Much of the ice sheet within the catchment of Thwaites Glacier is grounded below sea level on bedrock that deepens inland4, making it susceptible to rapid and irreversible ice loss that could raise the global sea level by more than half a metre5. The rate and extent of ice loss, and whether it proceeds irreversibly, are set by the ocean conditions and basal melting within the grounding-zone region where Thwaites Glacier first goes afloat6, both of which are largely unknown. Here we show—using observations from a hot-water-drilled access hole—that the grounding zone of Thwaites Eastern Ice Shelf (TEIS) is characterized by a warm and highly stable water column with temperatures substantially higher than the in situ freezing point. Despite these warm conditions, low current speeds and strong density stratification in the ice–ocean boundary layer actively restrict the vertical mixing of heat towards the ice base8, resulting in strongly suppressed basal melting. Our results demonstrate that the canonical model of ice-shelf basal melting used to generate sea-level projections cannot reproduce observed melt rates beneath this critically important glacier, and that rapid and possibly unstable grounding-line retreat may be associated with relatively modest basal melt rates.Thwaites Glacier is one of the fastest-changing ice–ocean systems in Antarctica Despite observations from a hot-water-drilled access hole showing warm ocean waters beneath Thwaites Glacier Eastern Ice Shelf, the basal melt rate is strongly suppressed due to the low current speeds and strong density stratification. The evolution of the ice sheet is dynamically linked to the fate of the floating ice shelves found at its seaward margin10. By exerting a resistive force at the grounding line where the ice sheet first goes afloat, ice-shelf buttressing helps control the flow of grounded ice into the ocean11. Over recent decades, elevated ocean-driven basal melting has triggered rapid thinning of many West Antarctic ice shelves12, reducing the strength of ice-shelf buttressing11. The rate of ice-shelf mass loss has increased by 70% between 1994 and 2012 (ref. 12), precipitating a shift towards faster drainage of grounded ice into the ocean13. Several major grounding lines in the Amundsen Sea sector have retreated rapidly inland14, raising the possibility of an unstable collapse of the WAIS15.The response of the marine-based West Antarctic Ice Sheet (WAIS) to a warming climate contributes substantial uncertainty to twenty-first century sea-level projections16  and is particularly susceptible to marine ice-sheet instabilities3. Its grounding line has retreated 14 km inland since the late 1990s17 and, in some regions, is retreating by up to 1.2 km per year at present (ref. 18). Thwaites may have already entered a state of rapid and irreversible ice loss3, and its complete collapse within centuries would contribute 65 cm to the global sea level5. A full destabilization of the main glaciers in the Amundsen Sea sector would contribute 3 m to the global sea level over thousands of years19. The rate and extent of ice loss from Thwaites Glacier, and whether it proceeds irreversibly, is highly sensitive to the poorly understood ocean conditions and basal melt rate in the constantly evolving grounding-zone region6.Nowhere are these processes more apparent and potentially serious than at Thwaites Glacier, which drains about 10% of the WAIS20. Long-term basal melt rates at five different sites  and an oceanographic mooring consisting of a current meter and a temperature–conductivity sensor was deployed 1.5 m beneath the ice-shelf base to monitor the temporal evolution of ocean properties. Wider oceanographic context is provided by ship-based CTD profiles from 2019 and 2020 (ref. 22)  creates a strong barrier to vertical mixing  is unstable with respect to density  and the water column might be susceptible to diffusive convection. Although this double-diffusive process could provide a limited source of energy for vertical mixing26, with an average density ratio of only 0.2 and a Turner angle of −57°, the temperature gradient is too weak to sustain a thermohaline staircase −1  thermocline outside the ice-shelf cavity )−1 Fig. . The graity Fig. . The sou29. At the borehole, glacial meltwater is found throughout the water column, with the concentration exceeding 10 ± 2 ml l−1 at the ice base . Instead, this trajectory is indicative of freshwater sourced from subglacial discharge at the grounding line  transport to the ice base and, ultimately, the basal melt rate, despite strong thermal forcing7 . Although the retreat rate is spatially variable, the grounding line has continued to retreat over the period covered by our melt rate observations (2019–2021), widely reaching 0.4 km year−1 throughout the butterfly region, with a maximum >1.5 km year−1 . The temperature and conductivity sensors were manufacturer-calibrated before deployment and the stated accuracy of the sensors are ±0.002 °C and ±0.0003 S m−1, respectively.A Sea-Bird Scientific SBE 49 FastCAT CTD profiler was used to observe the water-column structure between 9 January and 12 January 2020. A total of 15 individual CTD casts were completed, sampling at a rate of 16 Hz. Before each deployment, the CTD was stored in a warm bath (approximately 5 °C) to minimize icing in the conductivity cell during the profiler’s traverse of the approximately 90-m air-filled portion of the borehole. The CTD data were processed using standard routines in the Sea-Bird data-processing software version 7.26.7.129 and each profile was averaged into 0.1-m vertical bins. Absolute salinity and conservative temperature were computed using the Gibbs SeaWater (GSW) Oceanographic Toolbox for TEOS-10 (refs. 42. Consisting of a Nobska Modular Acoustic Velocity Sensor (MAVS) differential acoustic travel-time 3D velocity sensor, an RBRcoda fast-response temperature sensor and an RBRconcerto inductive conductivity sensor, the turbulence instrument cluster was scheduled to operate in burst mode, sampling at 5 Hz for 15 min every 2 h. For each 15-min burst, the average temperature, conductivity and velocity values were received over an Iridium satellite link. In this study we use the mean values from 4,459 individual bursts collected between 23 January 2020 and 1 February 2021. An analysis of the full 5-Hz turbulence data awaits a future study. The stated uncertainty in the velocity components is 3 mm s−1, whereas the stated uncertainty in the temperature and conductivity data are ±0.002 °C and ±0.0003 S m−1, respectively. A small offset in the conductivity data caused by proximity effects associated with the inductive sensor was removed through regression against the CTD conductivity data.A moored turbulence instrument cluster was deployed about 1.5 m beneath the ice base to observe the small-scale turbulent fluctuations in the ice shelf–ocean boundary layer−1 during the CTD deployment period; Extended Data Fig. −3 ± 10−4 °C km−1 for temperature and 1.1 × 10−3 ± 10−5 g kg−1 km−1 for salinity. The effect of tidal flow has been ignored in these calculations, as the tidal flow speeds are an order of magnitude weaker.CTD profiling beneath TEIS was largely