Records of Alpine microseismicity are a powerful tool to study landscape-shaping processes and warn against hazardous mass movements. Unfortunately, seismic sensor coverage in Alpine regions is typically insufficient. Here we show that distributed acoustic sensing (DAS) bridges critical observational gaps of seismogenic processes in Alpine terrain. Dynamic strain measurements in a 1 km long fiber optic cable on a glacier surface produce high-quality seismograms related to glacier flow and nearby rock falls. The nearly 500 cable channels precisely locate a series of glacier stick-slip events (within 20–40 m) and reveal seismic phases from which thickness and material properties of the glacier and its bed can be derived. As seismic measurements can be acquired with fiber optic cables that are easy to transport, install and couple to the ground, our study demonstrates the potential of DAS technology for seismic monitoring of glacier dynamics and natural hazards. In this study, Walter and colleagues deploy a 1 km long fiber optics cable on a glacier surface. Via the use of distributed acoustic sensing, the authors are capable of monitoring glacier dynamics and Alpine mass movements. Over the past 1–2 decades, advances in sensor and digitizer technologies have increased portability of seismic instrumentation. Seismic monitoring in poorly accessible Alpine and Polar regions is therefore becoming increasingly feasible. The resulting data focus on processes near the Earth’s surface rather than on traditional seismology subjects like the deeper crust and mantle.1 and cryoseismology3. This has filled critical observational gaps for investigation of mass movements such as bedload transport in torrents4, rockfalls5, debris flows6, and avalanches7 as well as the stability of rock structures8 and landslides9. In glaciated regions, seismic studies have proved the existence of seismogenic glacier sliding and provided time series of iceberg production2 and subglacial water flow10, which are difficult to obtain with traditional glaciological measurements.Seismic studies in Alpine terrain have cultivated new sub-disciplines like environmental seismology5. Capturing, for instance, precursory signals before failure requires sensors in the immediate vicinity of the unstable mass9, and coincidental recordings from nearby permanent seismic stations are rare and difficult to interpret11. Therefore, comprehensive, large-scale seismic monitoring essential for early warning or scientific purposes remains largely impossible because the required sensor coverage is usually infeasible.The sub-second time scales at which seismometers monitor ground unrest constitute an unrivaled temporal resolution. However, only specialized and dense sensor networks can locate mass movements with reasonable uncertainty12. Seismic waves dynamically straining the fiber can thus be recorded over distances of several tens of kilometers, with a bandwidth ranging between quasi-static and tens of kHz13. Advanced interferometric techniques applied to ultra-stable laser light injected into fiber-optic cables may even sample cables hundreds of kilometers long14.In other fields of seismology, the advent of distributed acoustic sensing (DAS) is currently revolutionizing seismic sensor coverage. DAS technology uses fiber-optic cables into which an interrogator injects a sequence of laser pulses. The time series of back-scattered signals can be transformed into strain rate sampled every few meters along the fiber15) and has been shown to record waveforms of regional and teleseismic earthquakes17. In sea basins, seismograms measured with fiber-optic cables capture local earthquake signals, which are too weak to be recorded by sparse ocean bottom seismometers14. DAS measurements of anthropogenic noise can furthermore be used to characterize the Earth’s near-surface structure19. Though individual DAS channels may have a lower signal-to-noise ratio (SNR) than conventional seismometers, the presence of unused fibers in telecommunication networks (dark fibers) suggests that a vast resource of already installed seismic sensors could be harnessed for monitoring with an unprecedented sensor coverage and density19.Fiber-optic technology such as DAS has started to complement geophone chain deployments in active exploration surveys  at an average surface slope of 10° . Between 20 and 26 March 2019, we carried out DAS measurements close to the glacier’s central flow line at an altitude of ~2500 m a.s.l. , 46.5968, 8.382 (WGS84)). At our study site, the surface velocity of Rhonegletscher is ~35 m/a and the ice thickness reaches 200 m as determined from interpolation of radar transects21 and hot-water drilling in summer 201822. Throughout the 1-week-long DAS field deployment the glacier ice was covered by ~3 m of snow.The study site is located on the ablation zone of Rhonegletscher (Switzerland), a temperate glacier (ice at pressure melting point) with an area of ~15.5 kmThree on-ice seismometers were deployed close to the central flow line of the glacier and form an equilateral triangle with ~220 m side lengths Fig. . Each coFrom 21–25 March 2019 we deployed the SILIXA iDAS™ fiber otpic system at Rhonegletscher. The interrogator was placed into a tent, powered by a Honda 20i generator (2 kW maximum power output) and connected to a 1-km long polyurethane mantled fiber-optic cable containing four single-mode and two multimode fibers. We utilized two single-mode fibers to increase spatial redundancy by splicing the end of one fiber together with the other fiber. This resulted in a total fiber length of 2 km. Approximately 820 m of the cable were placed into a shallow trench (few cm deep) carved into the snow and subsequently covered with loose snow Fig. . Of thes13. This gauge length is smaller than the seismic wavelengths of primary interest and thus does not alter seismic arrival-time measurements .Figure The signal strength and SNR of DAS records vary spatially as shown for the case of the stick-slip event in Supplementary Fig. Figure 26. For this we furthermore assume a source mechanism consistent with bed-parallel slip along the glacier flow line, which agrees with compressive first motions on all on-ice stations .The bedrock S-velocity tuned with the 2D ray tracing is 3200 m/s. The ice thickness needed to match the arrival times is 151–181 m. This is smaller than the 172–202 m estimated on the basis of interpolated radar lines  and thus cannot be explained with a direct phase propagating through the ice. We therefore interpret this first arrival as the critically refracted P-wave. The polarity of this refracted P-wave is opposite of the direct P-arrival of the on-ice stations Fig. , because28. Here, we use matched-field processing to determine the rockfall’s back azimuth and the apparent velocity at which the seismic waves propagate throughout the cable layout based on signal arrival-time differences (see Methods).The rockfall Fig.  induces 29, which is consistent with superficial rock impacts on the ground. Besides the unstable moraine from where the rockfall in Fig. Figure 30. The propagation of coherent noise signals is mostly . Especially when water saturated, such till layers have low seismic velocities compared with ice and bedrock35. Stick-slip seismicity could thus provide important information about the basal boundary conditions of fast polar ice streams. The fact that stick-slip patches tend to produce repetitive events such as shown here and in previous studies26 furthermore suggests an application for monitoring: Small changes in basal seismic velocities revealed by repeating stick-slip events could help identify changes of basal resistance as a result of evolving subglacial water pressures .In the present study, identification of the indirect phases emitted by the stick-slip source would not have been possible without the DAS system. In our case, the resulting estimates of ice thickness and seismic velocities of the glacier bed substrate were subject to uncertainties resulting from a poor azimuthal sensor coverage of the source hypocenter. However, with longer cable segments and better sensor coverage, our measurements offer new perspectives in cryoseismology. Without the need of active sources, DAS measurement can characterize the subglacial environment. In our case, seismic velocities within the glacier bed are higher than ice as expected for a mountain glacier resting on granite bedrock. This is confirmed by proglacial terrain, which until recently was covered by the tongue of the Rhonegletscher  that accounts for picking and velocity model uncertainties. Sensor location uncertainties are not accounted for in this method. To overcome this limitation for the DAS channels, we divide the channel location uncertainty of ±2 m by an assumed P- and S-wave velocity (vp = (3800 ± 200) m/s, vs = (1900 ± 100) m/s; see below), which translates into a location uncertainty of 0.5 ms × vp and 1 ms × vs. Finally, we add this location uncertainty linearly to the picking uncertainty of 2 ms for the S-wave and 10 ms for the P-wave. In total, the picking uncertainty used for the DAS records is 11 and 3 ms for P- and S-waves, respectively.We applied a probabilistic nonlinear hypocenter location scheme (NonLinLocvp = (3800 ± 200) m/s and vs = (1900 ± 100) m/s. These values are slightly higher than what was used in a previous seismic study on an Alpine glacier23, but agree with the 2D velocity model used for ray tracing (see discussion below). Note also that as a result of the surface crevasse zone with near-vertical fracture orientation, significantly lower seismic velocities have been determined near the surfaces of glaciers43, including Rhonegletscher44. However, we consider this effect negligible for our basal source, whose seismic rays cross significantly less near-surface fractures than seismic rays emitted by shallow sources. Similarly, we neglect the effect of the snow layer on travel times, because travel time uncertainties associated with highly variable seismic velocities in snow45 are likely comparable to uncertainties associated with our homogeneous velocity model assumption.In order to locate the impulsive stick-slip event shown in Fig. 21 is spatially interpolated, and an uncertainty of at least 10% of the ice thickness should be assumed. Previous source locations have shown that accurate 3D bedrock topography models allowing for critically refracted waves within the bedrock may slightly improve the hypocenter location accuracy over the homogeneous halfspace of ice assumed here46.We estimate the uncertainty of the body wave velocities to ±5% and use a homogeneous velocity model over the entire domain and refrain from including underlying bedrock as ice thickness is known only approximately below our study site: Ice thickness from radar measurements21. We also rotate the along-cross-section coordinates 5° counterclockwise to compensate for the glacier surface slope and achieve a flat glacier surface. Although this profile includes the bed overdeepening beneath the study site, it does not capture transverse variations in bed height (up to 60 m) and surface height (up to 4.5 m) at the study site , this would decrease the arrival time or lower the S-velocity within ice. The phase moveout shown in Fig. Generally, in future studies, longer cable segments could provide better sensor coverage. This would mitigate the location-velocity tradeoff and provide observational constraints for ray tracing or full waveform modeling allowing for 3D variations of bed topography. As a result, bed topography and seismic velocities would be better constrained.f between 10 and 30 Hz (0.2 Hz steps), MFP matches a data vector N sensors . The steering vector 50 but is formally performed using the cross-spectral density matrix (CSDM), which is defined as the outer product† is the complex conjugate operation. The inner product between MFP exploits signal coherence within the sensor array of the DAS system to calculate source back-azimuth and apparent seismic velocities. Since the DAS records contain noisy channels, we only use channels with SNR exceeding 8.5 . For the event shown in Fig. We split the continuous DAS record of the eastern triangle side into 30-min-long time windows, which we spectrally whiten to reduce the influence of transient and monochromatic seismic sources. The 47 channels are then cross-correlated to yield 1081 pairs. Stacking all 30-min time windows over a full day (24 March 2019) and channels within a 10-m radius further increases the cross-correlation SNR.51. Our cross-correlations of axial strain rates along the eastern straight cable portion are most sensitive to Rayleigh wave sources that locate along the cable axis beyond the cable ends30. For simplicity, we neglect the influence of the noise source distribution, and assume that the interferometric wavefield is proportional to the empirical Green’s function. In this case, the causal and acausal wavelets of the cross-correlation represent Rayleigh waves traveling in opposite directions between the station-pairs, where the weak acausal signal  shows that at wavenumbers smaller than 0.04 m−1 (wavelengths longer than 160 m) and frequencies below 10 Hz, the cross-correlations no longer produce Rayleigh wave estimates (Supplementary Fig. A two-dimensional Fourier transform over time and wavenumber Supplementary InformationPeer Review File


