerned.Substitution in the 2 position of ring A was beneficial when compared to the reference compound the left . The prethe left . Expandi1 (14) was synthesized to fulfil the interactions that both of these chlorine positions made. 14 showed an improved Kd of 12 μM with the highest LE of 0.43 (5OTR) where 14 (in blue) is overlapped with the two binding poses of 1 (green). The final compound 15, merging both the dichloro functionality on ring B and the 2-ethyl group on ring A, was synthesized and showed an improved Kd of 7 μM (LE = 0.41) with its binding mode overlapping well with that of the merged fragments (5OTZ). Although compounds 3 and 15 showed a comparable LE (0.39 vs. 0.41 respectively), compound 15 was chosen as hit to bring forward due to its ability to occupy a larger part of the αD pocket . In order to pick up additional H-bond interactions the pyrrole derivative 19 was chosen over 16 and we hypothesized that the heterocycle should go further up into the channel. Whilst the imidazole derivative 20 did not show significant improvement, the benzimidazole derivative 22 was found to be the most potent compound with an IC50 of 58 μM. 21 and 22 with CK2α, respectively. As expected, Met163 flips upon binding of the more extended compounds compared to the co-crystal structure of 1 , with both electron withdrawing and donating groups in position α and β of the benzimidazole improving the inhibition activity in respect to the derivative 23. The α and β methoxy derivatives 26 and 29 were found to be the most promising in the respective α and β substituted series with IC50 of 8 and 7 μM, respectively  (23 (compounds 31–34) was investigated in order to improve the moderate solubility of the related CAM4712 in water. Unfortunately, compounds 31–34 resulted in loss of activity and were therefore not pursued further.Alternative substitutions around the middle ring were also investigated and compared to compound ctively) . Substit35 and 36 were designed via a merging strategy so that they contained the most promising substitution patterns. The methoxy derivatives 35 and 36 were synthesized and tested but, disappointingly, gave worse results than CAM4712 . This data validated our fragment merging strategy to improve upon both affinity and inhibition. Unfortunately, as with the other compounds in the series, once the benzimidazole group had been added CAM4712 was not soluble enough for ITC. Therefore, an ITC competition study was performed to confirm the binding mode and to estimate the affinity of CAM4712 for the αD site  Inhibition of binding in the αD or ATP site by CAM4066 to CK2α was inhibited by 20 μM CAM4712. From this the affinity of CAM4712 was estimated to be 4.0 μM  Inhibition of binding in the αD site:15 was titrated into CK2α in the presence of 20 μM CAM4712  Inhibition of binding to Lys68:37) binds to the conserved Lys68 in the ATP site and occupies the right-hand side of the pocket . The binding of compound 37 was not inhibited by CAM4712 . This result predicted that it would be possible to generate a crystal structure of CAM4712 and 37 bound simultaneously to CK2α and this was confirmed by a crystal structure showing both compounds binding simultaneously to CK2α .2-Hydroxyl, 5-methyl benzoic acid  Inhibition of binding in the ATP site/hinge region:CX4945, which from the analyses of crystal structures would clash with CAM4712 in the hinge region, was titrated into CK2α in the presence of CAM4712 . Whilst the binding of both compounds to CK2 resulted in the flipping of the Met163, CAM4712 did not H-bond the conserved Lys68 in the ATP binding site. Instead, a low-energy hydrophobic π–π interaction between the His160 and the benzimidazole was introduced . It is also similar to pro-CAM4066 ,CAM4066, which had to be administered as the prodrug pro-CAM4066. The target engagement by CAM4712 was analysed by following the CK2α dependent phosphorylation of Ser129 of Akt1. This showed good inhibition of the phosphorylation of Ser129 by CAM4712 as well as by its close analogues 23 and 26 which confirms that these compounds inhibit CK2α in the cellular environment (The efficacy of ironment .CAM4712 showed a 10-fold decrease in potency compared to CAM4066 and therefore the selectivity of CAM4712 was screened against a panel of 140 kinases at a concentration of 30 μM (4 × IC50). CAM4712 showed good selectivity against the 20 closely related CMGC kinases in the panel .CAM4712 being less potent than CAM4066. Nevertheless, CAM4712 showed a more selective profile than other CK2α inhibitors  and target engagement was demonstrated. This second generation of αD pocket inhibitor overcomes the limitations of our first inhibitor, including the fact that it does not need to be administered as a pro-drug to exert anti-proliferative activity. We have also shown that it is not necessary to interact with the ATP site directly, but effective inhibition of the kinase and displacement of ATP can be achieved by blocking the mouth of the ATP site with no need to interact with conserved features of the ATP binding site. These results demonstrate an entirely new approach to CK2α inhibition and will allow the future development of drug-like molecules, lead compounds and chemical tools that utilise the novel properties of the αD site.In conclusion, we have developed a series of second-generation CK2α inhibitors that target the αD site. This was achieved by first optimising the fragments that bound in the αD site, followed by identification of groups that grow towards the mouth of the ATP site to provide potent inhibitors of CK2α. In our previous work we demonstrated that selectivity could be achieved anchoring the inhibitors in the αD pocket and with this work we achieve inhibition with ligands that do not target the active site. There are no conflicts to declare.Supplementary informationClick here for additional data file.