carried out in three separate sessions with approximately 19 h between the first and second sessions and 47 h between the second and third sessions. Dividing the mean absolute difference in temperature and salinity between each session pair by the lateral advective distance over the time separating each pair  measurements for the entire time series. The vertical motion of the ice within the layer from one return to the next was derived from the phase of the cross-correlation and the reliability of that estimate was indicated by its amplitude. As all displacements are measured with respect to the antennas, the vertical displacement of any individual layer is the effect of the integrated strain in the ice above. The ice-shelf thinning rate, as measured from the antennas, is obtained by using a depth interval that tracks the return from the ice base.By using both amplitude and phase, ApRES can monitor the changing distance between the antennas and the ice-shelf base with millimetre-scale precision. This raw Lagrangian ice-shelf thinning includes both the basal melt signal and the ice-column vertical strain that results from ice flow and snow compactionFor tracking the ice base to obtain the total thinning rate, an assumption in ApRES data processing is that, over the period of the time series, the topography of the ice base local to the radar does not change at length scales at or longer than the wavelength of the radar waves in ice, in this case, at length scales greater than about 0.5 m. This requirement was not met at the borehole site nor at various periods of the time series from the upstream sites. However, it was possible to use the first multiple echo, which is the result of the radar signal travelling from the transmit antenna to the ice base, back to the ice surface and then back to the ice base, before finally returning to the receive antenna. The range to the multiple is largely immune to local topographic evolution in the ice base, presumably because of the much larger effective footprint. The multiple is very much weaker than the first basal return, but its phase can be reliably tracked. For the downstream ApRES deployment, and those sections of the other deployments when the first basal return was not changing its form, the melt-rate time series from the first and second returns yielded a satisfactory match. Short-term variations in derived melt rate (3 to 5 days) were much stronger from the multiple, possibly resulting from snow-accumulation events.Vertical profiles of vertical ice velocities, averaged across the entire time series, were calculated by dividing the return into 4-m layers, cross-correlating as described above and calculating the mean vertical velocity for each layer. From these profiles a depth interval was selected from the lower part of the ice column from which to calculate the integrated non-melt contribution to the thinning rate. The selection of the depth interval was based on the strength of the time-averaged correlations and, for the downstream site, the borehole site and the first upstream site, was 300–472 m, 304–436 m and 300–550 m, respectively. The strain from one measurement to the next was averaged across the depth interval and the time series differentiated and low-pass-filtered to provide a time series of vertical velocity variability at timescales of 5 days and longer. Although the strain rate in the ice column was expected to evolve slowly as the ice moved downstream, we assumed that non-tidal short-term variations would not be present. The vertical profiles of vertical velocity showed an approximately linear gradient through the selected depth intervals and an offset vertical velocity was determined by extrapolating that variation to the depth of the basal reflector. That offset was then added to the vertical velocities to yield the final non-melt contribution to the time series of ice-shelf thinning rate. The 2019 site, and the initially grounded site, were processed slightly differently. The internal reflections from near the ice base were good enough to allow a deep depth interval to be selected and cross-correlated to yield an integrated vertical strain directly. Intervals from 520 to 580 m and from 515 to 550 m were used for the 2019 and initially grounded sites, respectively, with a minor correction to accommodate a small, approximately linearly increasing strain rate near the base.The final melt-rate time series was calculated by subtracting the non-melt contribution from the thinning rate and then low-pass-filtering at a cutoff of 15 days.−1, ±0.005 °C and 0.1 dbar for conductivity, temperature and pressure, respectively, which translate into uncertainties of ±0.008 g kg−1 for SA and ±0.018 °C for Θ. All sensors were factory calibrated before deployment and then cross-compared with the SBE 49 CTD profiler to remove offsets in conductivity and temperature of 0.0286 S m−1 and 0.0236 °C. The CT sensor recorded at a frequency of 5 Hz, whereas the pressure sensor recorded at 1 Hz. Pressure measurements were interpolated to match the 5-Hz CT data. Hydrographic data were post-processed by removing outliers that exceeded more than two standard deviations from the mean, as well as data points collected when the vehicle speed was lower than 5 cm s−1. A three-point weighted-mean filter was also applied to the conductivity and temperature data.Icefin was equipped with a Neil Brown Ocean Sensors conductivity–temperature (CT) sensor and a Valeport ultraP pressure sensor. The stated manufacturer accuracies are ±0.001 S m−1, the uncertainty in velocity recorded by the ADCP in the direction of travel is ≤5 mm s−1. The uncertainty in velocities perpendicular to the direction of travel is typically much lower. The velocity data were recorded at a rate of 1 Hz and were post-processed by removing data points when the vehicle pitch or roll is greater than 30°. A 30-s running mean filter was applied to all data points and measurements were filtered for gradients greater than one standard deviation from the mean in vehicle speed, pitch, roll and individual bin velocity. Finally velocities were bin-averaged into 1-m depth bins and velocities were excluded if they exceeded one standard deviation of the mean for each bin.Ocean current speeds were measured using a LinkQuest NavQuest 600 Micro Doppler Velocity Log, which doubles as an ADCP. The ADCP provides measurements of the current speed in 2-m bins at a variable distance from the vehicle, controlled by gradients in the pitch, roll, heading and speed of the vehicle. Uncertainty in the current velocity is typically 1% of the vehicle’s velocity in its direction of travel. As Icefin travels at speeds ≤50 cm sα = 0.03 and thermal anomaly time constant 1/β = 7.0. Water samples were taken from the CTD rosette and analysed using a Guildline Portasal salinometer to calibrate the primary and secondary conductivity sensors on the CTD profiler.A dual-sensors system based on a Sea-Bird 911 CTD was used for conductivity, temperature and pressure measurements outside the ice-shelf cavity from the RVIB Nathaniel B. Palmer in 2019 and 2020. Standard Sea-Bird software Seasave version 7.26.1.8 was used for data collection and conductivity cell