Ki, 16.9 µM), CYP2C9 , and CYP2C19  and the transport activity of OAT3 . It exhibited time-dependent inhibition on CYP3A4 . Other metabolizing enzymes and transporters such as CYP1A2, CYP2A6, CYP2B6, CYP2D6, UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A9, UGT2B7, OAT1, OATP1B1, OATP1B3, OCT1, OCT2, P-glycoprotein, and BCRP, exhibited only weak interactions with AB-PINACA. These data suggest that AB-PINACA can cause drug-drug interactions with CYP3A4 substrates but that the significance of drug interactions between AB-PINACA and CYP2C8, CYP2C9, CYP2C19, or OAT3 substrates should be interpreted carefully.Indazole carboxamide synthetic cannabinoid, AB-PINACA, has been placed into Schedule I of the Controlled Substances Act by the US Drug Enforcement Administration since 2015. Despite the possibility of AB-PINACA exposure in drug abusers, the interactions between AB-PINACA and drug-metabolizing enzymes and transporters that play crucial roles in the pharmacokinetics and efficacy of various substrate drugs have not been investigated. This study was performed to investigate the inhibitory effects of AB-PINACA on eight clinically important human major cytochrome P450s (CYPs) and six uridine 5′-diphospho-glucuronosyltransferases (UGT) in human liver microsomes and the activities of six solute carrier transporters and two efflux transporters in transporter-overexpressing cells. AB-PINACA reversibly inhibited the metabolic activities of CYP2C8 ( N-[(2S)-1-amino-3-methyl-1-oxobutan-2-yl]-1-pentyl-1H-indazole-3-carboxamide) was originally developed by Pfizer Inc. as a synthetic cannabinoid receptor agonist -mephenytoin, 4′-hydroxymephenytoin, bufuralol, 1′-hydroxybufuralol, d9-1′-hydroxybufuralol, 1′-hydroxymidazolam, N-desethylamodiaquine, 4′-hydroxydiclofenac, 13C2,15N-acetaminophen, medium 199, fetal bovine serum (FBS), collagen-coated 24-Transwell plates, and poly-d-lysine-coated 96-well plates were purchased from Corning Life Sciences . 3H-Methyl-4-phenylpyridinium (2.9 TBq/mmol), 3H-para-aminohippuric acid (0.13 TBq/mmol), 3H-estrone-3-sulfate (2.12 TBq/mmol), 3H-estradiol-17β-d-glucuronide (2.22 TBq/mmol), 3H-digoxin (1.103 TBq/mmol), and Optiphase scintillation cocktail were purchased from PerkinElmer Inc. . SN-38 was a product of Santa Cruz Biotechnology . SN-38 glucuronide, troglitazone, diclofenac, mycophenolic acid β-d-glucuronide, chenodeoxycholic acid 24-acyl-β-glucuronide, and N-acetylserotonin β-d-glucuronide were purchased from Toronto Research Chemicals . All other chemicals were of the highest quality available. LLC-PK1-BCRP cells (LLC-PK1 cells stably expressing BCRP) were obtained from Dr. A.H. Schinkel . LC-MS grade acetonitrile, methanol, and water were the products of from Fisher Scientific Co. .AB-PINACA and midazolam were purchased from Cayman Chemical Company . Acetaminophen, 2PO4 buffer (pH 7.4), ultrapooled human liver microsomes (0.2 mg/mL), 10 mM MgCl2, 1.0 mM NADPH, and two sets of a cocktail of eight CYP probes ), and AB-PINACA (0.1–100 μM) were incubated for 15 min at 37 °C in a shaking incubator  with or without preincubation with NADPH (30 min). After 15 min, the reaction was quenched by adding 100 μL of ice-cold methanol containing internal standards (IS) , followed by centrifugation at 13,000× g for 8 min at 4 °C. Supernatant from A and B sets (50 μL each) were mixed and 5 μL of the mixed supernatants were analyzed by LC-MS/MS. IC50 values were calculated by mean values of triplicate assays.The effects of AB-PINACA on CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, and CYP3A4 activities were measured in ultrapooled human liver microsomes as described in our previous report . Human lKi values and inhibition modes for the inhibition of AB-PINACA on CYP2C8-, CYP2C9-, and CYP2C19-catalyzed hydroxylation of amodiaquine, diclofenac, and [S]-mephenytoin were measured as described in our previous report -mephenytoin for CYP2C19  were incubated at 37 °C in a shaking incubator. After 15 min, the reaction was quenched by adding 100 μL of ice-cold methanol containing 13C2,15N-acetaminophen (IS) for CYP2C8 or d9-1′-hydroxybufuralol (IS) for CYP2C9 and CYP2C19, followed by centrifugation at 13,000× g for 8 min at 4 °C.The s report . Human lKi and kinact) for time- and concentration-dependent inhibition of AB-PINACA against CYP3A4 activity in ultrapooled human liver microsomes were evaluated according to our previous report estrone-3-sulfate and AB-PINACA (0–100 μM) on the basal side. Aliquots (100 μL) of transport samples were mixed with 200 μL of Optiphase scintillation cocktail. The radioactivity of the probe substrate in the cocktail mixture was measured using a liquid scintillation counter. The B to A transport of digoxin and ES mediated by MDR1 and BCRP, respectively, was calculated by subtracting the transport of probe substrate in LLC-PK1-mock cells from that in LLC-PK1-MDR1 and LLC-PK1-BCRP cells and the B to A transport rate of probe substrate was calculated from the slope of the B to A transport of probe substrate versus time graph -mephenytoin with IC50 values of 32.5 μM, 17.4 μM, and 20.0 μM, respectively, in human liver microsomes, but 30-min preincubation of AB-PINACA with NADPH did not significantly change the inhibitory potential of AB-PINACA toward CYP2C8, CYP2C9, and CYP2C19. However, preincubation of AB-PINACA with NADPH resulted in a 2.7-fold IC50 shift of CYP3A4-catalyzed midazolam 1′-hydroxylation   , Table 1N-deethylation, CYP2C9-catalyzed diclofenac 4′-hydroxylation, and CYP2C19-mediated [S]-mephenytoin 4′-hydroxylation with different substrate concentrations and varying concentrations of AB-PINACA and the results were transformed into Lineweaver–Burk and Dixon plots to determine the inhibition mode of AB-PINACA on the catalytic activities of CYP2C8, CYP2C9, and CYP2C19 along with the Ki values . AB-PINACA significantly inhibited OAT3-mediated ES uptake in a concentration dependent manner with an IC50 value of 11.8 µM. AB-PINACA did not significantly inhibit the transport activities of OCT2, OAT1, OATP1B1, OATP1B3, P-gp, or BCRP in the concentration ranges tested . Before docking, we removed each substrate from the CYP2C8, CYP2C9, CYP2C19, and CYP3A4 co-crystal structures, conducted structure optimization for refinement using the minimization protocol, and performed rigid- and auto-docking with standard protocol. The results yielded docking scores for AB-PINACA of −29.36, −26.99, −33.15, and −32.34 kcal/mol against CYP2C8, CYP2C9, CYP2C19, and CYP3A4, respectively  by Ki (17.6 μM) was 2.3 mL/μmol/min. Similar time-dependent inhibition of CYP3A4 has been reported for EAM-2201 , a halogenated naphthoylindole synthetic cannabinoid [kinact/Ki, 10.3 mL/μmol/min), an indazole carboxamide synthetic cannabinoid [AB-PINACA inhibited CYP3A4-catalyzed midazolam 1′-hydroxylation in a time-dependent manner . The ina1) by Ki .6 μM wasnabinoid . The calnabinoid . These fnabinoid .N-deethylation , and CYP2C9-catalyzed diclofenac 4′-hydroxylation  with a mixed mode of inhibition  of CYP2C8 -1-pentyl-1H-indazole-3-carboxamide) showed time-dependent inhibition on CYP3A4, which was similar to AB-PINACA. AB-FUBINACA (N-(1-amino-3-methyl-1-oxobutan-2-yl)-1-(4-fluorobenzyl)-1H-indazole-3-carboxamide) did not inhibited CYP3A4 but reversibly inhibited CYP2B6, CYP2C8, CYP2C9, CYP2C19, and CYP2D6. AB-PINCACA competitively inhibited CYP2C8, CYP2C9, and CYP2C19 in this study. We also elucidated that the pentyl group at indazole ring and amino-3-methyl-1-oxobutane moiety interact with the active sites of CYP2C8, CYP2C9, CYP2C19, and CYP3A4 through the hydrogen bonding and hydrophobic interaction via molecular docking results (We investigated the in vitro inhibitory effects of AB-PINACA, AB-FUBINACA, and APINACA, structural analogues of indazole carboxamide synthetic cannabinoids on the CYPs, UGTs, and transporter activities in this study and the previous reports ,19. APIN results . ConsideKi values of 145.7 μM and 8.3 μM, respectively (Ki values of AB-PINACA for OCT1 and OAT3 is suspected for AB-PINACA intoxication [AB-PINACA inhibited OCT1-mediated methyl-4-phenylpyridinium uptake with a mixed mode of inhibition and also competitively inhibited OAT3-mediated estrone-3-sulfate uptake with ectively . Althougxication . Thus suKi values.The CYP, UGT, and transporter-mediated drug interaction potentials of AB-PINACA were evaluated based on in vitro inhibitory effects of AB-PINACA on major CYP and UGT enzyme activities and the transport activities of eight drug transporters. Clinically significant drug interaction potentials between AB-PINACA and CYP2C8, CYP2C9, CYP2C19, CYP3A4, or OAT3 substrates should be evaluated carefully, although the plasma concentrations of AB-PINACA reported in drug abusers were much lower than the


Dephosphorylation of Thr1007 of KCC2, and Thr207 and Thr212 of NKCC1 were previously demonstrated to activate KCC2 and to inactivate NKCC1. In addition, application of the two agents resulted in dephosphorylation of the T-loop and S-loop phosphorylation sites Thr233 and Ser373 of SPAK, a critical kinase in the WNK-SPAK/OSR1 signaling module mediating phosphorylation of KCC2 and NKCC1. Taken together, these results suggest that reciprocal regulation of KCC2 and NKCC1 via staurosporine and NEM is based on WNK-SPAK/OSR1 signaling. The key regulatory phospho-site Ser940 of KCC2 is not critically involved in the enhanced activation of KCC2 upon staurosporine and NEM treatment, as both agents have opposite effects on its phosphorylation status. Finally, NEM acts in a tissue-specific manner on Ser940, as shown by comparative analysis in HEK293 cells and immature cultured hippocampal neurons. In summary, our analyses identified phospho-sites that are responsive to staurosporine or NEM application. This provides important information towards a better understanding of the cooperative interactions of different phospho-sites.The pivotal role of KCC2 and NKCC1 in development and maintenance of fast inhibitory neurotransmission and their implication in severe human diseases arouse interest in posttranscriptional regulatory mechanisms such as (de)phosphorylation. Staurosporine (broad kinase inhibitor) and N-ethylmalemide (NEM) that modulate kinase and phosphatase activities enhance KCC2 and decrease NKCC1 activity. Here, we investigated the regulatory mechanism for this reciprocal regulation by mass spectrometry and immunoblot analyses using phospho-specific antibodies. Our analyses revealed that application of staurosporine or NEM dephosphorylates Thr CCCs are divided into K+-Cl- outward cotransporters (KCC1-4), Na+, K+, Cl- inward