Mycobacterium tuberculosis complex (MTBC), with human disease resulting from infection by M. tuberculosis sensu stricto and M. africanum. Recent progress in genotyping techniques, in particular the increasing availability of whole genome sequence data, has revealed previously under appreciated levels of genetic diversity within the MTBC. Several studies have shown that this genetic diversity may translate into differences in TB transmission, clinical manifestations of disease, and host immune responses. This suggests the existence of MTBC genotype‐dependent host–pathogen interactions which may influence the outcome of infection and progression of disease. In this review, we highlight the studies demonstrating differences in innate and adaptive immunological outcomes consequent on MTBC genetic diversity, and discuss how these differences in immune response might influence the development of TB vaccines, diagnostics and new therapies.In 2015, there were an estimated 10.4 million new cases of tuberculosis (TB) globally, making it one of the leading causes of death due to an infectious disease. TB is caused by members of the  Mycobacterium tuberculosis complex (MTBC), is a chronic disease that affects humans and animals M. tuberculosis sensu stricto (Mtb) and M. africanumM. bovis, a pathogen of cattle and badgers M. microti, found in rodents M. pinnipedii infects sea lions M. caprae in goats M. orygis which infects wild buck ; M. mungi, which was isolated in mongoose populations M. suricattae in meerkats , caused by members of the ats Fig. . ZoonotiWith the development of genotyping techniques a phylogeographic genetic structure for the MTBC began to emerge Differences in virulence between MTBC strains were reported as early as the 1960s, with the observation that MTBC isolated in India were attenuated in the guinea pig model when compared with those isolated in the UK Evidence of significant differences in the transmission and manifestations of disease of MTBC lineages worldwide  are involved in the initial interaction between MTBC and host cells, including toll like receptors (TLRs), C‐type lectin receptors (CLRs), nod like receptors (NLRs) and scavenger proteins C‐type lectins are one important class of receptor that function in innate immunity to TB. Alveolar macrophages highly express Mannose‐receptor , while dendritic cell specific intercellular‐adhesion‐molecule‐3 grabbing non‐integrin  is mostly highly expressed on dendritic cells (DC) M. canettii have also been shown to differentially affect TLR2 and TLR4 expression on macrophages. Human MDMs produced higher levels of TNF‐α and IL‐10, and expressed lower levels of TLR2 and TLR4 when stimulated with lipid fractions from a lineage 2 strain when compared with H37Rv and M. canettiiet al. (2013) also demonstrated that activation of TLR4 was not specific to lineage 2 strains, with the Mtb Haarlem sub lineage strain, a member of lineage 4, also able to activate TLR4 with concomittant production of IFN‐β Toll‐like receptors (TLRs) are another set of PRRs that are important for sensing MTBC infection An interaction between host and bacterial genotype has been observed with respect to TLRs and susceptibility to disseminated TB disease M. africanum lineage but not strains from Mtb sensu stricto lineages. Furthermore, M. africanum strains were better able bind to wild‐type MBL than the H37Rv Mtb strain in vitro, suggesting that M. africanum uses this receptor as an entry ligand thereby promoting infection. This potentially explains why polymorphisms in MBL are protective against M. africanum but not Mtb sensu stricto Mannose Binding Lectin (MBL), a member of the collectin family, acts as an opsonin by binding pathogen cell surface carbohydrates These studies highlight the need to take human genetic variation into account when considering differential immunological outcomes associated with MTBC strain variation. Studies investigating association between polymorphism and human susceptibility to TB disease have yielded discrepent results  T cell response. CD4+ T cells, suggesting expansion of regulatory T cells + T cells had accumulated by 14 days, however, at 28 days, these cells began to decrease in the lungs of mice infected with the K strain compared to the H37Rv and H37Ra laboratory strains. Moreover, CD4+ T cells expressing RORγt, thought to represent a Th17 subset, were not present in the lungs of mice infected with the K strain while they were present in appreciable numbers in mice infected with laboratory strains, H37Rv and H37Ra. Similarly to the guinea pig study discussed above, higher numbers of CD4+CD25+Foxp3+CD223+IL‐10+ T‐regulatory (Treg) cells were observed in K Mtb strain infected mice when compared with H37Rv or H37Ra strains Characterization of the adaptive response to whole cell MTBC has revealed differences in the T cell response to different lineages. A low dose aerosol guinea pig model of infection was applied to examine differences in virulence of a set of lineage 2 MTBC isolates selected for their differences in transmission M. bovis, or PBMC from humans infected with Mtb, revealed that both the proportion of responders and the breadth of response, as measured by IFN‐γ production, were positively correlated with sequence conservation Unlike many bacterial pathogens, several genomics studies have revealed that MTBC T cell epitopes appear to be genetically conserved The translational implications of MTBC lineage variation is perhaps most apparent for the design of effective new TB vaccines. Escape from BCG vaccination is one of the factors proposed to have driven the wide geographic distribution of the lineage 2 genotype In terms of new vaccines, the recombinant BCG candidate (VPM1002) expressing listeriolysin but defective in urease production, was shown to confer better protection than standard BCG in BALB/c mice, hypothesized as a result of increased antigen translocation into the cytoplasm of infected macrophages and resultant antigen cross‐priming Streptococcus pneumoniae, T cell epitopes are evolutionarily conserved As previously discussed, several comparative genomics studies have shown that in MTBC, unlike other bacteria such as M. africanum infected individuals has been shown to affect the interpretation of IGRA results M. africanum and Mtb‐infected patients pre‐treatment. However, the kinetics of production of these cytokines following treatment was significantly different between the two groups, with IFN‐γ and GM‐CSF significantly higher in Mtb compared with M. africanum infected patients post‐treatment M. africanum is prevalent.Currently available tools for detection of MTBC infection rely on immunological sensitization and thus could theoretically be influenced by MTBC lineage associated immunological variation. The TST has traditionally been used detect latent TB infection (LTBI). The test, which requires subcutaneous injection of purified protein derivative (PPD) cannot differentiate MTBC infection from BCG vaccination or exposure to environmental non‐tuberculous mycobacteria (NTMs), and thus may yield false positive results ALOX5, a key mediator of the type 1 IFN response to TB infection, is associated with protection against TB caused by M. africanum strains but not Mtb senstu strictoMTBC lineage diversity may also constitute an important factor to consider in the development of host‐directed therapies (HDT). Some HDT aim to augment the host defense against TB by modulating host pathways involved in the elimination of MTBC Due to dysregulated host immunity, it has been suggested that HIV‐1 co‐infection may facilitate the survival of less “fit” MTBC lineages within a population pks1‐15 gene cluster resulting in production of a virulence associated phenolic glycolipid pks1‐15 deletion strain, which no longer produces the phenolic glycolipid, was similar to levels observed in PBMC co‐infected with the CDC1551 strain. This suggests that strain specific effects on HIV‐1 replication are associated with cell wall components of MTBC MTBC creates an inflammatory milieu that exacerbates HIV‐1 infection and increases viral replication 18F]‐fluoro‐D‐glucose (FDG) positron emission tomography/computed tomography (PET/CT) The studies discussed above indicate that genetic diversity within the MTBC can influence the host response to infection and during TB disease. However, the use of in vitro systems and animal models that do not completely recapitulate human TB makes it difficult to assess whether immunological variation associated with MTBC genetic diversity appreciably impacts clinical outcome, or whether TB control approaches should be tailored according to the infecting MTBC strain. The use of techniques such 2‐deoxy‐2‐[In addition to technical considerations associated with the immunological readout, many studies have characterized only a few representatives of a lineage, in many cases those that have been associated with well‐known outbreaks. Significant intra‐lineage diversity exists In terms of childhood TB, studies suggest that associations between particular MTBC genotypes and TB in children is likely to reflect ongoing transmission from adults Finally, definitively establishing genotype–phenotype relationships may benefit from allelic exchange type experiments that facilitate an understanding of cause and effect between a polymorphism and a particular phenotype The aforementioned caveats notwithstanding, the studies discussed here have uncovered important variation in innate and adaptive immune responses to different MTBC strains, that warrants further investigation. The use of new immunological, clinical, and genomics techniques provides the opportunity to pursue and throw greater light on their significant in tuberculosis response.The authors declare no financial or commercial conflict of interest.