thermal mass correction in 2019 and Seasave version 7.26.7.121 in 2020. Manufacturer-recommended values for cell thermal mass correction were used as follows: thermal anomaly amplitude, 56. Under Antarctic ice shelves, the presence of cold and fresh meltwater-laden waters above warm and salty modified Circumpolar Deep Water drives a double-diffusive process known as diffusive convection. Strong diffusive convection can lead to the formation of ‘diffusive staircases’, where well-mixed layers in temperature and salinity are separated by sharp interfaces58. Diffusive convection can still occur without staircase formation however. Diffusive convection can exert a first-order control on the rate of ice-shelf basal melting60.Double-diffusive convection occurs as a result of the difference in molecular diffusivities between salt and heatThe susceptibility of a water column to diffusive convection can be characterized through the density ratioα is the thermal expansion coefficient, β the haline contraction coefficient, Rρ is between 0 and 1, with the strength of diffusive convection increasing as Rρ approaches 1. The Turner anglewhich measures the degree of compensation between temperature and salinity gradients in terms of their effect on density stratification. −1) and the water column is susceptible to diffusive convection when Tu is between −45° and −90°.is related to the density ratio, in which arctan2 is the four-quadrant inverse tangent  present in the water column can then be calculated fromMeltwater fractions are calculated using the composite tracer methodxMW caused by uncertainty in the water-mass endmembers, 1,000 independent estimates of the meltwater fraction were made using a set of random endmember properties derived from the normal distribution described by the mean and standard deviation of each endmember property (Extended Data Table To quantify the variability in 27) and an absolute salinity of 0 g kg−1, whereas the conservative temperature of the subglacial discharge endmember is set to the pressure-dependent in situ freezing temperature for freshwater at the depth of the grounding line (−0.36 °C) with an absolute salinity of 0 g kg−1. The endmember values are consistent with those used in previous studies62.The conservative temperature and absolute salinity of the mCDW and WW endmembers were extracted from the ship-based CTD casts collected in front of TEIS Fig. . The proΘ–SA space, individual data points fall above the mCDW–MW mixing line, indicative of the presence of this fourth water mass  appears in the hydrographic data. In ΘSD and The value of this composite tracer in pure SD isSimilarly, the MW fraction for data points that lie outside the mCDW–WW–MW mixing triangle can be derived by constructing a composite tracer that is equal to zero for data points that lie along the mCDW–SD mixing line:Taking the value of this composite tracer in pure MW asthe MW fraction can be calculated as66:f is the Coriolis parameter, ρ0 is the fluid density, p is pressure, ν is the kinematic eddy viscosity and u and v are the horizontal velocity components, then the vertical structure of the horizontal velocity components through the boundary layer are given by the canonical Ekman solution66ug is the magnitude of the far-field geostrophic flow, z is boundary layer depth and d is the Ekman depth:Assuming a momentum balance between a steady, uniform, geostrophic flow beneath a flat ice base and the frictional stress exerted by the ice base against this flowu and v boundary-layer velocity profiles from Icefin to determine the value of the eddy viscosity and the Ekman depth beneath TEIS.Using the root mean square error as a cost function, we fit equations  and 16)16) to th40ρ is density, ab the basal melt rate, Li the latent heat of fusion of ice, c the specific heat capacity, κi the thermal diffusivity of ice, T the temperature and W is the vertical ocean velocity. The subscripts i, b and w refer to ice, ice–ocean boundary and ocean, respectively. The primes refer to turbulent fluctuations and the angled brackets to the time average. The first term on the right-hand side is the conductive heat flux into the ice, whereas the second term represents the vertical turbulent heat flux through the oceanic boundary layer. In the absence of direct turbulence measurements, or in regional or global models that do not resolve the vertical scales of the ice shelf–ocean boundary layer, the second term is quantified through a simple turbulence closure scheme that models the heat flux, Cd), a non-dimensional turbulent transfer coefficient for temperature (ΓT), the horizontal ocean velocity (U), and the thermal driving (Td), that is given as the difference between the in situ ocean temperature some distance from the ice–ocean boundary  and the temperature at the ice–ocean boundary (Tb) that is assumed to be at the in situ freezing point at salinity Sb and pressure Pb:The rate of ice-shelf basal melting is controlled by the divergence of the sensible heat flux at the phase change interfaceλ1, λ2 and λ3 are constants and the boundary salinity (Sb) is given by the salt balance at the phase-change interface:ΓS is the turbulent transfer coefficient for salt and the salinity of ice (Si) is taken to be zero for ice shelves. The turbulent transfer coefficients, ΓT and ΓS, assume that the thermal and saline diffusive sublayers  are controlled exclusively by current shear and thin with increasing current velocity; however, there are known to be many ice shelf–ocean environments where this is not the case8. The system of equations described by equations .The in situ freezing point at the ice–ocean boundary is given by the liquidus conditionquations –21) rep rep20\doBecause thermal driving is defined as the difference between the ocean temperature recorded by the mooring and the freezing temperature at the ice–ocean boundary, its magnitude is sensitive to the distance between the ocean mooring and the ice base. Basal melting increases the distance between the ocean mooring and the ice base as a function of time, and this drives an apparent increase in thermal driving without a change in the source water mass as the mooring effectively descends into warmer water. If we assume that the temperature profile through the boundary layer is fixed with respect to time, we can use the observed CTD profiles to estimate that the apparent change in thermal driving owing to ice-base recession is about 0.2 °C, or roughly 57% of the observed change (Extended Data Fig. Although the rate of basal melting beneath TEIS predicted by the three-equation melt-rate model is linearly related to the magnitude of the thermal driving, it is relatively insensitive to the apparent change in thermal driving owing to ice-base recession. Indeed, the mean predicted melt rate only falls by 8% when thermal driving is corrected for ice-base recession (Extended Data Fig. Any methods, additional references, Nature Portfolio reporting summaries, source data, extended data, supplementary information, acknowledgements, peer review information; details of author contributions and competing interests; and statements of data and code availability are available at 10.1038/s41586-022-05586-0.