cotransporters , the polyamine transporter CCC9, and the CCC-interacting protein CIP1 i) in neurons i causing GABA or glycine to elicit a depolarizing action that opens L-type voltage-gated Ca2+ channels necessary for proper synapse formation  in Wolfram Mathematica. For the boxplots, the box extends from the upper (Q1) to the lower (Q3) quartile. The line inside the box represents the median. The whiskers extend to the outermost data point that falls within upper inner and lower inner Quartile fences [Q1+1.5(IQR)] and [Q3-1.5(IQR)], respectively, where IQR = Q1-Q3 is the interquartile range.For dose-response analyses, we used the nls function from the stats package in R (version 3.5.1) to model the data points with the Hill-Langmuir equation,rnKCC2b cells  corresponding to SAYTYEK(T)LVMEQRSQI [residues 899–915 of human KCC2A], S959C); KCC2A phospho-Thr1007 (CYQEKVHM(T)WTKDKYM [residues 1032–1046 of human KCC3A] corresponding to TDPEKVHL(T)WTKDKSVA [residues 998–1014 of human KCC2A], S961C); SPAK/OSR1 (T-loop) phospho-Thr233/Thr185 antibody ; SPAK/OSR1 (S-motif) phospho-Ser373/Ser325 antibody ; SPAK ; NKCC1 phospho-Thr203+Thr207+Thr212 ; NKCC1 phospho-Thr212/Thr217 ; NKCC1-total antibody . Pan-KCC2 antibody (residues 932–1043 of human KCC2) was from NeuroMab (73–013). KCC2 phospho-Ser940 antibody was from ThermoFisher Scientific (PA5-95678). PKCδ phospho-Thr505 antibody (9374) and β-Actin (D6A8) antibody (12620) were from Cell Signaling Technology. Anti -β-Tubulin III antibody was from Sigma-Aldrich (T8578). Secondary antibodies coupled to horseradish peroxidase used for immunoblotting were obtained from Pierce. IgG used in control immunoprecipitation experiments was affinity-purified from pre-immune serum using Protein G-Sepharose.The following antibodies were purchased from Division of Signal Transduction Therapy Unit at the University of Dundee: KCC2A phospho-ThrrnKCC2b cells, as high amount of the cotransporter is advantageous for subsequent biochemical analyses. Immunoreactivity of HEKrnKCC2b cells against KCC2 was detected in all cells with clear labeling at the cell membrane and cytosol  . The loop diuretics furosemide, that specifically inhibits the function of cation chloride cotransporters  concentration [KCC2 and NKCC1 are key players for the development and maintenance of fast inhibitory neurotransmission. Their oppositely directed transport of Kntration , 55–60  results in transport activity that is equal or decreased compared to wild-type KCC2 (KCC2wt) [940 phosphorylation. Treatment of immature hippocampal neurons with either agent results in decreased phosphorylation of Ser940. However, treatment of HEKrnKCC2b cells with staurosporine decreases, whereas NEM increases phosphorylation of Ser940. This is in line with the different effect of these agents on transport activity of the phosphomutants Ser31A/D, Thr34A/D, Thr999A and Thr1008A/D [Another key regulatory KCC2 phospho-site is the PKC-mediated phosphorylation of Ser(KCC2wt) , 77, 111940 phosphorylation. In immature cortical neurons [940 phosphorylation, whereas it decreases Ser940 phosphorylation in immature cultured hippocampal neurons  affect : 0.1 mM –101 that: 0.1 mM  and regu: 0.1 mM . This ofrnKCC2b cells, immature hippocampal neurons) and NEM treatment  decrease Ser940 phosphorylation resulting in an equal or diminished transport activity compared to KCC2wt [940 is not the key regulatory phospho-site mediating the staurosporine and NEM-based stimulation effect on KCC2. This is in line with recent published analyses showing that NEM still enhances the transport activity of Ser940A (mimicking dephosphorylated state), indicating that other phospho-sites are important in NEM-dependent stimulation [Our data furthermore reveal that staurosporine Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S1 Fig(PDF)Click here for additional data file.S2 Fig(PDF)Click here for additional data file.


CYP3A5 genotyped human liver microsomes (HLMs), and further integrated the acquired parameters into the PBPK model to predict the drug–drug interaction (DDI) in patients with different CYP3A5 alleles. The results indicated STA was a time-dependent and reversible inhibitor of CYP3A4 while only a reversible inhibitor of CYP3A5; SIA inhibited CYP3A4 and 3A5 in a time-dependent manner but also reversibly inhibited CYP3A5. The predicted fold-increases of tacrolimus exposure were 2.70 and 2.41, respectively, after the multidose simulations of STA. SIA also increased tacrolimus’s exposure but to a smaller extent compared to STA. An optimized physiologically-based pharmacokinetic (PBPK) model integrated with CYP3A5 polymorphism was successfully established, providing more insights regarding the long-term DDI between tacrolimus and Wuzhi capsules in patients with different CYP3A5 genotypes.Wuzhi capsule (WZC) is commonly prescribed with tacrolimus in China to ease drug-induced hepatotoxicity. Two abundant active ingredients, schisantherin A (STA) and schisandrin A (SIA) are known to inhibit CYP3A enzymes and increase tacrolimus’s exposure. Our previous study has quantitatively demonstrated the contribution of STA and SIA to tacrolimus pharmacokinetics based on physiologically-based pharmacokinetic (PBPK) modeling. In the current work, we performed reversible inhibition (RI) and time-dependent inhibition (TDI) assays with  CYP3A5*1 allele with no functional enzyme activity [The pharmacokinetic drug–drug interaction (DDI) is usually caused by the interference of a perpetrator drug on the metabolizing enzymes or drug transporters of a victim drug. Genetic polymorphisms that alter these enzymes or transporters thereby can affect the magnitude of a DDI. Many clinical cases have demonstrated such influence. For example, tacrolimus metabolism inhibition is significantly greater in renal transplant recipients lacking CYP3A5 accounts for 40–50% of the variability in tacrolimus dose requirement [CYP3A5 , as known as CYP3A5*3 [CYP3A5*3 allele is considered “inactive” and classified as a non-expresser phenotype, while the “active” CYP3A5*1 allele infers to CYP3A5 expresser phenotype [CYP3A5 non-expresser patients than in CYP3A5 expressers [CYP3A5 non-expressers [CYP3A5 genotype on DDI between tacrolimus and other drugs remains unclear [As a first-line immunosuppressive agent for organ transplant patients, tacrolimus is mainly metabolized by CYP3A4 and CYP3A5 ,2. Its ci) value of 0.049 μM (the substrate is erythromycin) and inactivates erythromycin N-demethylation in a time–concentration-dependent manner [inact) and the inhibitor concentration causing half-maximal inactivation (KI) values were calculated to be 0.092 min−1 and 0.399 μM, respectively [i values of 5.83 and 4.8 μM for midazolam 1′-hydroxylation and 4-hydroxylation inactivation activity, respectively [Wuzhi capsule (WZC), a preparation of Schisandra sphenanthera ethanol extract, is often coadministered with tacrolimus to alleviate drug-induced hepatotoxicity in transplant recipients . WZC conectively ,11 showeectively . Clinicaectively . Thus, iCYP3A5 polymorphism [CYP3A5*1 allele carriers to achieve similar blood concentration levels with CYP3A5 non-expressers [CYP3A5 allele could contribute to the variability of interaction between tacrolimus and WZC. The first step was to investigate the inhibitory potency of STA and SIA on tacrolimus metabolism by CYP3A4 and CYP3A5 separately. CYP3cide (PF-04981517), a selective inhibitor of CYP3A4, is commonly used to determine contributions of CYP3A4 and CYP3A5 individually for CYP3A-cleared drugs [CYP3A5*1/*3 with CYP3cide to acquire the relevant parameters of CYP3A5, and HLM genotyped CYP3A5*3/*3 without CYP3cide for CYP3A4. The second step was to quantify the contribution of STA and SIA on DDI under different dosing strategies and estimate the impact of CYP3A5 polymorphism on using tacrolimus and WZC. In this regard, PBPK modeling and simulation was applied to accomplish these objectives by providing a quantitative framework to assess potential DDI [Our previous work developed a valid physiologically-based pharmacokinetic (PBPK) model to quantify the DDI between tacrolimus and WZC without considering morphism . It was morphism . The Clipressers . TherefoPF-049815, a seleced drugs ,19. In ttial DDI ,21 and itial DDI ,23,24.i values for the inhibition by STA on CYP3A4 and CYP3A5 of 0.15 and 0.11 μM, respectively . With the corresponding double-reciprocal plot, the derived kinetic constants from the inactivation experiments were estimated. The results indicated that STA showed a potent TDI profile on CYP3A4 A,B whileCmax, Tmax) are listed in R2 = 0.9171) between the predicted concentration and the observed concentration was noticed  and TDI (case #2) by STA increased the AUC of tacrolimus by 1.78- and 1.33-fold, respectively, in the single-dose simulation, and showed 1.86- and 1.76-fold increase in the multidose setting.Under the circumstances of case #3, the predicted AUC of tacrolimus in blood after a single oral dose of STA was 195.23 ng/mL·h as a 1.90-fold increase, and the multidose of STA could increase the AUC of tacrolimus 2.41-fold. RI or TDI increased the AUC of tacrolimus 1.48- or 1.50-fold, respectively, in the single-dose simulation, compared with 1.52- and 2.27-fold increases in the multidose setting. On the other hand, for simulation with SIA, there was a small contribution to AUC ratio (AUCR) with the values of 1.10 and 1.39 only via TDI in single-dose and multidose simulation, respectively. All of the above data are presented in CYP3A5 genotypes.Prior pharmacokinetic studies have noted that WZC increased tacrolimus’s concentration by inhibiting CYP3A4/5 enzymes and our previous work has quantified the contribution of STA and SIA to the interaction from the combined CYP3A4/5 perspective. In the present study, we managed to acquire the inhibitive potency of CYP3A4 and CYP3A5 separately. Although acceptable fold error values (less than two) were noted in all three dosing regimens during tacrolimus model verification, there existed some disparities on the high end in lower dosing (1 mg) and higher dosing (5 mg) groups. Therefore, we decided to use 2 mg dosing in our DDI prediction model to reduce variation. Integrating with the obtained parameters and pharmacogenetic factors, we established an optimized PBPK model to evaluate the DDI between tacrolimus and STA or SIA in patients with two different CYP3A5 alleles to measure the inhibitory effect of STA or SIA on separate CYP3A enzymes. Our results suggested that STA not only exhibited reversible inhibition on CYP3A4 (Ki = 0.15 μM) and CYP3A5 (Ki = 0.11 μM), but also showed robust inactivated inhibition on CYP3A4 . It was far less than that reported previously  with erythromycin being a probe substrate [In vitro studies supported the hypothesis of distinct binding domains for each substrate subgroup  [i values (0.15 or 0.11 μM) from RI were way less than relevant clinical STA concentration, increasing the likelihood of clinical DDI and toxicity. In comparison with STA, SIA showed a mild RI on CYP3A5 with Ki values of 8.74 μM while little inhibition on CYP3A4, and a weak TDI on CYP3A4  and CYP3A5. The inhibition values were less than the reported ones in rat liver microsome (RLM), which could be explained by the fact of different species of microsome and the types of probe substrate [RI and TDI assays were performed with CYP3cide and HLMs of different ubstrate . On the edipine) ,28. One 5 Ki = 0. μM, but ® Simulator, plasma concentration-time profiles of tacrolimus under the single dose of 1, 2, and 5 mg in both CYP3A5 expressers and non-expressers matched well with the in vivo profiles. In the DDI study with a single dose of STA, the blood AUCR of tacrolimus was 1.90 in CYP3A5 non-expressers, and the contribution of RI was comparable to TDI (1.48 versus 1.50). However, the AUCR was 2.17 in CYP3A5 expressers and the potency of RI was higher than TDI (1.78 versus 1.33). Similarly, the blood AUCR in CYP3A5 expressers was higher than non-expresser (2.70 versus 2.41) in the multidose setting CYP3A5. It could be concluded that TDI was more prominent in the long-term administration due to its irreversible time-dependent manner. Compared with STA, the blood AUCRs of tacrolimus after SIA use were higher in CYP3A5 expressers than non-expressers in both dosing simulations. A small contribution was observed in the single-dose group, and the multidose use had a moderate one to DDI. Overall, both STA and SIA could increase the blood exposure of tacrolimus. STA showed greater potency than SIA in both inhibition mechanisms, corroborating our previous study. What could be drawn from the present study exclusively was that CYP3A5 showed a lower binding priority than CYP3A4, given the uneven increases among the three cases. It also should be noted that the rises in blood exposure of tacrolimus in CYP3A5 expressers were higher than non-expressers, suggesting the CYP3A5 non-expressers were less prone to inhibitory effects caused by STA or SIA.After integrating metabolic data and abundance of enzymes into SimcypCYP3A5 on DDI between tacrolimus and other drugs. It was reported that the trough concentration/dose (C0/D) of tacrolimus was significantly increased in renal transplantation adult patients with CYP3A5 expressers after 1, 3, 6, 12 months of WZC use, while it had no statistical significance in CYP3A5 non-expressers [CYP3A5*3 resulting in the expression of inactive proteins, CYP3A5 genotype-dependent inhibition identified in the present study could be explained. However, the other studies have come to the opposite conclusion. Renal transplant pediatric patients with CYP3A5 non-expressers suffering from nephrotic syndrome showed a higher blood concentration of tacrolimus than CYP3A5 expressers after one-week WZC use [Some studies have demonstrated the impact of genetic polymorphism in pressers . Similarpressers . Our res WZC use . Moreove WZC use . A possiAs tacrolimus is expensive and requires long-term use while WZC is affordable, the coadministration could cut down the cost of treatment by 40–60% for each patient per year . ApplyinCYP3A5 polymorphism could help understand the pharmacokinetics of tacrolimus with WZC use. Patients with CYP3A5 expressers exhibit a much greater inhibition by the two principal ingredients (STA and SIA) of WZC. Thus, our study supports a strategy to evaluate the inhibitive potency on different CYP3A5 genotypes and integrate the information into PBPK modeling to investigate the magnitude of DDI.One important future direction of individualized medicine is how to interpret and utilize pharmacogenomic variation. Knowledge of CYP3A5*1/*3 HLM (lot: 0710232) and CYP3A5*3/*3 HLM (lot: 0710253) were obtained from Transheep Co., Ltd. . All other chemicals and reagents were the same as described in our previous study [Schisantherin A  and schisandrin A  were obtained from PUSH Bio-Technology Co., Ltd. . Tacrolimus  was from Aladdin Biochemical Technology Co., Ltd. . Ascomycin  and prednisolone  were purchased from Dalian Meilun Bio-Technology Co., Ltd. ; CYP3cide  was obtained from J&K Technology Co., Ltd. . us study .m/z), declustering potential (DP) and collision energy (CE) for detection were shown in R2 = 0.9995) and 6β-hydroxyl-testosterone (R2 = 1.000), respectively.Tacrolimus and 6β-hydroxyl-testosterone in all samples were detected using HPLC-MS/MS methods in our published study . In brieCYP3A5*3/*3 HLM was used to study the inhibition mechanism of STA/SIA on CYP3A4. Similarly, CYP3A5*1/*3 HLM along with CYP3cide was chosen to determine the inhibition mechanism of STA/SIA on CYP3A5. In order to obtain the Ki value of STA/SIA on CYP3A4, different concentrations of tacrolimus  together with STA  or SIA  were pre-incubated with CYP3A5*3/*3 HLM (0.2 mg/mL) and 0.1 M potassium phosphate buffer solution  for 3 min in a shaking water bath at 37℃. The reactions were started with the addition of nicotinamide adenine dinucleotide phosphate  and stopped 10 min later upon adding a double volume of ice-cold methanol. For the Ki value of CYP3A5, CYP3cide with a final concentration of 0.5 μM and CYP3A5*1/*3 HLM with a final concentration of 0.2 mg/mL were pre-incubated for 10 min in the presence of NADPH (1 mM) before tacrolimus and STA or SIA were added to the mixture. Reactions were terminated with the same method as the CYP3A4 group. All incubations were carried out in triplicate. Samples were processed and detected as described in our previously published article [i value.In this study, CYP3A5*3/*3 HLM (0.5 mg/mL) was added to the incubation with diverse concentrations of STA  or SIA . After pre-incubation for 3 min at 37 °C in a shaking water bath, the enzymatic reaction was started upon adding NADPH (1 mM) with various pre-reaction time . For TDI on CYP3A5, NADPH (1 mM) was added to the incubation which contained CYP3A5*1/*3 HLM (0.5 mg/mL) and CYP3cide (1.2 μM) after being pre-warmed for 3 min. The mixture was added with different concentrations of STA or SIA 10 min later and shared the same various incubation times as the CYP3A4 group. Next step, an aliquot (20 μL) of the primary pre-reaction mixture was transferred to 180 μL of the secondary incubation system which consisted of PBS (0.1 M), testosterone (200 μM) and NADPH (1 mM). The secondary reaction was incubated at 37 °C for 10 min and terminated by adding 400 μL ice-cold acetonitrile containing prednisolone as the internal standard (0.3150 μM for CYP3A5*3/*3 HLM incubation and 0.07875 μM for CYP3A5*1/*3 HLM incubation). In order to calculate the inactivation kinetic parameters, a linear regression of the logarithm of “the percentage of formation rate of 6β-hydroxyl-testosterone” against the pre-incubation time in the function of various concentrations of inhibitors (STA or SIA) was applied. The observed inactivation rate of the affected enzyme (kobs) was the negative slope of the linear regression. kinact and KI were calculated using Equation (1) [I represents the concentration of the inhibitor.Two-step incubation was performed to evaluate the TDI assay. First step, for TDI on CYP3A4, tion (1)  by the dAll incubations were carried out in triplicate. Samples were processed and detected as described in our previously published article .® Ltd., Certara, Sheffield, UK). The drug-related properties including physicochemical and in vitro PK parameters were listed in All PBPK simulations were constructed using the population-based ADME simulator  value of each tissue was tested at 10, 100, and 1000 as a default setting for screening purposes to identify the most significant contribution to tacrolimus tissue distribution volume. The result showed the Kp of adipose tissue set at the maximum value improved model performance, while there was no evident effect for any other tissues such as brain, gut, heart, kidney, lung, or liver. The sensitivity analysis of the Kp value of adipose tissue ranging from 250 to 1000 indicated the best fitting at 1000, which specifically reflected the distribution phase of CYP3A5 expressers and non-expressers (Tacrolimus human PBPK models of CYP3A5 expressers and non-expressers were slightly modified based on the model we published before . The intpressers . The estmax), and time to reach the maximal concentration in plasma (Tmax) were assessed against the observed data using Equation (2).All simulation trials were conducted with a healthy virtual population (fasted state) derived from the clinical study ,5,25. ThKi, KI, and kinact values of CYP3A4 and CYP3A5 by STA and SIA were integrated into this DDI model. To determine the contributions of RI and TDI to fold-increase in tacrolimus AUC in CYP3A5 expressers and non-expressers, we simulated three cases with different inhibitions: case #1 with only RI, case #2 with only TDI, case #3 with combined RI and TDI.The model is considered to have good fitness if the fold error is less than 2 . The DDICYP3A5 genotypes regarding the long-term combination use.Based on our previous work, the present study aimed to distinguish STA or SIA’s inhibitory effect on CYP3A4 and CYP3A5 of different genotypes. An optimized PBPK model integrated with *morphism was successfully established, providing more insights to evaluate DDI between tacrolimus and Wuzhi capsule in patients with different


We observed the variations in 90Sr and 137Cs, which were emitted from atmospheric nuclear tests and nuclear power plant accidents, due to their diffusion, deposition, and resuspension. In March 2011, the Fukushima Daiichi Nuclear Power Plant accident occurred in Japan, and significant increases in 90Sr and 137Cs were detected at our main site in Tsukuba, Ibaraki. Our continual observations revealed that the 137Cs monthly deposition rate in 2018 declined to ~ 1/8100 of the peak level, but it remained more than ~ 400 times higher than that before the accident. Chemical analysis suggested that dust particles were the major carriers of 90Sr and 137Cs during the resuspension period at our main site. Presently, the effective half-life for 137Cs deposition due to radioactive decay and other environmental factors is 4.7 years. The estimation suggests that approximately 42 years from 2011 are required to reduce the atmospheric 137Cs deposition to a state similar to that before the accident. The current 90Sr deposition, on the other hand, shows the preaccident seasonal variation, and it has returned to the same radioactive level as that before the accident.We have measured artificial radionuclides, such as  We have continuously monitored artificial radionuclides in atmospheric depositions for more than 63 years in the Kanto areas around Tokyo, Japan. Our long-term observations clarified the historical variations in artificial radionuclides in atmospheric depositions as a result of nuclear tests and their atmospheric transport and circulation from the 1950s to the 1970s8. For example, after the Partial Test Ban Treaty (PTBT) in 1963, atmospheric radionuclide deposition from the stratosphere, called global fallout, started to decline. However, the decline of the deposition rate was slowed because China and France continued nuclear tests until 1980. After the last nuclear test in 1980, the decrease rate increased until ~ 1990  into the atmosphere and ocean21. Studies have estimated the amount of radioactive materials released from the accident22 and their geographic distributions25. Other studies showed the chemical and physical properties of the carriers of radionuclides, such as glassy particles27 and sulfate28, and estimated the resuspension processes of 137Cs in the atmosphere36 through dust suspensions34 or emissions of bioaerosols36.In March 2011, an earthquake with a magnitude of 9.0 occurred and the subsequent tsunami severely damaged the Fukushima Daiichi Nuclear Power Plant (FDNPP). The accident resulted in enormous emissions of artificial radionuclides including 90Sr, 134Cs, and 137Cs, respectively38. However, the rate of decline due to environmental removal processes is complex and depends on the weather, environment, and physical and chemical properties of radionuclides. It is crucial to understand the time scale of environmental decay to predict the fate of radioactive materials from accidents and to evaluate their long-term influences on the environment and human health. Hence, this study aims (1) to show our long-term observation results, (2) to estimate the current resuspension carriers of radionuclides, and (3) to evaluate their environmental decay period. To achieve this goal, we measured the radioactivities of 90Sr, 134Cs, and 137Cs and stable elements and isotopes  of monthly atmospheric deposition samples collected at two sites in different environments: suburban site A and mountain site B   was much higher than those resulting from nuclear weapon tests  and the Chernobyl accident   was lower than that due to the nuclear tests in the 1960s   were 7.0 (1.2–22.5) mBq m−2 at site A and 25.0 (6.1–76.4) mBq m−2 at site B, while those for 90Sr amounted to 1.9 (ranging from not detectable (N.D.)