As a key persistent component of the atmospheric dynamics, the North Atlantic blocking activity has been linked to extreme climatic phenomena in the European sector. It has also been linked to Atlantic multidecadal ocean variability, but its potential links to rapid oceanic changes have not been investigated. Using a global ocean-sea ice model forced with atmospheric reanalysis data, here it is shown that the 1962–1966 period of enhanced blocking activity over Greenland resulted in anomalous sea ice accumulation in the Arctic and ended with a sea ice flush from the Arctic into the North Atlantic Ocean through Fram Strait. This event induced a significant decrease of Labrador Sea water surface salinity and an abrupt weakening of the Atlantic Meridional Overturning Circulation (AMOC) during the 1970s. These results have implications for the prediction of rapid AMOC changes and indicate that an important part of the atmosphere-ocean dynamics at mid- and high latitudes requires a proper representation of the Fram Strait sea ice transport and of the synoptic scale variability such as atmospheric blocking, which is a challenge for current coupled climate models. Sea ice is an important component of the Arctic climate system, affecting heat, freshwater and momentum fluxes between the ocean and the atmosphere. Huge amounts of sea ice and freshwater are transported through Fram Strait, which connects the Arctic Ocean and the northern North Atlantic sector, and influence water densities in these regions. Variations in Fram Strait Sea Ice Export (FSSIE) have been associated with the Great Salinity Anomaly (GSA) observed in the late 1960s to early 1970s in the North Atlantic12The atmospheric circulation over the Arctic plays a key role in the evolution of sea ice growth, movement and melting. Half of the variance in the summer sea ice extent over the past three decades has been influenced by lower atmospheric winds5688131416On interdecadal timescales, NAO also plays a role in modulating the AMOC variability20222325262526The study relies on the Finite-Element Sea-Ice Ocean Model (FESOM)30higher (lower) than 0.75 (−0.75) standard deviations. To emphasize the variation in the relationships between the two variables, the simultaneous and lagged relationship, with a lag of up to 5 years (FSSIE lags) when the FSSIE was higher than 0.75 standard deviations, is analyzed.Atmospheric blocking is a large-scale mid-latitude atmospheric phenomenon mostly associated with persistent quasi-stationary synoptic-scale high-pressure systems. It may cause large-scale circulation anomalies exerting a strong impact on weather patterns and is therefore often associated with significant climate anomalies3435v = , where h is the sea ice thickness and u, v denote the ice velocity components, multiplied with the sea ice concentration c, by solving the Poisson equation ΔΨ = −curl(c·v). This stream function accounts only for the horizontal advective sea ice transport, but not for contributions from source terms like freezing and melting. Advective sea ice transport occurs along the lines of constant stream function values. Positive (negative) stream function values characterize a clockwise (counter clockwise) circulation. We refer to this stream function as the advective sea ice stream function. The lagged (high) and in phase (high and low) composite maps for the blocking frequency, the Arctic sea ice thickness and the advective sea ice stream function were computed based on the simulated FSSIE time series  in the strength of the Beaufort Gyre (Transpolar Drift)  for highIn summary, reduced FSSIE is associated with a center of enhanced blocking activity over Greenland, coupled to a persistent center of enhanced blocking activity over Northern Europe . This ki37Enhanced FSSIE is associated with strongly reduced blocking activity over Greenland and enhanced blocking activity over the eastern North Atlantic . This path century witnessed a series of decadal-scale anomalies of salinity, temperature and sea ice cover in the northern North Atlantic12134The second half of the 20Although the causes of 1970’s GSA event are partially knownThe mean blocking frequency, averaged over the region −40°E–0°E, 48°N–58°N for the 1960–2001 period , shows tUsing a stand-alone global ocean-sea ice model, with an increased resolution around Greenland and in the deep water formation areas, here it is shown that an atmospheric blocking configuration, extending from Greenland to the northern part of Europe, modulates the sea ice accumulation in the Arctic Ocean and the FSSIE. Correspondingly, high blocking activity during the early-mid 1960s generated an anomalously large sea ice accumulation in the Arctic. The sea ice export through Fram Strait caused the 1970’s GSA event, which in turn weakened the Labrador Sea convection and AMOC.et al.The enhanced blocking activity stretching from Greenland to Western Europe is related to a warmer and more saline subpolar Atlantic Ocean39Here, we have shown that persistent atmospheric blocking in winter can lead to abrupt shifts in AMOC variability via excessive sea ice export through Fram Strait, which in turn could also affect the climate over Europe. A weaker AMOC is associated, among other things, with a reduced volume of warm water transported from the tropics towards Europe and hence with colder winters and an increased storminess over EuropeThe results presented here point to an internal source for abrupt climate changes, with implications for the prediction of AMOC shifts, since winters with enhanced blocking over Greenland tend to persist for years. They also indicate that an important part of the atmosphere-ocean dynamics at mid- and high latitudes requires a proper representation of the Fram Strait sea ice transport and of the synoptic scale variability such as atmospheric blocking, which is a challenge for current coupled climate models49As a measure of local blocking frequency, we have used the two-dimensional (2D) blocking indexwhere For each winter we calculate the ratio between the number of days when a certain grid point was blocked, i.e. the conditions GHGS > 0 and GHGN < (−10m/°.lat) are simultaneously satisfied for at least five consecutive days, and the total number of winter days (90 days). Because we have used Z500 data for 20°N–90°N, the blocking field extends from 35°N to 75°N.To identify the physical mechanism responsible for the connection between the winter sea ice export through Fram Strait and the atmospheric blocking, sea ice thickness, sea ice stream function, and wind-stress, we constructed the composite maps between the normalized time series of sea ice export through Fram Strait for the years when the values of the index were higher than 0.75 std. dev. This threshold was chosen as a compromise between the strength of the climate anomalies associated to sea ice anomalies and the number of maps which satisfy this criterion. Further analysis has shown that the results are not sensitive to the exact threshold value used for our composite analysis (not shown). We have computed composite maps, instead of correlation maps, because the former considers the nonlinearities included in the analyzed data. The significance of the composite maps is based on a standard t-test (confidence level 95%).How to cite this article: Ionita, M. et al. Linkages between atmospheric blocking, sea ice export through Fram Strait and the Atlantic Meridional Overturning Circulation. Sci. Rep. 6, 32881; doi: 10.1038/srep32881 (2016).