V. Whelan & K. M. Halanych Nature Communications 10.1038/s41467-023-36151-6 (2023)1 (R&M) we integrated precomputed site-heterogeneous amino acid substitution models and amino acid recoding into partitioned phylogenomics and showed that this improved model-fit and resistance to long-branch attraction . These better fitting modelling strategies revealed a shift in support away from Ctenophora (comb jellies), and towards Porifera (sponges), as sister to all other animals1. Whelan and Halanych2 (W&H) criticised our recoded analyses and interpretations on the placement of Porifera and Ctenophora in the animal phylogeny. Here we counter these criticisms, and bolster evidence for Porifera as sister to all other animals.In Redmond and McLysaght1) when assessing the performance of new approaches in R&M2. The site-heterogeneous models and recoding strategy that we employed have been tested and used elsewhere (see R&M1), and we deemed further validation redundant. However, we did test the ability of partitioned site-heterogeneous models and recoding to combat specific and well-characterised LBA problems in real datasets4 (see R&M1), finding that both improved upon standard partitioned phylogenomics1. Although we agree that new approaches should recover well-accepted clades, W&H’s argument against our approaches (particularly recoding), based on failure to recover Chordata (LEA[N/P] datasets1) and Deuterostomia (WEA17 dataset and reduced support in BEA dataset1), is weak. Whether deuterostomes are monophyletic is currently unresolved5, meaning deuterostome non-monophyly cannot convincingly cast doubt on our site-heterogeneous, recoded analyses of WEA17 (including recovery of Porifera-sister) or BEA. The LEA(N/P) datasets were not designed to assess chordate monophyly4, and Chordata was never recovered with strong support in any of our1, or W&H’s2, LEAN/LEAP analyses, but neither was any alternative1. Interestingly, despite each being expected to improve phylogenetic inference, (i) using closer-related outgroups has also disrupted Chordata in some past analyses of these datasets4, and (ii) partitioning-by-gene outperforms W&H’s better fitting partition scheme in recovering Chordata . Conspicuously, the problematic lineages, Ambulacraria and Cephalochordata, are represented by a single species each, and improved taxon sampling resolves this issue for a related dataset6. Thus, these datasets appear to harbour little signal either for or against Chordata, rather than recoding causing an inference problem. Given the numerous potential lineage-specific dataset and biological biases that can arise, we do not believe that data insufficient to recover one off-target clade with available modelling strategies cannot reliably be used to assess target relationships.W&H claim we did not adequately consider well-accepted relationships  do not suffer from this issue, and thus, in R&M we proposed that combining partitioning and recoding had reduced the number of complex alignment site patterns per partition such that simple F81 exchangeabilities fit better than GTR exchangeabilities when using site-heterogeneous models1. We test this here with unpartitioned SR4-recoded reanalyses of these datasets, as this might provide enough data for models that are both site-heterogeneous and GTR-based to fit best. As expected, topologies and branch supports are largely consistent with those recovered in our original RL2 analyses  under the best-fitting unpartitioned site-heterogeneous F81 models  exchangeabilities in partitioned analyses of recoded data, accounting for which reinforces Porifera-sister.As W&H noted, our site-heterogeneous recoded analyses showed poor resolution and inconsistencies between REA, WEA15 and WEA17, an issue they ascribe to recoding2. While improved ingroup taxon sampling can undoubtedly improve phylogenetic inference, it is noteworthy that the extra species do not break the long branch leading to the Ctenophora clade—the very branch which is purported to cause LBA . This branch is longer in WEA17 than in REA or WEA15  towards supporting Porifera sister is recovered more slowly for WEA17 (only apparent at the partition-specific level when not recoding), and most easily for WEA15 (which has the shortest ctenophore branch). This may explain why Porifera-sister is only recovered for WEA17 when the data are recoded8. Thus, W&H’s emphasis on ctenophore sampling inadvertently prioritizes a dataset (WEA17) with increased potential for ctenophore LBA. This is a major concern given that Ctenophora-sister is far more likely than Porifera-sister to be erroneously recovered in simulations affected by LBA-inducing systematic error10.W&H favour the WEA17 dataset as it includes more ctenophore speciesA15 Fig. . Regardl2. First, we disagree that we inappropriately dismissed Hernandez and Ryan’s11 concerns about recoding given our above points on the recovery of Deuterostomia and Chordata in our test datasets and evidence that W&H’s issues with recoded analyses in R&M do not in fact derive from recoding. Other simulation studies have supported recoding12, or are at least ambivalent13, and as we advocated in R&M, ‘a fuller understanding of the implications of recoding is needed’1. Second, our claim that REA and WEA15 contain paralog contamination referenced other work7 and personal communication was limited to WEA171, which has now been shown to support Porifera-sister without recoding when orthogroups with poor orthologous signal  are excluded14. We concede that personal communication was less than ideal, particularly as sorting orthologs from paralogs is at least somewhat dependent on the approach employed/investigator interpretation, but our comment also referred to the relatively high percentage of missing data for ctenophores in WEA17, which is directly observable in the dataset. W&H contend that if these datasets contain paralog contamination it would invalidate the findings in R&M2, yet it also would invalidate the original findings favouring Ctenophora-sister, a hypothesis derived from phylogenomics. Furthermore, phylogenomic analyses with site-homogeneous models, from which the strongest evidence and support for Ctenophora-sister emerges1, appear more easily misled by orthology errors than site-heterogeneous approaches15, with which we observe a shift towards support for Porifera-sister in R&M1. Lastly, we disagree that discussing W&H’s simulations16 comparing the Phylobayes17 CAT18 model with partitioning is irrelevant, as although we did not directly use the CAT model, we did employ partitioning with previously defined, precomputed variants of CAT19 using IQ-tree20 in R&M1.W&H claim we were unfairly critical of previous studies1) and do not apply an ‘objective lens’ in our interpretations2. Rather W&H’s assertion that ‘partitioning with linked branches and site-heterogeneous models recovered the Ctenophora-sister hypothesis’2 disregards the clear pattern observed in R&M1 for all datasets of increasing support for Porifera-sister (over Ctenophora-sister) as model fit increases, downplaying this as reduced support in ‘some’ datasets/analyses2. In summary, our primary conclusions remain firmly intact.Our points above refute W&H’s arguments that our use of recoding was spurious and we strongly reject the notion that we do not accurately present results favouring Ctenophora-sister  SR4 recoding (as contended by W&H2) or (ii) simple, flat F81 exchangeabilities (as proposed here and in R&M), which were always better-fitting than GTR exchangeabilities when combining partitioning, recoding and site-heterogeneous models in R&M1. The logic behind this is that when a single model is applied to the entire phylogenomic dataset rather than separate models to each partition, then there may be enough data for site-heterogeneous models with GTR, rather than simpler F81, exchangeabilities to fit best when recoding is applied. ModelFinder23 in IQ-tree was used to assess the best-fitting models under both the AIC and BIC. The site-homogeneous models F81 and GTR were tested, as well as pairings of each of these exchangeability matrices with SR4 recoded derivations (from R&M1) of the site-heterogenous C10, C20, C30, C40, C50, and C60 precomputed CAT models19. All site-heterogeneous models also incorporated 4 discrete gamma categories to help accommodate rate heterogeneity across sites. For example, the model ‘SR4C60GTR’  in a given dataset  the more difficult it will be to overcome potential LBA of Ctenophora towards the root of the animal tree when analysing that dataset. We extracted this branch length value from the maximum likelihood consensus trees resulting from standard partitioned phylogenomics of the REA, WEA15, and WEA17 datasets performed in R&M . Branch lengths were plotted in Fig. The long ancestral ctenophore branch  has been suggested to cause LBA between Ctenophora and non-animal outgroups, producing tree topologies supporting Ctenophora as sister to all other animalsFurther information on research design is available in the Reporting Summary