–6.0) mBq m−2 at site A and 26.0 (6.5–116.8) mBq m−2 at site B. The possible causes of the higher depositions rates at site B than those at site A are the differences in altitude  and local environmental effects .In March 2011, 90Sr and 137Cs in atmospheric depositions and that of 137Cs in aerosol samples rapidly decreased after the first surge in March 2011 , the second phase is dominated by tropospheric circulation and removal (from April to December 2011), and the third phase is dominated by resuspension (after January 2012). In the first phase, the direct discharge/emission of radioactive materials during the FDNPP accident and meteorological conditions governed the radionuclide concentration in the environment44. In the second phase, tropospheric transport of the radioactive materials remaining in the atmosphere after the FDNPP accident and their removal processes dominated atmospheric depositions29. The third phase (after January 2012) mainly depended on the resuspension of radioactive materials36. For comparison, the corresponding decrease rates  resulting from the Chernobyl accident were shorter than those resulting from the FDNPP accident  and ~ 1/4500 (3.0 Bq m−2), respectively, with regard to the peak levels after the accident. But these levels were still ~ 400 and ~ 130 times, respectively, higher than those before the accident  mBq m−2 and 33.8 (3.1–117) mBq m−2 at sites A and B, respectively  at site A. On the other hand, it peaks twice in May and September at site B  at site A and during the fall season (September and October) at site B , no seasonal variations in 137Cs at either site were observed  within the collected samples before the accident suggest that mineral dust particles are the dominant carriers of these radionuclides at site A  emitted through, for example, geochemical and biological cycles. The correlations between dust components  and radionuclides  observation exhibited the presence of inorganic salt particles including KCl, NaCl, and CaSO4 in dried deposition samples  for 90Sr and 137Cs deposition after the accident  of the 137Cs deposition were 195 days and 4.7 years, respectively, at site A, and those at site B were 148 days and 5.9 years, respectively. Interchange of the predominant short- and long-lived components occurred during the period between September and December 2013  excluded the direct emission period and 2) extended observation data by the end of 2018. The effective half-life of the long-lived component of 137Cs at site A after the FDNPP accident is shorter than that before the accident (8.5 years). There are two possible reasons for the difference between the effective half-lives before and after the accident. First, the dominant resuspension processes are different before and after the accident. Second, the elapsed time after contaminations is different between the pre and postaccident periods, i.e., more than 30 years had passed for the analysis period before the accident since the last atmospheric nuclear test, on the other hand, only 8 years had passed since the significant pollution after the FDNPP accident.The effective half-lives of the short- and long-lived components . A better understanding of the carriers, resuspension processes, and environmental circulation conditions of radionuclides is needed to confirm the above estimates. The radionuclide decreasing trend may change in the future if resuspension process, biological recycling, and their carriers changed. Finally, our observations only pertain to atmospheric deposition and provide limited features of the environmental radionuclide cycle. The contamination in other fields, such as surface soils, forests, and oceans, will exhibit different effective half-lives. Nevertheless, our continuous observations of the radionuclides in atmospheric deposition before and after the accident enable the evaluation of the atmospheric phases and the changes in various processes to regain the environmental conditions before the nuclear power plant accident.The above estimated effective half-lives imply that, based on the atmospheric 50. Sampling trays were placed on the rooftop of one-story (1980–2011) and six-story buildings (2011–) on the MRI campus. Site B was established in 2007 at the top of the Mt. Haruna (1390 m above sea level), Gunma, Japan. Sites A and B are 170 and 250 km away, respectively, from the FDNPP.We collected monthly atmospheric deposition samples at two sites: a suburban site in the Kanto Plain  and a mountain site in the northwestern corner of the Kanto Plain   and evaporating dishes followed by weight measurements. After March 2011, we collected aerosol samples using high-volume air samplers  on quartz fiber filters  at a flow rate of 700 L per minute to observe the atmospheric radioactivity concentration.Atmospheric deposition samples, which include both rain (wet deposition) and dry deposition, were collected using the above plastic trays with a total open area of 1–4 m6 s after the FDNPP accident. After the radiocesium measurement, 90Sr was radiochemically separated, purified, and solidified as Sr-carbonate precipitates. After leaving the sample for several weeks in order to achieve 90Sr and 90Y radioequilibrium, the β-activity was measured with an alpha/beta counting system  using a maximum live time of 103 min. The detection limits were 1.55 and 39.6 mBq m−2 for 90Sr and 137Cs, respectively, in the deposition samples, which were, obtained by multiplying each counting error measured in 2018 by three. Details on the sample preparation and measurement methods have been described in a previous study51.The activity of radiocesium was measured by Ge semiconductor detectors  coupled with a computed spectrometric analyzer (Oxford-Tennelec Multiport or Seiko EG&G 92x) using a maximum live time of 109Be, 133Cs, 232Th, and 238U) were measured by inductively coupled plasma atomic emission spectrometry  and inductively coupled plasma mass spectrometry , respectively, based on aliquots of the samples (3.6% in mass) during the acid decomposition processes. The detection limit and quantification values were estimated as three and ten times the standard deviation of ten measurements of 10 ppb standards. An SEM  with an energy-dispersive X-ray spectrometer  was adopted for chemical and physical analysis of the dried deposition samples.The stable elements  and isotopes (Supplementary Information.


Numerous data obtained by different research laboratories indicate that specific IgE production is triggered independently of specific IgG or IgA ones and so it is not linked to fully matured germinal centers formation in the secondary lymphoid organs. The aim of this study was to clarify whether specific IgE production is triggered by low antigen doses administrated in tertiary tissues enriched by lymphoid structures.Ovalbumin (OVA) in different doses (100 ng to 10 μg) was administrated three times a week for 4–5 weeks intraperitoneally (i.p.) or subcutaneously (s.c.) to female BALB/c mice in the wither region which is enriched in fat-associated lymphoid clusters or in the foot pad region not containing them.OVA-specific IgE was predominantly induced by low but not high antigen doses and only after immunization into the withers. IgE isotype switching was triggered exclusively in the withers adipose tissue but not in the regional lymph nodes while mature IgE expressing cells were observed both in the withers and lymph nodes. Anti-proliferative genotoxic stress inducing drugs shifted the balance from IgG1 towards IgE production.Tertiary lymphoid structures possess unique environment where B-cell antibody isotype switching to IgE predominantly occurs. This phenomenon is partially explained by hampered proliferation of B-cells in these structures. There is currently a significant increase in allergic diseases around the world, especially in the developed countries . These dIn order to develop new methods of ASIT dedicated to stimulate allergen-induced B-cell switching to IgG4 but not IgE isotype, it is clearly necessary to understand specific mechanisms triggering selective B-cell immunoglobulin class switching to these isotypes. For the development of the general strategy to eliminate IgE-producing B-cells and their precursors, it is important to know the exact site where B-cell IgE isotype switching occurs. We have previously shown that in young (1–8 years old) patients with allergy to house dust mite specific IgE production is not associated with specific IgG, IgG4 or IgA1 production , insteadLifestyle can significantly affect the severity of atopic diseases. One of the characteristics of the developed country population is the high prevalence of obesity . EpidemiDespite a large number of successively developed mouse allergy models, most of them require high allergen doses –17 sometThe aim of this work was to clarify the hypothesis that B-cell IgE class switching in response to low, but not high, administrated antigen doses occurs mostly in the specific milieu of TLSs but not in SLOs. In clinical practice allergens usually pass through the respiratory barrier tissues where inducible bronchial-associated lymphoid tissue or nasal polyps represent TLSs , 30. In We demonstrate here that direct IgE class switch in response to subcutaneously administrated allergen occurs in local TLSs, and adipose tissue provides unique environment for B cell activation.To reconstitute typical clinical situation in which allergens enter mucosal tissue over a long period, mice were repeatedly immunized s.c. with low (10 or 100 ng), intermediate (1 μg) or high (10 μg) antigen doses in the regions which are enriched in TLSs such as withers (s.c.w.) and peritoneal cavity (i.p) or lack them .The results Fig.  a show tSpecific IgG1 production Fig. .Fig. 2SIt is well appreciated that in humans type I hypersensitivity reactions mediated by mast cell and basophil mediators are linked exclusively with allergen-specific IgE antibodies. However, in mice IgG1- and even IgG2a- mediated type I hypersensitivity becomes possible because of basophils degranulation in response to IgG immune complexes . In addio) was associated with the specific IgE or IgG1 production. Results  [Ebi2) [Aicda) [germline ε and germline γ1 transcripts respectively) [Aicda, germline ε and germline γ1 genes were triggered exclusively in the withers tissue but not in the regional lymph nodes. It is interesting that even high antigen doses have not triggered germline transcripts expression in the lymph nodes while they have triggered Aicda expression   or extra) [Ebi2) . We have [Aicda)  and the ctively) . As showion Fig. , a.Fig.cd19 expression. Both doses of antigen induced comparable levels of extrafollicular B-cell activation marker (Ebi2) but low doses induced more pronounced germinal center formation in the withers characterized by Bcl6 upregulation. However, it is not surprising that high antigen doses induce significant accumulation of both types of activated B-cells in lymph nodes. Predominant expression of transcripts corresponding to B-cell activation in low dose group was observed not only when expression was normalized to Gapdh but also when it was normalized to Cd19 (data not shown). B-cell IgE class switching depends on IL-4 and less on IL-13 which could be produced by either T helper 2 or ILC2 cells. Both cell types express transcription factor GATA3 [Gata3 and Nmur1 genes expression in the withers tissue. Only high antigen doses triggered expression of Gata3 in the lymph nodes  and Il33 (both groups) but not TSLP was induced  promoter following (after their splicing) by CH sequence antibody, coding postswitch transcripts expressed after Ig isotype switching starts at their 5ʼ end with a specific pre-rearranged VDJ sequences followed by common intronic non-coding sequences corresponding to iEμ (intronic Enhancer μ) and Iμ chromosomal regions and specific CH coding exons [H coding exon. Indeed, data from Fig. postswitch ε and postswitch γ1 transcripts was induced in the regional lymph