However, in-air XRF analysis requires chemical fixation modifying the cell’s chemical composition. Here, we describe first nanoscopic XRF analysis upon cryogenically frozen (-150°C) fibroblasts at the ID16A-NI ‘Nano-imaging’ end-station located at the European Synchrotron Radiation Facility (ESRF) in Grenoble (France). Fibroblast cells were obtained from skin biopsies from control and Friedreich’s ataxia (FRDA) patients. FRDA is an autosomal recessive disorder with dysregulation of iron metabolism as a key feature. By means of the X-ray Fundamental Parameter (FP) method, including absorption correction of the ice layer deposited onto the fibroblasts, background-corrected mass fraction elemental maps of P, S, Cl, K, Ca, Fe and Zn of entire cryofrozen human fibroblasts were obtained. Despite the presence of diffracting microcrystals in the vitreous ice matrix and minor sample radiation damage effects, clusters of iron-rich hot-spots with similar mass fractions were found in the cytoplasm of both control and FRDA fibroblasts. Interestingly, no significant difference in the mean iron concentration was found in the cytoplasm of FRDA fibroblasts, but a significant decrease in zinc concentration. This finding might underscore metal dysregulation, beyond iron, in cells derived from FRDA patients. In conclusion, although currently having slightly increased limits of detection (LODs) compared to non-cryogenic mode, SR based nanoscopic XRF under cryogenic sample conditions largely obliterates the debate on chemical sample preservation and provides a unique tool for trace level elemental imaging in single cells close to their native state with a superior spatial resolution of 20  FXN gene 4-, is precipitated as the bright blue and highly water-insoluble complex, potassium ferric ferrocyanide KFeIIIFeII(CN)6 or Prussian blue:For Prussian blue staining, collected fibroblasts were grown on glass slides and air-dried, fixed in methanol for 10 min and washed with distilled water. Equal volumes of hydrochloric acid  and potassium ferrocyanide  equipped with centrifuge, light microscope, analytical balance, fridge at 4°C and freezer at -20°C. Under previously described culture conditions , fibroblasts were grown upon Si3N4 wafers considered as ideal support for XRF analysis , 5x5 mm2 size, 1.5 mm2 membrane area, 500 nm membrane thickness and 200 μm frame thickness). After verification of firm attachment and spreading of the fibroblasts using optical microscopy, wafers were taken to the sample preparation laboratory using an autoclavable ‘bio-carrier’ red box. There, Opti-MEM™ cell culture medium was removed, wafers briefly washed in 0.25 M freshly prepared ammonium formate solution to remove salts and trace metals from the medium . As vitreous ice upon the fibroblasts causes X-ray absorption, excess water is removed before plunge freezing via blotting. Wafers were blotted and then plunge frozen in liquefied ethane automatically using a Leica EM GP™. After plunge freezing, a cryogenic workflow is maintained until and during the XRF measurements. Wafers are transferred into the liquid nitrogen bath of a Leica™ EM VCM (Vacuum Cryo Manipulation System) where they are clamped in a pre-cooled gold-coated VCT sample holder. The sample holder can then be loaded into the Leica™ EM VCT500 (Vacuum Cryo Transfer system). Finally, sample shuttle is attached to the ID16A-NI vacuum chamber and the gold-coated copper cube holding the silicon nitride membrane with deposited fibroblasts is transferred onto the sample stage kept at -150°C. An external visible light microscope system with long working distance positioned outside the vacuum chamber is used to bring fibroblast cells in the focus of the X-ray nanobeam.For SR nano-XRF analysis, fibroblast cell cultures were transported to the ESRF and immediately brought to incubator at 37°C with 5% CODuring our analyses, fibroblasts generally remained well-preserved during scanning XRF, nevertheless even under cryogenic conditions we did observe sample radiation effects such as slow freeze-drying of the sample or abrupt ablation of the sample, typically occurring for cells covered by a thinner ice layer of a few micron. Under these circumstances, parts suddenly detach from the fragile, glass-like frozen cell layer when struck by a high intensity X-ray nanobeam. Higher sensitivity of the sample with respect to radiation damage was related to a lower ice layer thickness covering the cell, i.e. a cell covered by an ice layer of only a few micron is less protected than a cell covered by several tens of microns of ice.m long beamline for hard X-ray microscopy, providing quantitative 3D characterization at the nanoscale of the morphology and the elemental composition of specimens in their native state [11photons/s confined within a beam of 27 nm horizontally by 21 nm vertically full-width at half-maximum (FWHM). Incident energy upon the X-ray focusing optics housed within the high vacuum sample chamber is 17.1 keV having a spectral bandwidth ΔE/E of 1%.; also 33.6 keV excitation energy can be provided. Beamline techniques include full field holography, ptychography and X-ray fluorescence. Measurements are always performed vacuum whereas cryogenic analysis is optionally. In the latter case, temperature of the sample holder clamping the sample wafer is continuously monitored and generally remains constant at -150°C. Main fields of application are life science and nanotechnology. A positioning stage provides nanoscopic positioning and fast continuous scanning with maximal speed of 4 μm/s. A diode is present behind the sample determining the flux density for normalization purposes. The X-ray nanobeam can be provided in ‘high-dose’(HD) and ‘low-dose’ (LD) mode; in HD-mode, a thin Au foils of 4.6 μm is removed, increasing the photon flux by a factor of 3.1 times to 2.1011photons/s with respect to LD-mode.Scanning nano-XRF experiments were performed at the ID16A-NI (Nano-Imaging) beamline (UPBL04) at the European Synchrotron Radiation Facility (ESRF). ID16A-NI is a 185 ve state . The beamm2 active area each. Minimal detector distance was set at 40.2 mm, limited by the experimental geometry at that time. Due to the vertical alignment of the detectors, not all SDD detectors could be placed in the synchrotron ring plane where Compton scatter is minimal under 90° detector angle. In order to have the sample surface perpendicular to the X-ray beam and still being able to collect X-ray fluorescence emerging from the sample, the angle between sample surface and detector was set to approx. 