The lipid-regulating drug gemfibrozil is a useful medication for reducing high cholesterol and triglycerides in the blood. In addition to oxidation, it undergoes extensive glucuronidation to produce gemfibrozil acyl glucuronide, which is a known mechanism-based inactivator of cytochrome P450 (CYP) 2C8. Such selective and time-dependent inhibition results in clinically important drug–drug interactions (DDI) with the drugs metabolized by CYP2C8. Similarly, the acyl glucuronide of clopidogrel, a widely used antiplatelet agent, is a potent time-dependent inhibitor of CYP2C8 that demonstrated significant DDI with the substrates of CYP2C8. Current progress in atomic-level understanding mostly involves studying how different drugs bind and undergo oxidation in the active site of CYPs. It is not clear how an acyl glucuronide metabolite of the drug gemfibrozil or clopidogrel interacts in the active site of CYP2C8 and selectively inhibit the enzyme. This mini-review summarizes the current knowledge on some of the important clinical DDI caused by gemfibrozil and clopidogrel due to the inhibition of CYP2C8 by acyl glucuronide metabolites of these drugs. Importantly, it examines recent developments and potential applications of structural biology tools to elucidate the binding and orientation of gemfibrozil acyl glucuronide and clopidogrel acyl glucuronide in the active site near heme that contributes to the inhibition and inactivation of CYP2C8. A totse drugs ,13,14. Tse drugs . Mechanid–glucuronides [Notably, CYP2C8 is the only known P450 enzyme capable of metabolizing glucuronides with a potential role as a regulatory linkage between the Phase I and Phase II drug metabolizing enzymes. The conjugation of Phase I metabolites with the Phase II glucuronic acid leads to an increase in molecular weight of the compound by 176 Da and enhances water solubility for secretion into the bile and elimination from the body in the urine . The weluronides . ImportaiK value ranging from 9.3 to 270 µM and an IC50 of 95 µM as shown previously, whereas the 50IC of gemfibrozil glucuronide was 20–52 µM for enzyme inhibition [Gemfibrozil, a fibric acid derivative, is used in the treatment of hypercholesterolemia and hypertriglyceridemia . It is ohibition ,26.iK value of 5.8 µM and 24 µM, respectively, and its inhibitory activity towards several other human CYP isoforms was much weaker [In addition, gemfibrozil also inhibits other CYP2C subfamily of human enzymes that include CYP2C9 and CYP2C19 with a h weaker . Importah weaker ,29,30,31h weaker . Dosing Clinically relevant and harmful DDI occur due to changes or elevation of the plasma concentrations of one drug that is metabolized by an enzyme, which is inhibited by another drug. Gemfibrozil has been shown to inhibit CYP2C8-mediated metabolism of another statin drug cerivastatin . The plaThe binding of gemfibrozil glucuronide in the active site of CYP2C8 has been probed using computational docking. The results revealed the location of glucuronosyl moiety away from the heme with the carboxylate group hydrogen bonding to the distal amino acids. The ortho-methyl group of gemfibrozil glucuronide oriented toward the heme more preferably compared to the meta-methyl group as illustrated by ligand docking . EmployiAs the list of acyl glucuronides that have been identified continue to evolve, clopidogrel acyl glucuronide is another well-characterized example of a glucuronide conjugate that inactivates CYP2C8 through mechanism-based inhibition. Clopidogrel is a widely used antiplatelet drug to treat cardiovascular and cerebrovascular diseases. It binds the P2Y12 receptor and inhibits platelet activation and aggregation to prevent heart attack and stroke . Clopidotorsades de pointes [iK of 9.9 µM and inactk of 0.047 min−1 [Together, co-administration of clopidogrel affects a wide variety of CYP2C8 substrates of diverse sizes and shapes that include dasabuvir, paclitaxel, pioglitazone, desloratadine, etc. The plasma exposure of dasabuvir increased primarily due to the inhibition of CYP2C8 by clopidogrel-acyl glucuronide ,58. Thus pointes . Further pointes ,61. Simi pointes . The clo47 min−1 , and the47 min−1 . Such in1T) relaxation measurements to determine the ticlopidine protons from the heme iron of CYP2B4 and establish the preferred orientation of the drug toward the heme [Application of various structural and biophysical tools to probe the structure-function relationship of cytochrome P450 enzymes in the presence or absence of ligands is fairly common, However, the complex of acyl glucuronide and CYP2C8 has remained elusive, but successful use of a combination of these methods may provide a useful, synergistic strategy to assess and interpret the basis of interactions that should extrapolate to observed inhibition of the enzyme. Specifically, probing the affinity, on- and off-rates, and thermodynamics of ligand binding using various biophysical techniques that include isothermal titration calorimetry and/or surface plasmon resonance will be helpful in understanding the inactivator binding and mechanism of inactivation. In addition, several structural biology approaches that include NMR, X-ray crystallography, and cryo-EM can potentially be used to analyze the glucuronide bound CYP2C8 complex. For example, utility of NMR was previously explored via longitudinal  at 3.2 Å resolution and a GPCR-peptide ligand complex (150 kDa) to 3.8 Å resolution ,68,69. SLastly, any attempt to use the structural and biophysical data for drug interaction studies with gemfibrozil or clopidogrel and CYP2C8 will have to take into consideration the effect of UGT1A1 that catalyzes glucuronidation, as well as their inhibitory effect on OATP1B1 and OAT3 . This isAs the list of drug glucuronides continue to grow, it will be of prime interest to probe the binding of glucuronide in the active site of CYP2C8 and further investigate why glucuronidation converts some drugs to mechanism-based inhibitors of CYP2C8 leading to clinically relevant drug–drug interactions. Efforts in our laboratory have focused on studying structure-function relationship of CYP2C subfamily of enzymes using structural, biophysical, and functional methods ,74. This