nodes. It is not surprising that in a case of regional lymph nodes postswitch ε and postswitch γ1 transcripts were mainly induced by low and high antigen doses, respectively, regardless of the reference gene, i.e. Gapdh or Cd19  increase in OVA specific IgE production after prolonged (14th immunizations) treatment, though only high drug dose (20 μg/injection) demonstrated a significant effect. After short time treatment (7th immunizations) only etoposide effect was significant. In contrast, specific IgG1 production at short time period was not affected by these drugs but was slightly, though significantly (p < 0.05), dampened after long time treatment  stimuli, dendritic cells are either not activated or activated in a tolerogenic but not immunogenic way, and their maturation and following migration from tissues to SLOs are rare . Therefocd19 gene despite induction of genes corresponding to B-cell activation and Ig class switching. Because cd19 gene expression should correspond to the B-cell numbers [Low antigen doses are very weak immunological stimulus which does not engage PRRs and very weak triggers of FALCs immune cell activation. So, cells activated by low antigen doses in these structures would be stimulated occasionally in the presence of very low amounts of proliferation supporting stimulus  and cell attracting chemokines. The latter may intensify activation-induced B cell migration from tissues to SLOs. This hypothesis was confirmed by reduced expression of  numbers , 52 and  numbers  and so lpostswitch ε and γ1 transcripts expression indicates that despite the local tissue-restricted B-cell activation IgE production occurs mostly in SLOs, i.e. regional lymph nodes. It is low rather than high antigen doses induced high levels of specific IgE production, according to ELISA, which enhanced the expression of postswitch ε transcripts in the lymph nodes. Also the expression of postswitch ε transcripts in the lymph nodes normalized to Gapdh was relatively weak which probably corresponded to larger numbers of mature antibody producing cells. This reflects the presence and availability of a higher number of B-cell niches in the lymph nodes than in the withers. In mice, upon high dose antigen administration, the migration of IgE-switched B-cells from TLSs to SLOs does not occur or occurs relatively weakly, therefore, the IgE switched B-cells, that not completely differentiate to plasma cells, accumulate in TLSs. These cells are probably responsible for low level IgE production observed in high dose group.Similarly, the data on + B-cells have diminished expression of anti-apoptotic proliferation supporting transcription factor Bcl6 [+ B-cells have low levels of costimulatory molecules expression and were more apoptosis prone than IgG1+ B-cells [Supposing that (i) B-cell proliferation in TLSs, namely FALCs, especially in response to weak immunological stimulus, is hampered due to low numbers of B-cell supporting niches and (ii) it is FALCs B-cells that are switched to IgE production in a response to low antigen doses, it is logical to speculate that hampered proliferation in FALCs is responsible for shifting the balance towards IgE production. Indeed, in this work, we showed that anti-proliferative drugs, especially hydrophobic  etoposide, enhanced specific IgE production. But the effects of the same drugs on IgG1 production were opposite. These data are in agreement with the recent observations. For example, in reporter transgenic mice expressing IgE fused with fluorescent protein, the isotype switching occurs mostly in early stages of anti-helminthic immune response when B-cells proliferation in germinal centers is not so rapid as at the later stages , 26. IgEtor Bcl6 . In the  B-cells . However B-cells . The samIn conclusion, in our work we introduced a new low-dose reproducible mouse allergy model which can be an alternative to standard allergy models with high antigen dose usage. In this new model all mice produceed significant specific IgE and IgE isotype switching was triggered in the damaged tissue but not in the lymph nodes. So this new model may be more clinically relevant that commonly used high antigen dose and adjuvant based mouse allergy models. We hope that this model may be interesting and useful for further investigations of IgE production triggering. This model may be useful in studying the mechanisms responsible for the more severe course of atopic diseases in overweight individuals in which the excess of adipose tissue may correspond to the excess of numbers of FALCs in which B-cell proliferation relative to SLOs is hampered and IgE switching occurs more effective. Although the authors do not deny that the other mechanisms may also be responsible for IgE+ cells generation in such structures, for example chronic expression of pro-inflammatory cytokines and adipokines, that may be the subject for future investigations.In summary, low dose of administrated allergen triggers B-cell activation and IgE class switching exclusively in TLSs in the damaged tissue. High antigen doses weakly activate B-cells in TLSs and weakly, if any induce IgE antibody production. Hampered B-cell proliferation could shift the balance from IgG1 towards IgE switching in these structures. Despite this fact mature IgE producing plasma cells differentiate from immature precursors in SLOs to where they migrate after the activation in TLSs.Female wild type BALB/c mice (6–8 weeks old) were obtained from Andreevka Center . Mice were housed in SPF conditions about 2 weeks before experiments. During all experimental procedures mouse were kept in 12-h light dark cycle at room temperature in a special room in vivarium. Mice were housed in plastic cages (10–12 mice for 1 cage) with filings as a bedding material. Mice were fed ad libitum by granular feed. Mice were treated and their health status was monitored according to an approved by IBCh RAS IACUC protocol.n = 5) We used 2 experimental groups for each immunization protocol when studied the dependence of antibody production from the site of immunization and antigen dose (n = 5). The number of animals in groups was determined so that for U-test quantification p < 0,01 values could be obtained. Blood sampling from retro-orbital sinus was performed after 7th (17th day) or 14th (33th day) of immunization. Blood was collected, coagulated for 15 min at 37 °C and spinned . Serum was collected and stored at − 20 °C prior to use.Ovalbumin  was used as a model antigen. Mice received OVA by intraperitoneal (i.p.) and subcutaneous injections in foot pad (f.p.) or in the withers areas (W). OVA was administrated repeatedly 3 times a week for 4–5 weeks  in low (100 ng/injection) or high (10 μg/injection) doses in total volume of 100 μl saline. Before study the randomization procedure was performed. Two control groups were commonly used in the study, namely intact mice and saline immunized mice (n = 5) were immunized with low OVA dose with anti-proliferative drugs doxorubicin and etoposide (Sigma Aldrich) in doses of 2 and 20 μg/ml which corresponded to doses 0.1 and 1 mg/kg. These doses are too low in comparison with usually used for treatment of malignancies [n = 6–7).In some experiments, mice  Optical densities (OD) were measured with automatic 96-well plate reader  at 450 nm with subtraction of optical density at 620 nm that does not correspond to the reaction colored product. Antibody quantities were estimated as OVA-specific antibody titers defined as the serum dilution, in accordance according to 4-PL regression analysis.Microtiter plates  were coated with 50 μl of 5 μg/ml OVA solution in PBS (pH = 7.2) and incubated overnight at + 4 °C. Following extensive washing with PBS containing 0.05% Tween-20 (PBS-T) and subsequent blocking with 5% BSA in PBS, plates were incubated with serially diluted serum samples at + 4 °C overnight. Next day plates were subsequently exposed to primary biotin-labeled anti-mouse antibodies  to either IgEa (clone UH297) or IgG1 (clone RMG1 1) or IgG2a (clone RMG2a 62), and streptavidin-conjugated HRP. Plates were further processed with substrate solution . Serum samples were added in 1:100 dilution in duplicates with different dilutions of mouse IgE κ . In following steps biotin labeled anti-mouse IgE (UH297) and streptavidin-HRP were added subsequently as described above.For measurement of systematic anaphylactic response in mice after full immunization protocol, OVA in challenging dose (0.2 ml of 2.5 mg/ml solution) was administrated i.p. to animals. Body temperature was measured with infrared thermometer CEM DT-8806S,  as it was performed in  before OStandard Evans Blue based test was used to measure local anaphylaxis response. Briefly, abdominal region of each mouse was shaved and after that different doses stating from 10 μl of 1 mg/ml OVA were administrated subcutaneously in this area. After 3 min 200 μl of 0.5% Evans Blue solution were administrated in the tail vein. After 15 min reaction intensity was evaluated.After 14th immunization mice were sacrificed in laboratory by cervical dislocation. Lymph nodes were harvested, homogenized and cells were spinned in PBS , followed by the RNA extraction using phenol chloroform based extraction protocol. . In the case of white adipose tissue small pieces were harvested and washed from blood in the PBS and then subjected to RNA extraction with the same reagent. cDNA was prepared from the samples with RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific). Quantitative PCR was performed with SybrGreen I based kits from BioLabMix . Expression of target genes was estimated by normalizing to expression of house-keeping gene GAPDH and was calculated as 2^-d(dCt) in comparison with expression in the tissues of intact control mouse. In the case of postswitch ε transcripts for analysis of absolute IgE-producing B cell numbers in samples, normalization to GAPDH was performed. For measurement of IgE-producing B cell content in B cell fraction, normalization to CD19 was performed. Primers were designed and synthesized by EvroGen. The following primers were used:for GAPDH F: GGTGCTGAGTATGTCGTGGA; R: TGGAAGAGTGGGAGTTGCTG;IL-25 F: CCCAGCAAAGAGCAAGAACC; R: ATCCTCTAGCAGCACAAGCG;IL-33 F: GTCTCCTGCCTCCCTGAGTA; R: GTGGTGCCTGCTCTTCTGAA;TSLP F: CTGCCTGAATCAAACCTCACAA; R: TGACTGCCCGAACTGTCATT;CD19 F: TTCACTACTGAGCCTAAGCCTTG; R: CAACAGCCAGAGCCACACT;AICDA F: ACAGCACTGAAGCAGCCTT; R: GCCCAGCGGACATTTTTGAA;Bcl6 F: CAGTCCCCACAGCATACAGA; R: CCTCAGAGAAACGGCAGTCA;EBI2 F: CATAAAAGGACGCCTGCTCG; R: TTGCCAGTGGGGTAGTGAAA;germline ε F: GCACAGGGGGCAGAAGAT; R: CCAGGTCACAGTCACAGGAT;germline γ1 F: CACGGGAGATTGGGAAGGAG; R: TTTGGGCAGCAGATCCAGG;Gata3 F: TGGAGGAGGAACGCTAATGG; R: GATGTGGCTCAGGGATGACA;NMUR1 F: ATGACTCCTCCCTGCCTCA; R: GAGCACCAGCATATCGGACA;postswitch ε F: CAGGCTGCTGCTGGGTAG; R: GCTGGTGGTGACCTTGAGTT;postswitch γ1 F: ACATGCTCTGTGTGAACTCCC; R: AGGTCAGACTCCAGGACAGC. Ct values were determined for each sample. Reaction was performed in DT Prime Amplificator  according to the following protocol: initial denaturation at + 95 °C for 3 min and then 50 cycles with: 15 s denaturation at + 95 °C, 45 s annealing and elongation at + 60 °C.p < 0.05 were considered statistically significant. For correlation coefficients determination, Spearman test was used. The data from each individual animal was considered as a single point. Mean and standard deviations for each compared group were calculated.For comparisons between experimental groups and samples, nonparametric Mann-Whitney test was used. Levels of