18°. A shielding plate was attached to the detector to protect it from scattered X-ray photons from the mirror system; no collimator was used.Two pairs of three vertically aligned silicon drift detectors  were connected to left vacuum flange (downstream direction) of the sample chamber, orthogonal to the beam direction. All six SDDs are equipped with a beryllium window and have 50 ms dwell time per pixel and 55 nm step size was used. Since the total scanned area covers 44.9 x 32.7 μm2, each element map contains 816 x 595 pixels and was derived from 480k individual XRF point spectra. For potassium, we clearly observe the outlined fibroblast for SDD detector no. 3, 4 and 5 only  map using the following formula:mg NIST SRM 1577C powder was therefore pressed in a self-supporting (dry) pellet using a die with 13 mm diameter pushing rods, resulting in an areal density of 16.20 mg/cm2. Using a scalpel, a mm-sized fragment was removed and fixed to a silicon nitride (Si3N4) membrane. The Si3N4 wafer was then manually plunge frozen in the Leica™ EM VCM and transferred into the sample chamber under identical conditions.After optimization of the experimental set-up upon an FRDA fibroblast, the reference material NIST SRM 1577C ‘bovine liver’ was analyzed. NIST SRM 1577C powder contains most of the elements present in single cells and is particularly well-known for its excellent homogeneity on the (sub-) micrometer scale . Anotherμm2 was scanned in 25 nm steps with 100 ms dwell time, resulting in an element map of 100x100 points. Second, an XRF map was obtained with size approx. one order of magnitude larger, i.e. 25 x 25 μm2 in 250 nm steps with identical dwell time of 100 ms was measured. nm steps and b) 25 nm steps. Homogeneous Compton scatter maps of both scans indicate a constant total areal mass over the scanned area. Other elements present in NIST SRM 1577C which are not shown in μm step size), larger ‘isles’ spanning several tens of microns and smaller areas of approx. 5 μm with increased intensity (indicated by arrows) are present. Calcium hot-spots are present with sizes of 1.75 up to 3.25 μm. Excellent homogeneity is observed for iron and zinc, albeit with the sporadic presence of some hot-spots. We did however not observe the presence of micrometer sized nuggets containing cupper as reported in another study, scanning a 10 x 10 μm2 area of NIST SRM 1577C, using 200 nm steps and 500 ms [nm step size) exhibits much less heterogeneities for potassium and calcium. For iron and zinc, some more intense areas of approx. 100 nm are observed. Note however that this improved homogeneity is observed within a 2.5 x 2.5 μm2 area.For investigating the homogeneity of NIST SRM 1577C, two XRF maps were collected: first, a randomly selected area of 2.5 x 2.5 d 500 ms . The nanppm) of NIST SRM 1577C for the elements P, S, Cl, K, Ca, Mn, Fe, Cu, Zn and Rb when using the XRF sum spectrum of the largest XRF map collected . LODs were calculated based on: 1) the sum spectrum of all SDD detectors available (no. 0 to 5), 2) half of the detector array providing the better XRF signal  and 3) SDD detector no. 5 only providing optimal iron elemental maps (see earlier). Measurements were performed in ‘high dose’ (HD) mode (2.1011photons/s). From the graph, we clearly observe a higher LOD when using a single SDD detector compared to using all detectors, e.g. 8 ppm instead of 2 ppm for iron, corresponding to a downturn factor of almost 5. Note that this is almost double the theoretical factor of ppm is derived for iron, corresponding to a molar LOD of 140 μM or to an absolute LOD of 1 ag, equivalent to 12000 iron atoms. For the sake of completeness, LODs  for the other detectable elements of biological interest  using detector no. 5 only are provided in th order. Batch processing was performed using the MICROXRF2, written in IDL programming language. Raw elemental maps (expressed in counts or cts) were then normalized to 1s measuring time, incoming beam intensity ‘I0’ (a.u.) and dead time ‘DT’ (in %). In this manner, normalized element maps are obtained (expressed in cts/s). As incoming beam intensity detector was not properly obtained during our measurements, elemental maps were normalized to the transmitted beam intensity ‘It’ since absorption effects of the cell on the transmitted intensity map could be considered negligible. For additional information concerning the normalization procedure we refer to the Materials and Methods section in another work [Spectral analysis was performed using the AXIL  , includiher work .ice for which 50% of the original fluorescence of a probed element i remains, the following relation must apply:After cryofixation and blotting—an action which removes the excess of water before submersion into a cryogen—an ice layer generally remains covering the fibroblasts. Since the whole procedure can be effected by the human hand or robotically, the ice layer thickness varies with the position of the cell on the membrane . For accurate calculation of the mass fractions of the lower atomic number elements such as P, S, Cl, K, and Ca, where absorption of the lower energetic fluorescent X-rays plays a significant role, the thickness of the ice layer covering the cells is an important quantity which needs to be determined. For calculating the ice layer thickness dα fluorescent photons are absorbed . For phosphorus, sulphur and chlorine, 50% of the original fluorescent intensity is already absorbed by a thin ice layer of only 2.9, 4.4 and 6.3 μm; 99% of the fluorescence of these elements is absorbed by an ice layer of 20, 29 and 42 μm thick, respectively. Also for potassium and calcium, 50% of the Kα fluorescence is absorbed after 12 and 17 μm of ice, respectively. Manganese, iron and zinc having higher Kα fluorescent energies are less problematic: 90% of the fluorescence still penetrates through an ice layer thickness of 10, 13 and 32 μm, respectively.dice above the single fibroblasts, i.e. using the total X-ray transmission (I0/It) on the one hand and by using the K-Kα/Kβ ratio on the other hand. For the ‘I0/It’ method, the X-ray beam intensity was measured using a diode with the sample in the X-ray focus, resulting in an intensity ‘Isample’ measured; thereafter without the sample in the X-ray focus, providing an intensity ‘I0’. As the primary X-ray beam is attenuated both by the silicon nitride membrane and the ice layer on it, the following equation applies according to Lambert-Beer’s attenuation law:ice:Two methods were examined for calculation of the ice layer thickness 3N4 membranes have a thickness of 500 nm (or 500 .10−7cm), a density of 3.4 g/cm3 and a total X-ray cross section of 4.583 cm2/g at 17.1 keV and the total X-ray cross-section ice is 1.186 cm2/g at 17.1 keV, the following formula is obtained for determining the ice layer thickness Tice (in cm):I0 represents the total measured XRF intensity while having the sample out of the beam, Iice the total XRF intensity of the sample within the beam, −7 refers to the 500 nm thickness of the Si3N4 membrane. This method is however has inaccuracies as: 1) it takes into account non-present absorption from ice behind the wafer 2) the measurement in only performed at one specific position on the cell and 3) it considers absorption along the direct beam path which is different from the fluorescence path towards the detector. Therefore, a second more indirect way was used to determine the ice layer thickness by using the K-Kα/Kβ signal: due to the fact that the Kα fluorescent is lower in energy than the Kβ radiation and K is richly present in