Ice hockey is a dynamic and competitive sport that requires a high level of neuromuscular and cardiovascular function. An objective assessment of skating helps coaches monitor athletes' performance during training sessions and matches. This study aimed to estimate the temporal and spatial parameters of skating by proposing an optimized configuration of wearable inertial measurement units (IMUs) and validating the system compared to in-lab reference systems. Ten participants were recruited to skate on a 14 m synthetic ice surface built in a motion-capture lab. Eight original event detection methods and three more adopted from gait analysis studies were implemented to detect blades-off and skate-strikes. These temporal events were detected with high accuracy and precision using skate-mounted IMUs. Also, four novel stride length estimation methods were developed to correct the estimated skaters' position using IMUs' readouts. The stride time, contact time, stride length, and stride velocity were obtained with relative errors of 3 ± 3%, 4 ± 3%, 2 ± 6%, and 2 ± 8%, respectively. This study showed that the wearable IMUs placed on skates and pelvis enables the estimation of temporal and spatial parameters of skating with high accuracy and precision, which could help coaches monitor skaters' performance in training. Players with higher neuromuscular and cardiovascular abilities, capable of starting quickly and skating at higher speeds, are more likely to possess the puck and win face-to-face competitions in matches5. Accurate assessment of hockey players' skating movements during training sessions can help coaches continuously monitor players' performance with the aim of improving it during training. Spatial  and temporal  parameters of skating serve as mobility biomarkers7 and are recognized as significant metrics to characterize any repetitive activity like forward ice striding. These parameters, traditionally, were obtained in human motion laboratories using stationary motion-capture (MoCap) systems. However, the application of these instruments is limited since they are not available in every ice rink, and their captured volume is confined to a small part of the ice rink, which can disrupt the natural skating patterns of ice skaters10. Thus, wearable and garment-embedded technologies are preferable for on-ice skating performance assessments13.Ice hockey requires high levels of aerobic and anaerobic fitness, well-coordinated body motion, and efficient functioning of the neuromuscular and cardiovascular systems14. Also, Stetter et al. studied the feasibility of using wearable accelerometers to identify skating parameters such as ST and CT to differentiate players in terms of their skill level15. However, these studies investigated the skating parameters using 3D accelerometers rather than inertial measurement units (IMU). IMU has been applied to measure human motion for clinical outcome evaluation18, sport biomechanics evaluations25, and movement modalities detection26. They have the potential to obtain temporal and spatial parameters during hockey skating.Buckeridge et al. used a portable system composed of accelerometers, EMG modules, and force sensors to assess on-ice hockey player performance28. The second step is to estimate the participant's trajectory in each stride necessary to calculate the spatial parameters29. Finally, temporal and spatial parameters can be calculated by the detected temporal events and the participants' trajectories. Participants' trajectory can be calculated using double-time integration of the participant's acceleration in a global reference frame. However, due to the cumulative error in the numerical integration of IMU readings, the obtained trajectory can be drifted and erroneous30. There are two types of error in calculating the stride length: the noise on the acceleration time series and the drift in the sensor orientation (used for double-integration of acceleration to estimate trajectory) obtained by the gyroscope readouts. In gait analysis application, it has been suggested to correct foot velocity and, subsequently, foot trajectory time series by assuming zero velocity and minimum foot height during foot-flat periods6. However, a similar period to the foot-flat with zero velocity in all directions is absent in ice hockey skating strides, making foot velocity and trajectory estimation more challenging in hockey skating than on-land gait. This study addresses these challenges toward improving the accuracy of on-ice measurement of spatial and temporal parameters of ice skating using a set of IMUs fixed on the participant's skates, shanks, and pelvis on a synthetic ice surface. Synthetic ice surface as an alternative to real ice with comparable forward skating mechanism31 has the potential to be used in in-lab testing and training, particularly where ice access is limited.The computation of the temporal and spatial parameters, in the first step, requires the detection of skating temporal events. The accuracy of event detection using IMUs can vary significantly depending on the extraction method usedThe objective of this study is to: (1) detect temporal events of skating using skate-mounted IMUs, (2) estimate the skate trajectory using IMUs, (3) calculate the temporal and spatial parameters of skating using the obtained temporal events and corrected skate trajectory, and (4) experimentally validate the obtained results against those measured by in-lab motion-capture systems on a synthetic ice surface.Ten able-bodied individuals  among participants, six male and four female) were recruited to participate in this study. All participants were free from injury and capable of skating comfortably. The study was approved by the research ethics board of the authors' current institution (Pro00092821), and all methods were performed in accordance with the relevant guidelines and regulations. All participants were informed of the experimental procedures and gave informed written consent before the test.32, NL, full-scale ranges are: acceleration: 33, Novel, DE) were placed in the skates  were used to track the trajectory of retro-reflective markers. After 10 s of standing still, the participants skated forward alongside the synthetic ice rink for 14 m. At the end of the forward skating trial, they also stood for 10 s quietly. During each trial, the IMUs, motion-capture cameras, and pressure insoles recorded their motions and ground reaction forces simultaneously. All the systems' sampling frequencies were 100 Hz, and each skating trial was repeated five times.Tests were carried out at an indoor synthetic ice rink  and obtained the events during five trials of each participant. These detected SS and BO were validated against those detected using the pressure insoles, with a 5 N threshold  and blades-off (BO) instants using the IMUs' readouts. In addition to the originally proposed methods of temporal event detection, described in Table old Fig. .Table 1D6. However, during contact time in ice skating, the skate keeps sliding on ice, making the ZUPT algorithm impractical in this application. In this study, four novel methods were proposed to eliminate the accumulating errors in the calculated velocity and trajectory of skaters:(i)38, using the IMU orientation obtained by the Xsens software package,The accelerometer readout was transferred into the north-east-up (NEU) reference frame(ii)The gravitational acceleration was subtracted from the transformed acceleration to obtain the free acceleration of the sensor in the NEU frame,(iii)The skater's velocity was obtained using time integration of the free acceleration. The obtained velocity, however, was not zero in the resting periods due to the accumulated error of the IMU readouts,(iv)The accumulated error was removed, and the obtained velocity and acceleration time series were corrected using the assumption of zero velocity and acceleration in the resting periods. To this end, four original methods were developed and implemented. These methods removed sensors' acceleration bias (S1), acceleration bias and estimated noise profile S2 and S3), and estimated velocity error time series (S4) using the assumption of zero acceleration and velocity in the resting periods The corrected velocity was used to obtain the skater position trajectory used to obtain the SL for each stride see “” below.All these four algorithms (S1–S4) used IMU readouts during the resting period. The effect of resting period duration was investigated on the stride length estimation error to recommend a minimum resting period duration. To this end, IMU readouts collected during time windows from 0.1 to 10 s, by the increment size of 0.1 s, out of the two 10-s resting periods originally considered for data collection before and after the motion, were used in S1–S4. Then, the shortest resting period duration that would not significantly affect the stride length estimation error compared to longer resting periods was explored.The spatial parameters were calculated using the IMUs placed on the skates, shanks and pelvis. Any inherent signal bias in the sensor readouts is prone to be accumulated in the integration process and corrupt the obtained velocity and trajectory of the skater. In the gait analysis application, the zero-velocity update (ZUPT) technique has been used to force