Retroperitoneal fibrosis (RPF) is a rare disease with a poor prognosis characterized by systemic inflammation and fibroinflammatory tissue. Idiopathic RPF (IRPF) accounts for approximately two-thirds of RPF cases.A 56-year-old female patient with abdominal distension was admitted to Changxing Hospital. Laboratory tests revealed mild anemia and elevated CA125, while IgG4 and autoantibodies were within the normal ranges. Computed tomography (CT) revealed a gallbladder-occupying lesion, pancreatic cyst and retroperitoneal mass, which may have contributed to bilateral ureteral compression and hydronephrosis. The initial diagnosis was gallbladder carcinoma with lymph node metastasis. Then, abdominal adhesiolysis, cholecystectomy and partial hepatectomy were performed. Histologically, there were fibrosis and inflammation in the retroperitoneal tissue without any malignant cells in the retroperitoneal or gallbladder tissue. Finally, we confirmed the diagnosis of idiopathic retroperitoneal fibrosis, chronic cholecystitis and pancreatic cyst. The patient recovered well following the CT scan, in which dilatation of the bile duct was reduced, and effusion of the bilateral upper ureter was no longer significant.This atypical case illustrates that RPF can be combined with other biliary tract diseases. The coexistence of other diseases conceals the symptoms of RPF, which increases the difficulty of image identification. A high degree of suspicion is necessary for routine clinical work. As more cases are reported, further advances in the diagnosis and treatment of RPF can be expected. Retroperitoneal fibrosis (RPF) is a rare disease with a poor prognosis characterized by systemic inflammation and fibroinflammatory tissue. Idiopathic RPF (IRPF) accounts for approximately two-thirds of RPF cases, and the remaining third are secondary to specific causes such as medications, neoplasms, radiotherapy, infection, surgery and malignancies , 2. UnfoA 56-year-old woman with a history of rheumatism complained of right upper abdominal distension half a month prior. Abdominal distension was not accompanied by pain, exacerbated after meals and relieved after rest. Decreased urine output, nausea, vomiting, fever or chills were denied by the patient. Physical examination revealed the following: temperature, 37.2 °C; blood pressure, 123/70 mmHg; heart rate, 70 per minute; and respiratory rate, 18 per minute. There was no evidence of jaundice. Cardiac, pulmonary and neurologic abdominal examinations were all unremarkable. Dermatologic examination demonstrated pigmentation of both lower extremities. Laboratory tests showed elevated indirect bilirubin , decreased albumin , mild anemia with hemoglobin , and elevated CA125 , while other tumor markers, creatinine, autoimmune and serum IgG4 were all within normal ranges. On the abdominal computed tomography (CT) scan, a rim of soft-tissue density in the retroperitoneum extended and encased the upper end of the bilateral ureter, causing bilateral hydronephrosis by ureteral obstruction , C-reactive protein (CRP), anti-nuclear antibody (ANA) titer, and anti-neutrophilic cytoplasmic antibody (ANCA) titer . In futuBecause imaging evidence is critical to the diagnosis of RPF, CT and MRI are considered to be of more importance than other devices . HistoloThe greatest limitation of our case was that a biopsy was not taken for the initial pathological diagnosis. In summary, this atypical case illustrates that RPF can be combined with other biliary tract diseases. The coexistence of other diseases conceals the symptoms of RPF, which increases the difficulty of image identification. A high degree of suspicion is necessary for routine clinical work. As more cases are reported, further advances in the diagnosis and treatment of RPF can be expected.


To this end, we used two qualitative, weight-of-evidence methods. Method 1 employs nine criteria to select assessments for PDQA and help control for confounders. To conduct PDQA, Method 2 uses three US Environmental Protection Agency standards—the temporal, geographical, and technological representativeness of sampling. Our Method 1 results reveal four current toxic-site assessments ; at all of these sites the main risk drivers are solvents, volatile organic compounds, including trichloroethylene. Our Method 2 results indicate that all four assessments violate most PDQA standards and systematically underestimate health risk. These results reveal environmental injustice, disproportionate health threats to children/minorities/poor people at all four sites. Although preliminary, our conclusion is that alleviating harm and environmental-health inequalities posed by urban-infill-toxic-site pollution may require improving both the testing/cleanup/redevelopment requirements of the New Urban Agenda and the regulatory oversight of assessment and remediation performed by private redevelopers.Most hazardous-waste sites are located in urban areas populated by disproportionate numbers of children, minorities, and poor people who, as a result, face more severe pollution threats and environmental-health inequalities. Partly to address this harm, in 2017 the United Nations unanimously endorsed the New Urban Agenda, which includes redeveloping urban-infill-toxic-waste sites. However, no systematic, independent analyses assess the public-health adequacy of such hazardous-facility redevelopments. Our  Children, minorities, and poor people are disproportionately represented among those living near hazardous-waste sites; as a result, they face at least five times higher toxic exposures and environmental-health inequalities than other citizens . They beIn poor- and middle-income nations, at least 200 million people are at risk from thousands of mostly urban hazardous-waste sites , only 3%Globally these mostly urban, toxic-waste sites will soon threaten even more people because, by the year 2050, 70% of the world’s population will live in cities, up from the current 55% . All memTo promote urban-infill-toxic-site redevelopment, the World Bank ,10, the Despite the UN’s New Urban Agenda and the World Bank’s and EU’s apparent promotion of semi-privatized cleanup, information about the public-health consequences of semi-privatized toxic-site cleanups is largely anecdotal. On one hand, semi-privatized assessments/cleanups are cheaper and faster but, on the other hand, critics charge that semi-private testing/remediation often fails government scientific-data audits, is “typically driven by purely [private] financial motives” , and freNo one has conducted an independent, systematic, proactive analysis of whether semi-privatized urban-infill-toxic-site testing/remediation actually satisfies scientific data-quality-assessment (DQA) standards, a necessary condition for protecting public health and environmental justice/equality. The objective of this analysis is to conduct a preliminary investigation see the , a preliPerhaps one reason no one has yet conducted an independent analysis of the quality of vapor-intrusion studies of toxic sites is that VI assessment is relatively new. In the 1960s, scientists recognized radon and petroleum-landfill VI, but not until the 1970s did they understand that VOCs are also VI-inhalation carcinogens. Similarly, not until the late 1980s did the US Environmental Protection Agency (EPA) warnings to assess VOC VI; it did so in its Risk Assessment Guidance for Superfund sites and its draft VI Guidance .In assessing toxic-site VOC testing, we follow US EPA and define representativeness in terms of three standards: the “temporal, geographical, and technological correlation” or representativeness between sampling data and the true population of interest  (p. 11).Testing our hypothesis (see above), that toxic-site sampling may fail PDQA and thus threaten public health and environmental justice, is important partly because pollution is the largest environmental cause of global disease and premature death . Even inThis PDQA of the representativeness of toxic-site-VOC sampling relies on two main observational, weight-of-evidence (WoE) methods.  and trichloroethylene (TCE) have been known at least since 1969, when disproportionate numbers of children in Woburn, Massachusetts began dying of leukemia [Second, although global data are not available, the majority of US toxic sites, at least 88,000 [Third, despite these VOC public-health risks, and the ubiquity of urban sites contaminated with solvent VOCs, VI field investigations are common in only 5 countries: Australia, Canada, Denmark, the United Kingdom, and the United States [Regarding Criterion 3 in leukemia , subsurfleukemia . Second,t 88,000 , could ct 88,000 ; other nd States .Regarding Criterion 4 in Regarding Criterion 5, to help avoid the confounder of inadequate law/regulation/guidance causing flawed testing, this criterion specifies evaluating only California hazardous-site sampling. Why? California toxic-site laws/regulations generally are stricter/more comprehensive than federal laws , and, alTo operationalize Method 1′s preceding assessment-selection criteria 4–5 in Criterion 6 in Criterion 8 requires that assessments to be evaluated have publicly accessible soil-gas data sheets and other documents on the California Department of Toxic Substances Control (DTSC) Envirostor website . This an3 TCE airborne exposure can “produce an adverse developmental effect” on the blood, kidneys, and liver and on the cardiovascular, immune, nervous, and respiratory systems [In order to select especially health-threatening hazardous-waste locations, Criterion 9 of  systems ,37  Are samples less than 3 years, preferably within 1 year, old?ast year . TherefoThe second representativeness standard, geographical representativeness, measures whether data collection is in “the desired geographical location” [(RepQ2) Were data collected near contaminant sources?(RepQ3) Do samples trace the full horizontal/vertical extent of vapor-con-taminant plumes?(RepQ4) Do samples provide a 3-D delineation of all subsurface contamnants?(RepQ5) Are data dense enough to provide isoconcentration-contour maps for all toxins?(RepQ6) Do data provide at least two sub-slab-sampling locations per building?ocation” , that isocation” ; (ii) toocation” ; (iii) tocation” ; (iv) toocation” ; and (v)ocation” . Followiocation” ,42,43,44The third representativeness standard, technological representativeness, measures whether the scope of sampling technology adequately captures at least 4 aspects of the data source or target system: (i) the design conditions imposed on sampling; (ii) the material stability (input/output) of sampling products; (iii) the process scale, sampling output per time/space; and (iv) the site-specific operating conditions [(RepQ7) Are method-reporting limits sensitive enough for a screening evaluation?(RepQ8) Are samples from permanent/semi-permanent soil-gas wells?(RepQ9) Does soil-gas sampling output show seasonal/temporal varia-tions?