all cells, the ratio R of both fluorescent intensities can be used to calculate the thickness of the ice layer through which they travel. When no absorption occurs, R can be calculated from the X-ray radiative rates of K-Kα and K-Kβ as follows:Since the used Siα and K-Kβ ratio R0 for the case without absorption occurring and the case R with absorption occurring through an ice layer:Therefore, the following relation applies for the K-Kcm2) is obtained:After rearrangement, the following equation for the ice layer thickness  were generally higher: 14 μm relative to 7.6 μm for the control fibroblast case ‘SL’ and 50 μm relative to 4.0 μm for the FRDA fibroblast case ‘DJS’. This is most probably due to the additional amount of ice behind the Si3N4 membrane which is also taken into account when using the ‘I0/It’ method. The percentage of absorbed Kα fluorescence for a specific element using the calculated ice thickness of the control and FRDA fibroblast using both methods (‘I0/It’ and ‘K-Kα/Kβ ratio’) is shown in 0/It’ method cause absorption up to 90%, even for calcium; for the ‘K-Kα/Kβ ratio’ method this is only a mere 20%.By using the K-Ki emanated from a homogeneous cell layer, the Fundamental Parameter equation applies0 is the incoming photon flux, G the geometry factor, ωi the mass fraction of element i, (ρd)cell the areal density of the cell, Qi, Kα the production cross-section of the element i in the cell at the incoming photon energy. The term χcell(i) is a factor which corrects for absorption effects of exciting and fluorescent X-rays within the frozen cell layer:μ0,cell is the total X-ray cross-section of the cell layer at the incoming photon energy E0. The total X-ray cross-section of the virtual cell layer for a particular element i is represented by μi,cell. Both quantities are defined by the mass fraction ωi of a particular element i multiplied by the photo-electric X-ray cross section τi at the incoming excitation energy E0 or fluorescent energy Ef,, respectively:For determining the fluorescence radiation for a particular trace element All photo-electric and total X-ray cross sections were obtained from the Xraylib database , 30 callf,orig according to the law of Lambert-Beer:In our experiment cells are additionally covered by an ice layer, absorbing its original fluorescence Iρd)ice the areal mass of the deposited ice layer above the cell, expressed in g/cm2. The ice layer thickness above the cell dice can now be determined as discussed in previous section using 0/It method) or Eqs α/Kβ ratio method). By assuming that ρice = 1g/cm3, an ice layer thickness of x μm is equivalent to an areal mass of x 10−4g/cm2. The factor χice,i in 0,ice and μi, ice in analogy to ice(i) is easier to calculate than χcell(i) due to the divalent composition of ice:Hereby is SRM of 16.20 mg/cm2 was measured under identical conditions as the fibroblast cells  and for NIST SRM 1577C ‘bovine liver’ (red curve).I0, G and ωi,cell(ρd)cell actually represents the areal concentration (in g/cm2) of a certain element i in the cell, we obtain after rearrangement that:By dividing In which the first term between brackets symbolizes the self-absorption correction factor for the SRM while the second term stands for the absorption correction due to the ice layer. μg/cm2), the mass fraction maps can be obtained assuming a mean cell thickness of 10 μm, resulting in a total areal mass of the cells of 1 mg/cm2. Dividing the areal concentrations maps by the total estimated areal mass of the cells finally provides the mass fractions maps in ppm:From the areal concentration maps  subtraction of background mass fraction from the cytoplasm mass fractions (2) cluster areas of cell and cytoplasm were not taken perfectly adjacent since stray fluorescence of the cell can contribute to the background and (3) isolation and removal of XRF point spectra from ‘hot spots’ within the cell identified as inorganic (dust) particles and therefore artificially increasing the element concentration. Masks were then multiplied with the quantified maps of each element from which a mean concentration value could be obtained. Due to the rather large background for the elements Fe, Cu and Au induced by fluorescence from the sample holder and vacuum chamber, the mean concentration value of the background was subtracted from the mean cell concentration values. A flowchart illustrating the entire quantification workflow for an FRDA fibroblast raw elemental map is provided in nm, currently a world’s first. Human fibroblast cells were cultured on the synchrotron site, after which the support membranes were automatically plunge frozen, mounted and transferred to the cryocooled sample stage of the ID16A-NI ‘Nano-imaging’ end-station, all in an uninterrupted cryogenic workflow. Although we did observe some radiation damage effects upon the cryogenically frozen fibroblasts such as slow freeze-drying or ablation of thinner micrometer regions, nanoscopic XRF scanning under cryogenic conditions generally maintained the elemental distributions of fibroblast cells for more than several hours, even under extremely high flux conditions of 2x1011photons/s. Owing to this high flux, relative limits of detection (LODs) for our experiment reached 7.6 ppm in scanning mode for iron, equivalent to absolute LODs of 140 nM or approx. 12.000 atoms. LODs were generally higher compared to non-cryogenic mode due to the higher production of scattered X-ray photons for frozen cells, increasing the background signal and secondary fluorescence from sample holder and vacuum chamber. Elemental maps of the fibroblasts also showed the virtual presence of copper, nickel and gold confined within microscopic areas, which were caused by the presence of microcrystals in the shock-frozen fibroblasts—supposedly containing amorphous ice only—diffracting incoming X-ray photons.Trace level elemental distributions within cryogenically frozen (-150°C) fibroblasts from control-disease and Friedreich’s ataxia (FRDA) patients were obtained using synchrotron radiation (SR) based nanoscopic hard X-ray fluorescence (XRF) using an X-ray nanobeam with unprecedented spatial resolution of 20 α/Kβ fluorescence ratio of potassium, the ice layer thickness covering each fibroblast could be determined, typically ranging from a few μm up to 100 μm. Ice layer thickness varied strongly with the blotted sample and position. The Kα/Kβ fluorescence ratio of potassium was used for estimating the thickness of the ice layer covering the cells because of its prominent presence in the fibroblasts. Background-corrected mass fraction elemental maps for the elements P, S, Cl, K, Ca, Fe and Zn were finally determined for the control and FRDA fibroblast using the Fundamental Parameter (FP) quantification method. Within high resolution (55 nm) XRF maps, iron-rich hot-spot areas were detected in the cytoplasm of both a control and FRDA fibroblast, which were estimated to have a concentration of approx. 