the foot velocity to be zero during the stationary foot-flat period in each strideST was computed from one SS to the subsequent SS of the same skate, and CT, the time when the skate is in contact with the ice, was calculated from one SS to its subsequent BO. Also, SL in each stride was calculated using the estimated sensor trajectory. SL, then, was defined as the two norms of a 2D vector containing the travel distance in the mediolateral  and precision (SD) of the errors were computed. To compare the SL estimated by the methods described in “This study was approved by the research ethics board of the authors' current institution (Pro00092821), and all methods were performed in accordance with the relevant guidelines and regulations. All participants were informed of the experimental procedures and gave informed written consent before the test.In total, 184 SS and 186 BO were identified and then compared with those obtained by the pressure insole. The finding from method T4 revealed that temporal event detection with an average 0.01-s error using IMU readouts is achievable, equivalent to one sampling period Table . Also, TThe skaters' speed was entclass1pt{minimaST and CT were calculated based on the temporal events identified by T1 and T3: the best methods in detecting SS and BO, respectively. Also, SL was calculated based on the trajectory estimated by the velocity correction method (method S4: the best method in SL estimation). Finally, SV was calculated as the average of the sensor velocity estimated by method S4. Using the IMU readout, ST, CT, SL, and SV were estimated with 3 ± 3%, 4 ± 3%, 2 ± 6%, and 2 ± 8% relative error, respectively, compared to those obtained from in-lab reference systems' recordings. Other than the bias errors mentioned above, the Bland–Altman plot . Also, almost the same accuracy in calculating ST has been obtained compared to the other studies on long-track speed skating (3.6%)11. Finally, the accuracy of CT and ST obtained by IMU in this study was comparable with those reported in a previous study on ice skating15 investigating the accuracy of CT and ST estimated on the real ice . The relatively higher errors in this study compared to the previously mentioned study were due to three factors:6.Lack of a foot-flat period in ice skating: The proposed methods for skater position estimation benefited from velocity correction only once at the end of each measurement trial, while the ZUPT technique in gait analysis can be implemented during the foot-flat period in each gait cycleLack of familiarity with the synthetic ice: The skaters, even higher calibre skaters, may not have much experience with synthetic ice skating, and thus, they skated less consistently compared to real ice.31, where the players tend to skate. Hence, skating on a 14 m-length ice rink could result in more inconsistency in the participants' skating.Skating on a shorter skating area requires faster acceleration and deceleration compared to skating on a standard ice rinkIn this study, for the first time, the measurements of the wearable IMUs were used to obtain the temporal and spatial parameters of forward skating  using various methods, and the results were validated against those obtained by the reference system, i.e., pressure insole and motion-capture cameras. 11 methods were implemented to obtain the temporal events with high accuracy and precision in forward striding in ice skating using wearable IMU readout. Also, four methods were implemented to correct the stride length estimation using IMU readout. The accuracy of temporal and spatial parameters in skating (on average less than 4% of relative error) in this study was comparable with those reported in gait analysis  their slippage in the skates during long-term dynamic motions and (ii) the inconvenience of carrying a data logger and batteries in a belt connected to the insoles via cables.IMU-based systems have been widely used for temporal event detection in gait analysis and have advantages over other devices such as pressure insolesThe temporal events (SS and BO) detected using IMU readouts showed high accuracy and precision . The methods (T1–T4) that used the upward and forward accelerations of the skate to detect SS and BO were more effective than those that used angular velocities to detect SS and BO . Implemented methods from gait analysis (T9 and T10) strongly depend on the false positive or false negative detection and the chain of the events detected by these methods. Although these methods were comparably precise in skating event detection, false positive or false negative detection (T9 and T10) of one event influence the proceeding event detections. As a result, the effectiveness of these methods (T9 and T10) requires a repetitive skating pattern leading to repetitive time series all over the trial, which may not be easily achievable, particularly in lower calibre players. In summary, the most effective methods were T1, T2, and T4 in finding SS events and T3, T2, and T1 in detecting BO events in skating when skate-mounted IMUs' readouts were used. Also, the skate-mounted IMUs' readout outperformed the shank-mounted IMUs' readout in temporal events detection . As a result, only the two skate-mounted IMUs are recommended for detecting bilateral skating temporal events.38. Furthermore, removing the acceleration's DC offset  considerably reduced the SL estimation error, and none of the added noise estimation methods, i.e., removing white noise and discreet wavelet transform (DWT) details, in S2 and S3 further reduced the SL estimation errors. This was evident by the Pearson's correlation coefficient of 0.99 between SL obtained by S1 and SL obtained by S2 and S3. In summary, the acceleration obtained by the pelvis-mounted IMU corrected by S4, among the other IMUs and methods, obtained the most precise SL estimation. Also, according to the Bland–Altman plots  used in the developed event detection methods. Third, our proposed methods were validated only in 14-m trials and forward skating slower than 2.63 m/s. Future studies are needed to investigate the validation of our proposed methods for the measurement of temporal and spatial parameters of ice skating in longer distances, faster skating, and other skating types such as turning. Yet, this study showed that methods S1–S3 by using only 0.5-s resting periods could significantly enhance the stride length estimation precision, at least in short-term  forward striding experiments. Fourth, identification, modelling, and eliminating other sources of error during skating can promise better precision and accuracy in IMU position estimation in miscellaneous skating types in longer experiments. Finally, the effectiveness of IMU pelvis-based methods for temporal event detection can be further investigated in future studies.Development and validation of the proposed methods to estimate the temporal and spatial parameters of ice skating have potential limitations. First, unlike event detection algorithms developed for gait analysisFor the first time, novel methods were proposed for estimating the temporal and spatial parameters of ice skating using wearable IMUs and their accuracy and precision were validated against the in-lab reference systems. Among different sensor configurations, the optimal set of IMUs for this purpose consists of two skate-mounted IMUs  and one pelvis-mounted IMU . The proposed methods estimated the ST, CT, SL, and SV of the ice skaters with relative errors close to those previously reported for gait analysis. The proposed methods detected SS and BO within 0.01-s accuracy using a skate-mounted IMU readout. We improved the SL estimation precision between 53 and 88% using a pelvis-mounted IMU readout. The next step toward developing this wearable technology is to validate the 3D joint angles obtained by the IMUs and validate the measurements on ice. In summary, IMU technology can be utilized for developing metrics for real-time performance assessment of ice hockey players during training sessions and matches. This assessment can potentially help athletes, trainers and coaches by continuously monitoring skating performance and potentially reducing the risk of injuries.