(RepQ10) Is soil-gas sampling conducted under “steady-state conditions”?nditions . Governmnditions . Followienvirostor.dtsc.ca.gov/public/profile_report?global_id=19970020; (2) the 10-acre heavy-manufacturing site, Monrovia, California, Envirostor ID 60002828 envirostor.dtsc.ca.gov/public/profile_report?global_id=60002828 and cityofmonrovia.org/home/showdocument?id=21816; (3) the 51-acre former Raytheon Missile site, in California’s Canoga Park neighborhood, Envirostor ID 80001366 envirostor.dtsc.ca.gov/public/profile_report?global_id=80001366; and (4) the 18-acre former Santa Fe Railyards, Boyle Heights, California, Envirostor ID 19400008 envirostor.dtsc.ca.gov/public/profile_report?global_id=19400008.Four Los Angeles County hazardous-waste sites meet our preceding 9 criteria for selecting high-risk locations. Because the developers claim they have completed and submitted sampling at all four locations assessed in this study, all testing results are supposedly final at each of these four sites. These are (1) the 9-acre former US Naval Ordnance Testing Station, Pasadena, California (NOTSPA), Envirostor ID 19970020 To evaluate the four soil-gas assessments revealed by Method 1, we used 10 representativeness questions (RepQ) based on US EPA DQA standards and California DTSC soil-gas “requirements” see . These 1Temporal representativeness is important because only recent sampling ensures that site toxins have not migrated either deeper or further offsite, thus threatening additional victims. Dated sampling can reveal only some of the locations of toxic waste, as it might not capture later contaminant migration. At Pasadena’s former NOTSPA, a US military weapons-testing site, the latest soil-gas sampling was in 2007 , submittNone of the soil-gas data for the four sites includes sampling near all sources, many of which remain unknown, because no assessments conducted a full-site, soil-gas survey, randomized sampling, or grid-based sampling. Consequently, Pasadena documents identified only 5- and 15-ft sources for only 2 (carbon tetrachloride and perchloroethylene) of roughly 35 contaminants . MonroviCBRE/TCC conducted no Canoga Park sitewide soil-gas survey, despite California DTSC’ repeatedly requesting it ; consequAs indicated, no sites had full soil-gas surveys, randomized sampling, or grid-based sampling. As a result, no assessments trace all-contaminant extent (RepQ3), their 3-D contours (RepQ4), or all-contaminant, all-depth isoconcentration contours (RepQ5) for the Pasadena , Monrovi3 carbon tetrachloride at location NMSD3, up to 307,500 times above their own screening/health-protective level (0.067 µg/m3) . Insteable 3 in ). Insteable 3 in . Likewise, because Monrovia assessors tested only shallow soil, only at one small area of the site, they failed to meet RepQ3, RepQ4, and RepQ5 . Because3 at location SG2011-1) 200 times above their own health-protective/screening level (1300 µg/m3) [Instead of following RepQ3, Canoga Park assessors sampled only three soil-gas locations, each only 20% of the way down to groundwater   ; yet the0 µg/m3) . However0 µg/m3) . TCFM ob0 µg/m3) ). In sumBecause of VOC volatilization at open-air-sampling spots and especially shallow, sandy soil at all four sites ,41,43,44None of the four sites satisfies the technological-representativeness standard of having method-reporting/detection limits at least as sensitive as screening/health-protective levels . 3, up to three orders of magnitude less protective than DTSC’s required health-protective/screening levels, e.g., 0.067 µg/m3 for carbon tetrachloride [3 [3 for perchloroethylene, 0.48 µg/m3 for trichloroethylene) [3 for perchloroethylene) [3 for perchloroethylene [3 for perchloroethylene and trichloroethylene [3 for perchloroethylene and 0.48 µg/m3 for trichloroethylene [For all site contaminants, CBRE/TCC’s Pasadena reporting/detection limits are 20 µg/mchloride . CBRE/TCoride [3 , up to tthylene) . CBRE/TCthylene) , in docuthylene) , are up ethylene . CBRE/TCethylene , are up ethylene . In shorNone of the four sites satisfies the technological representativeness standards of employing only permanent/semi-permanent wells (RepQ8) and measuring sitewide, same-location, same-contaminant seasonal/temporal variations (RepQ9) .Given only temporary wells, the preceding table shows that neither Pasadena   magnitude of contamination because no tests sampled all of the highest-contamination areas (see the authors’ Table 8). That is, the preceding results show that assessors failed to conduct all required near-source sampling (RepQ2), under-slab sampling (RepQ6), and long-term sampling (RepQ8), including sampling showing seasonal/temporal variations (RepQ9). Likewise, no results showed the greatest/highest extent of contamination because tests at most sites used outdated samples (violating RepQ1) that failed to account for contaminant migration, and they did not trace contaminant extent (RepQ3), 3-D-contaminant outlines (RepQ4), and isoconcentration contours (RepQ5) (see the authors’ Table 8). Results likewise show that assessors also employed insensitive tests (RepQ7) and conducted no tests under steady-state conditions (RepQ10), all of which failed to capture the highest levels of most site pollutants. Instead, this systematic contaminant underestimation, at all four sites, indicates that pollutants could be migrating offsite, into buildings (VI), or into groundwater and thus causing public-health harm. The fact that all 10 PDQA violations cause risk underestimation suggests that the results are systematically biased; normal or expected errors likely would cause a mix of over- and under-estimates.One of the most significant aspects of these 39 sampling-representativeness violations is that all of them appear to cause risk underestimation, and none appears to cause risk overestimation. No site results revealed the highest/worst Flawed sampling that causes such risk underestimates is especially worrisome because whenever toxic-site testing underestimates health risks, by failing to conduct complete, adequate sampling, the resulting hazardous-site cleanup is likely also to be incomplete; this is because toxic-chemical threats that are not detected, through testing, cannot be remediated. Yet if toxic-site health threats are not remediated, obviously people could be harmed.First, because the Pasadena [Consider four specific public-health threats from the toxic sites because of flawed testing and resulting risk underestimation. Pasadena  and MonrPasadena  toxic siPasadena . As alrePasadena ,36.Second, the Pasadena–Monrovia toxic-site-apartment-residence developments are the only of the four sites built immediately adjacent to a freeway; both sites abut a 10-lane, Los Angeles, East–West, diesel-truck artery [k artery , Interstk artery .Not only are residents of these two sites (Pasadena and Monrovia) likely to be disproportionately children, minorities, and poor people, as Third, our results likewise show that the public-health consequences of the preceding risk/contaminant underestimates could be especially dangerous at the Pasadena [Pasadena  and CanoPasadena  sites. Tfourth public-health threat is that, despite the fact that the four site assessments together violate 39 of 40 DQA “requirements,” at every site CBRE/TCC misrepresented its non-representative sampling as safe  [A CBRE/TCC’s 2018 Canoga Park misrepresentations are especially disturbing because its own documents repeatedly and explicitly contradict its written claims to state regulators and near-site residents. CBRE/TCC petitioned the regulator for no further cleanup action, on grounds that site TCFM (a) is not “a carcinogen and is low in toxicity”, (b) is “below all available Environmental Screening Levels (ESLs)”, and (c) exhibits “no significant increase of concentrations with depth” . HoweverFirst, CBRE/TCC’s 2018 low-toxicity claim is false because TCFM is an ozone-depleting and global-warming chemical, which has been banned internationally since the 1986 Montreal Protocol. It is also a cardio-/neuro-/pulmonary toxin that causes reduced human-lung capacity, bradycardia, and heart arrhythmias after only 15-second, 16,000 µg/m3 airborne exposures; at higher doses, TCFM causes cognitive malfunction, pneumonia, lung inflammation, hemorrhage, pleuritis, and pericarditis [3 TCFM dose is lower than 86% of Canoga Parks’ soil-gas levels that could enter indoor air [carditis . Yet thidoor air . CBRE/TCdoor air .Second, CBRE/TCC’s 2018 below-all-screening-levels claim is false because when the Canoga Park sampling was conducted in 2018, the 2018 EPA TCFM-screening/health-protective level was 730 µg/m3 [3 level by three orders of magnitude [3 TCFM [30 µg/m3 . Yet thiagnitude . Moreove [3 TCFM , and 93% [3 TCFM .Finally, CBRE/TCC’s 2018 claim that contaminants exhibit no increases with depth is false because when the Canoga Park sampling was conducted, all three soil-gas-sampling locations showed increasing-with-depth TCFM [3 (6 to 47 ft); from 170,000 to 260,000 µg/m3 (5.6 to 33 ft); and from 140,000 to 170,000 µg/m3 (6 to 35.5 ft). Respectively, these Canoga Park samples show 1600%, 150%, and 120% TCFM increases with depth [pth TCFM  (Table 1th depth —yet anotThe preceding examples of misrepresentations, non-representative sampling, and health-threatening pollution underestimates will disproportionately affect all four toxic-site neighborhoods, as subsequent sections show that all four sites are environmental-justice communities. The failure to adequately assess/sample toxic-site contaminants, as shown by our PDQA results, exacerbates already-existing, inequitable pollution and health harm.As subsequent paragraphs show, all four sites are environmental-justice communities, home to disproportionate numbers of children, Hispanics, and poor people (see the authors’ Monrovia’s beside-Interstate-210 census blocks likewise house the poorest two quintiles of California residents . These cIn the Canoga Park neighborhood of Los Angeles, poverty is 85% higher  and 44% The Boyle Heights neighborhood of Los Angeles has a poverty rate 130% higher than California generally, its Hispanic population is 147% higher than California generally, and its rate of households with age-18-and-under children is 42% higher than for California as a whole . Boyle Hlimitations of this analysis, this article provides only a preliminary DQA, as it covers only soil-gas sampling at only four semi-privatized urban-infill-development, VOC-contaminated sites. Our preliminary assessment also identifies only DQA representative-sampling violations. As such, it provides neither a full DQA nor any quantitative evaluation of health harm resulting from site-risk underestimates and failure to follow government soil-gas sampling “requirements” that are needed to ensure data quality [Regarding  quality .Future research might examine non-urban-infill, non-semi-privatized, non-California, non-US, non-CBRE/TCC assessments/cleanups, including those with non-VOC contaminants, to see whether future results are consistent with our results. Researchers also might examine exposures/potential health risks associated with “remediated” toxic sites, including any association between DQA, representativeness, or other sampling violations and resulting health harm, environmental injustice, and environmental-health inequality.future policy, if our results can be replicated, it might be desirable to add representative-sampling/other technical, scientific requirements, including third-party testing/remediation oversight, to the implementation and lender documents mandated by the UN’s New Urban Agenda [Regarding n Agenda . Future Our results confirm our hypothesis that interested parties’/redevelopers’ semi-privatized testing of urban-infill, hazardous-waste sites contaminated with volatile organic compounds may be non-representative, fail data-quality analysis, fail to adequately assess carcinogenic vapor intrusion into buildings, and therefore fail to address pollution threats and environmental injustice/inequality. We tested our hypothesis by determining whether sampling/assessments conducted by the world’s largest commercial developer, CBRE/TCC, met government-mandated “requirements” for volatile-organic-compound sampling. All but one of the CBRE/TCC toxic-site redevelopments assessed in this study violated all 10 of the government-mandated “requirements” for ensuring the temporal, geographical, and technological representativeness of hazardous-site sampling. The one remaining CBRE/TCC toxic site violated 9 of the 10 government-mandated sampling requirements. Assessing the consequences of the preceding 39 of 40 possible violations of government requirements for scientific sampling, our discussion showed that these violations uniformly cause health-risk underestimation. As a result, these violations cause at least four distinct public-health threats to children, minorities, and poor people, as well as a worsening of environmental inequities. That is, all the CBRE/TCC toxic-site redevelopments are located in environmental-justice communities, neighborhoods with disproportionate numbers of children, minorities, and poor people.If our results can be replicated at other toxic sites, then society may need to devise and require new safeguards in order to ensure protective toxic-site redevelopment. In particular, society may need to re-examine how to ensure that semi-privatized testing and cleanup serves not mainly private profits but also public health, environmental justice, and environmental-health equity.