210 ppm. Highly pronounced iron enrichments as observed in FRDA fibroblasts by Prussian blue staining were however not found, which may be related to the fact that fibroblasts were kept in culture for several cycles, imposing an unnatural environment compared to in vivo conditions. As metal metabolism of fibroblasts may already change after a few cell cycles, immediate XRF analysis after skin biopsy is recommended. Another reason could be that given the limited sample throughput of SR based XRF, only FRDA fibroblasts without iron accumulation were scanned; in this respect pre-selection of the cells of interest using fluorescence-activated cell sorting (FACS) would be a valuable approach. From medium resolution (200 nm) XRF maps, mean mass fractions of the cytoplasm within 5 control and 5 FRDA fibroblasts were calculated. Also here, no significant cytosolic iron increase for FRDA fibroblasts was observed. However, provided the multi-element character of SR based XRF, we detected a significant decrease in zinc for the FRDA fibroblasts, which may be related to impaired homeostasis of this/these element(s) for FRDA. While Zn2+ is not redox active under physiological conditions, zinc deficiency causes increased oxidative damage to DNA, proteins, and lipids, indicating an indirect antioxidant role for zinc as a required element in CuZnSOD or MnSOD superoxide dismutase (SOD) [For accurate quantification of the (FRDA) fibroblast elemental maps, estimation of the ice layer thickness turned out to be critical, especially for lighter elements such as P, S, Cl, K and Ca. By using the Kse (SOD) .in vivo state. Owing to the current availability of nanoscopic XRF analysis under cryogenic conditions, the debate on the validity of elemental distributions obtained on chemically fixed cells–prone to contamination and metal relocation- becomes largely unnecessary. Similar to XRF analysis under ambient temperature, a wide range of biological elements can be probed simultaneously , sometimes even with better sensitivity for the lighter elements due to the required vacuum conditions in cryogenic mode. In contrast to staining reagents or visible light fluorescent probes, X-ray fluorescence allows to measure the sample directly, without the use of any chemicals. Future challenges of nanoscopic XRF imaging under cryogenic conditions include improving sensitivity by better shielding of the detector array from the surrounding sample environment or by installation of a dual detector setup, providing natural shielding against background fluorescence. The observed presence of microcrystals within the vitreous ice matrix causing diffraction, secondary fluorescence and increased background could be overcome by optimization of the shock-freezing methods preventing the formation of these crystals. More accurate estimation of the ice layer thickness should lead to more reliable quantification for lower atomic number elements such as phosphorus, sulphur, chlorine and potassium. Another possibility is to produce a well-defined (or even constant) ice layer covering the cells by more reproducible blotting procedures or the fabrication of cryosections [rd generation synchrotron radiation sources (ESRF-EBS and PETRA IV), an increase in brilliance of 10 to 100 times is to be expected. This will result in up to 10 times more sensitive nanoscopic XRF imaging in the same time frame currently used, which will enable visualization of (ultra) trace elements in single cells, e.g. nickel and selenium. On the other hand, when settling with the current sensitivity, single cells will be scanned in a few minutes rather than several hours.We highlight synchrotron radiation based nanoscopic XRF analysis under cryogenic conditions as a new method of choice for life science investigations requiring the ultimate trace level subcellular elemental distribution or aiming at a quantitative study of several cells close to the sections . Due to S1 Figμm2. Elemental maps were obtained at ‘High dose’, using a step size of 55 nm and a dwell time of 55 ms. Elemental maps showing clear fibroblast structure are indicated using green rectangles. Virtual presence of Ni, Cu and Au through secondary fluorescence is indicated with red rectangles.The total scanned area covers 44.9 x 32.7 (TIF)Click here for additional data file.S2 Figμm2. Elemental maps were obtained at ‘High dose’, using a step size of 55 nm and a dwell time of 55 ms. Cell border and nucleus are indicated on the iron elemental map of detector no. 5 for clarity using a dashed and full black line, respectively. Within the same elemental map, the presence of iron containing hot-spots is indicated with a red striped circle.The total scanned area covers 44.9 x 32.7 (TIF)Click here for additional data file.S3 FigFor each detector, the corresponding dead time map is shown.(TIF)Click here for additional data file.S4 Fignm (a) and 25 nm (b), 100 ms dwell time.Elemental maps were obtained under cryogenic conditions at ID16A-NI ‘Nano-imaging’ beamline at the ESRF in Grenoble (France). ‘High dose’ mode, pixel size: 250 (TIF)Click here for additional data file.S5 FigLODs were calculated in ‘High Dose’ mode. Relative LOD is calculated for integrated signal from all 6 detector (blue), integrated detector signal from detectors no. 3-4-5 (orange) and from detector no. 5 only (grey). Colored curves are added to guide the eye.(TIF)Click here for additional data file.S6 Figcm2/g. A curve was added to ‘guide the eye’. The total absorption cross-section of the incoming photon energy (17 keV) within both matrices  was determined as well.Results are expressed in (TIF)Click here for additional data file.S7 FigCorrection factor for self-absorption in NIST SRM 1577C (blue), in ice (blue) and total absorption correction factor (green).(TIF)Click here for additional data file.S8 FigI0 map (in a.u.) and the raw element intensity maps (in cts), normalized element intensity maps are produced (in cts/s). Using the Fundamental Parameter equation, the normalized element maps are converted into concentration maps (in ppm or in μg/cm2). Absorption effects in NIST SRM 1577C and in the ice layer covering the cells are corrected for. Background is subtracted from the entire concentration map. Cluster maps can then be generated containing regions of interest (ROIs), such as entire cell, cytoplasm and background, omitting hot-spots. In combination with the quantitative element distributions, background corrected mean concentrations of ROIs are obtained.From the incoming (ICR) and outgoing count rate map (OCR), the dead time percentage map is determined (in %). Together with the (TIF)Click here for additional data file.S1 TableLODs were calculated in ‘high dose’ mode. Only a single detector channel was used (XIA05).(XLSX)Click here for additional data file.S2 Tableα fluorescence for a specific element using the calculated ice thickness of the control and FRDA fibroblast using the ‘I0/It’ and ‘K-Kα/Kβ ratio’ method (right main column).Ice layer thickness for which 10, 50 and 99% of the fluorescent photons of the elements P, S, Cl, K, Mn, Fe and Zn are absorbed (left main column). Percentage of absorbed K(XLSX)Click here for additional data file.


GWAS in a separate cohort of 48 TIH patients and 2,922 controls from the 1958 British birth cohort identified an additional 14 regions associated with TIH. We identified a suggestive association with a variant in SLCO2A1, which encodes a prostaglandin transporter in the distal nephron. Resequencing of SLCO2A1 revealed a nonsynonymous variant, rs34550074 (p.A396T), and association with this SNP was replicated in a second cohort of TIH cases. TIH patients with the p.A396T variant demonstrated increased urinary excretion of prostaglandin E2 and metabolites. Moreover, the SLCO2A1 phospho-mimic p.A396E showed loss of transporter function in vitro. These findings indicate that the phenotype of TIH involves a more extensive metabolic derangement than previously recognized. We propose one mechanism underlying TIH development in a subgroup of patients in which SLCO2A1 regulation is altered.Thiazide diuretics are among the most widely used treatments for hypertension, but thiazide-induced hyponatremia (TIH), a clinically significant adverse effect, is poorly understood. Here, we have studied the phenotypic and genetic characteristics of patients hospitalized with TIH. In a cohort of 109 TIH patients, those with severe TIH displayed an extended phenotype of intravascular volume expansion, increased free water reabsorption, urinary prostaglandin E Cardiovascular disease is the leading cause of mortality worldwide , 2 and hHowever, some patients given thiazides develop thiazide-induced hyponatremia (TIH) . Severe https://doi.org/10.1172/JCI89812DS1). The characteristics of the TIH cases and controls from cohorts 1 and 2 are presented in Two cohorts of patients of mixed European descent who were hospitalized with symptomatic TIH were recruited (P < 10-5) lower serum potassium on treatment compared with off treatment. Cohort 2 hyponatremic TIH cases on thiazides also had lower blood pressure, lower blood glucose, and a lower serum concentration of chloride, magnesium, calcium, zinc, and vitamin D relative to cohort 2 normonatremic thiazide controls.Cohort 1 hyponatremic TIH cases on thiazides had lower serum potassium compared with cohort 1 normonatremic thiazide controls . In cohoTwenty-four hour urine volume and osmolarity were significantly lower in hyponatremic TIH cases on thiazides compared with normonatremic thiazide controls . FractioSerum abnormalities resolved following thiazide cessation with the exception of levels of chloride and zinc . AlthougCohort 2 TIH cases reabsorbed 48% more free water when on thiazides compared with when off thiazides, which is in marked contrast with cohort 2 controls, who showed only a 9% increase in free water reabsorption while on thiazide . This suIncreased fractional renal excretion of uric acid is observed in the syndrome of inappropriate antidiuretic hormone secretion (SIADH) and is caused by arterial blood volume expansion , 19. MeaMean plasma antidiuretic hormone (ADH) concentration in cohort 2 hyponatremic TIH cases on thiazides was significantly lower than in cohort 2 normonatremic thiazide controls and normonatremic TIH cases off thiazides . AlthougP < 10–5. After quality control filters were applied (see Methods), 502,663 SNPs from 48 cohort 1 hyponatremic cases on thiazides and 2,905 controls remained for association testing. The genomic inflation factor (λ = 1.007) and the resultant quantile-quantile (QQ) plot (P < 10–5)  plot  were not < 10–5) . Of thesSLCO2A1 showed association with TIH at P = 3.92 × 10–6 (odds ratio [OR] = 2.58). Targeted resequencing of SLCO2A1 in cohort 1 TIH cases and cohort 1 controls confirmed the presence of the nonsynonymous variant encoding p.A396T (rs34550074) in complete linkage disequilibrium (2r = 1) with the sentinel GWAS SNP rs4854769. Association with rs34550074 between cohort 1 cases and the carefully phenotyped normonatremic cohort 1 controls on thiazide was observed at P = 0.0005 , driven by the association with p.A396T. Sanger sequencing also confirmed the presence of rs34550074 in cohort 1 hyponatremic TIH cases on thiazides   and 0.18 in normonatremic thiazide controls (test of association:  = 1.70) . When daSLCO2A1)  (the protein product of SLCO2A1)  20). Co. CoSLCO2SLCO2A1) . PGT staSLCO2A1) . ExpressSLCO2A1) .Species conservation of rs34550074 is shown in 2 (PGE2) and PGE2 metabolite (PGE2M) concentrations from 24-hour urine samples were significantly higher in cohort 2 hyponatremic TIH cases on thiazide compared with cohort 2 normonatremic thiazide controls . We focused on SLCO2A1 (encoding PGT) because augmented renal water reabsorption seemed most relevant to the principal abnormality of hyponatremia from our phenotype studies. PGT is known to regulate this process, and there was direct association between a protein-altering variant in SLCO2A1 (p.A396T) and TIH. This association was replicated in a second independent cohort. SLCO2A1 p.A396T was twice as frequent in TIH cases compared with control and general populations, with approximately half of all TIH patients carrying this variant. In vitro data suggest that this variant may reduce PGT activity and lead to the higher urinary PGE2 concentrations observed in TIH patients who carry the variant allele. However, since half of TIH patients do not carry the SLCO2A1 variant and at least a quarter of unaffected patients do, other genetic or environmental determinants must also contribute to the development of TIH.Having defined the phenotype of TIH, we set out to identify possible genetic predisposition to this condition. Given the small number of cases available in our initial cohort, we chose to prioritize GWAS associations with suggestive evidence for follow-up using a less conservative One potential criticism of this study is the limited sample size in the discovery cohort. This was inevitable, since we were careful to include only those with severe thiazide-induced hyponatremia and excluded those who lacked mental capacity or who had relevant comorbidities.2 and intravascular volume expansion? And how might a reduction in SLCO2A1 activity cause TIH? SLCO2A1 is expressed at the apical membrane of the renal collecting duct, where it scavenges newly synthesized PGE2 away from luminal EP4 receptors  to generate solute-free water directly and also act by reducing effective vascular volume and thus solute delivery from the end-proximal tubule . We suggTwo cohorts of patients of mixed European descent hospitalized with symptomatic TIH were recruited . Cohort A GWAS was performed using 48 cohort 1 TIH cases genotyped using the Illumina Omni1quad array and 2,922 general population controls from the British 1958 birth cohort , genotypP < 10–5, followed by association testing (http://www.ebi.ac.uk/ena). Replication for one SNP of interest identified in the sequencing analysis of cohort 1  was undertaken using Sanger sequencing in the second cohort of TIH cases and controls  underwent resequencing of the genes nearest to SNPs that were associated with TIH at  testing . Next Gecontrols . Rare vacontrols . Rare vac-α test , also imFormalin-fixed paraffin-embedded human tissue sections were obtained from the Cambridge Human Research Tissue Bank. After antibody labeling, confocal imaging was performed .3H-PGE2 was assayed as described previously (Human SLCO2A1 cDNA (encoding PGT) was modified by site-directed mutagenesis using standard methods so as to generate cDNAs encoding amino acids Ala, Thr, or Glu at position 396. DNA sequences were confirmed by direct sequencing. Human embryonic kidney (HEK) cells were transiently transfected with one of the 3 cDNAs, and the timed uptake of eviously .t test. Where comparisons between more than 2 groups were undertaken, 1-way ANOVA and Bonferroni’s post hoc test were used. The data were analyzed using Graph Pad Prism V6.05. P < 0.05 was considered significant. Statistical methods used for GWAS and resequencing experiments are contained within the relevant Methods and Significance between groups was assessed using a 2-tailed Student’s This study was conducted in line with the standards of ICH/Good Clinical Practise sections 8.2.8 and was given approval by the Queen’s Medical Centre Ethics Committee  and the UK National Research Ethics Committee . Written informed consent was received from participants prior to inclusion in the study.JSW, SC, LVW, SKC, VEJ, EE, RL, KS, WJ, NS, APH, and MG performed experiments and analyzed data. SK, MJ, JC, and MG recruited patients. KMO, VLS, MDT, IPH, and MG designed the study. All authors contributed to the drafting and revision of the manuscript, which was led by MG and IPH.