A minimally invasive technique with various screw configurations without open surgery is currently used for the fixation of transverse patellar fractures. Percutaneous crossing screw configuration has been reported to have a good bone union rate in patellar fractures. However, the difference in mechanical stability of the fractured patella between different screw-included angles has not been fully investigated. Hence, this study aims to compare the mechanical stability of parallel and crossing screw fixations with different screw-included angles for the fixation of transverse patellar fractures during level walking.A finite element knee model containing a patella with a transverse fracture is created. Two headless compression screws with different angles  are used to fix the fracture. The loading conditions of the knee joint during level walking are used to compare the stability of the fractured patella with different fixation screw configurations.The results indicate that the maximum fracture gap opening distance increased with an increase in the included angle. Two parallel screws yield the smallest gap distance among all screw configurations. The maximum gap opening distances at the anterior leading edge of the fractured patella with two parallel screws and two screws having an included angle of 90° are 0.73 mm and 1.31 mm, respectively, at 15% walking cycle.Based on these results, the superior performance of two parallel screws over crossing screw fixations in the fixation of transverse patellar fractures is established. Furthermore, the smaller the angle between the crossing screws, the better is the stability of the fractured patella. The patella, embedded in the patellar tendon, plays an important role in transferring the quadriceps force to the tibia to develop the extensor mechanism of the knee joint . The extTo date, several surgical approaches have been proposed for the management of patellar fractures, including tension band wiring, cerclage, modified tension bands, pins or screws, and combined approaches –10. AccoA percutaneous crossing screw configuration without an anterior wire has also been proposed to achieve a good union rate for patellar fractures . HoweverA knee joint model containing the patella, distal femur, proximal tibia, and fibula was developed based on the computer tomographic images of a healthy man with a body weight of 70 kg and height of 170 cm. The images were obtained when the subject was lying supine; hence, the knee model was created in the full extension position. The areas of the cortical and cancellous bone in the images were isolated out by the higher gray values than the surrounding soft tissues. The 3D model of the bones was then developed by stacking the isolated bony areas. It was subsequently imported into the CAD software, SolidWorks 2019, to create the cartilage and meniscus. The thickness of the cartilage of the patella, distal femoral, and proximal tibia was set at 1 mm based on the literature . The spaTo investigate the effect of the included angle of the screws in crossing fixations, three crossing angles were considered: 30°, 60°, and 90°. Furthermore, in the crossing screws, there are two different screw configurations: the anterior screw inward and anterior screw outward. In addition to cross-fixation, traditional parallel screw fixation was used for comparison. Two parallel screws were placed in the middle third of the patella in the front plane. The proximity of the screws was approximately half of the thickness of the patella. Additionally, the currently used anterior wire in a figure of eight with two parallel screws was used for validation. The diameter of the anterior wire was set at 1 mm. The length of the screws ranged from 35 to 40 mm, and the thread length was 12 mm. The thread was controlled to avoid passing through the fracture site; hence, compression and separation of the fractured patellar fragments under loading were allowed. In total, eight different configurations were used  was used to mesh the complex model, including the bone, cartilage, screw, and wire. The ligaments of the knee joint were reconstructed using tension-only springs in the ANSYS Workbench. In total, five ligaments  between 452 053 and 3 208 329 nodes  between 452 053 and 3 208 329 nodes. Additionally, the difference in the contact area between the patellar fragments was 1.4%  Fig. . The conThis novel study demonstrates loading on a fractured patella with different screw configurations in level walking. The present simulation could demonstrate the difference in fracture gap deformation between the stance and swing phases of the walking cycle. The loading conditions of the patella differ during walking, and the direction of the muscle force changes during knee flexion and extension. In most previous studies, a worst-case loading condition of the fractured patella was assumed, and the gap opening distance was demonstrated at specific knee flexion angles , 32, 34.Patellar fracture is a common fracture encountered by orthopedic surgeons in clinical practice. The treatment strategy for fractures is still evolving because new devices and surgical approaches are continuously being proposed. In recent years, the conventional approach, that is, tension band wiring, has gradually decreased, while the combined approach has become popular . Some spCrossing screw fixation with a minimally invasive technique for transverse patellar fractures was proposed to achieve a good union rate in previous studies . In a prIn the present simulation, the results indicated that the stability of the fractured patella with crossed screw fixation was lower than that with parallel screw fixation. However, crossed screw fixation with an included angle of 90 °was reported to achieve good clinical outcomes . MechaniThe use of an anterior wire has been shown to achieve higher stability than that without it , 38. HowThe present quasi-dynamic simulation has several limitations. First, inertial force was not considered in the simulation. Second, the motion and force data for the boundary conditions were not directly measured from the subject who accepted the CT scan and provided CT images. Third, only the quadriceps force was considered, and the muscle forces from the hamstring and gastrocnemius were not considered. Fourth, the ligaments around the knee joint were simplified as a spring, and all the materials were simplified as linear elastic, isotropic, and homogeneous.This novel FE simulation of knee model can represent the mechanical responses of a fractured patella with different screw fixations during level walking, and the fracture gap opening distance and contact area between fragments are demonstrated. Based on these results, the two parallel screws are noticed to have superior performance than the crossing screw fixations in the management of transverse patellar fractures with headless compression screws. Furthermore, the smaller the angle between the crossing screws, the better the stability of the fractured patella when two headless compression screws are used.


This repository contains lactobacilli strains from eight different species. As many other lactobacilli are known to inhibit human pathogens, we hypothesized that some urinary lactobacilli will have similar abilities to inhibit the growth of typical uropathogens and thus, provide a link between the urinary microbiome and the predisposition to the rUTI. Therefore, we screened the urinary lactobacilli in our repository for their ability to inhibit model uropathogens in vitro. We observed that many urinary isolates strongly inhibit model strains of gram-negative Escherichia coli and Klebsiella pneumoniae but demonstrate less inhibition of gram-positive Enterococcus faecalis. The observed inhibition affected model strains of uropathogens as well as clinical and multidrug-resistant isolates of those species. Our preliminary analysis of inhibition modes suggests a combination of pH-dependent and cell-dependent inhibition. Overall, inhibition strongly varies among species and strains of urinary lactobacilli. While the strength of the inhibition is not predictive of health outcomes in this limited repository, there is a high level of species and strain diversity that warrants future detailed investigations.The human urinary microbiome is thought to affect the development and progression of urinary tract infections (UTI), particularly recurrent UTIs in aging populations of women. To understand the possible interactions of urinary pathogens with commensal bacteria inhabiting the aging bladder, we conducted an initial functional assessment of a representative set of urinary lactobacilli that dominate this niche in postmenopausal women. We created a repository of urinary bladder bacteria isolated  Escherichia coli, Klebsiella pneumoniae, and Enterococcus faecalis (Urinary tract infections (UTIs) are among the most abundant bacterial infections worldwide, with over 6 million annual diagnoses in the U.S. alone . The majfaecalis . The risfaecalis , as predfaecalis .Lactobacillus crispatus and Lactobacillis iners, appear to correlate with urinary health  and 1492R (5’-GGTTACCTTGTTACGACTT-3’) and Q5 polymerase mastermix (NewEngland Biolabs). The resulted PCR product was purified and submitted for Sanger sequencing (Genewiz).via a well-diffusion assay using solid MRS agar plates. Lactobacilli strains were grown over two nights in 10 mL of MRS broth and uropathogenic strains were grown overnight in 4 mL of LB broth, both stationary at 37°C. Surface of the MRS agar plates was then inoculated with 100 μL of the uropathogenic culture. Wells were punched in the surface (Ø6 mm) and removed with sterile tweezers. 50 μL of the bacterial culture or filtrates to be tested were added to the wells. The plates were then grown overnight at 37°C, unless mentioned otherwise. To measure the size of the zones of inhibition, all plates were first imaged, and the zones were quantified by the graphical program GIMP (www.gimp.org) and analyzed in MS Excel.Inhibition of a number of substances was measured 600) of the strains was then taken, and 20 μL of the uropathogen culture was placed into each of the tubes needed for the experiment. The OD600 was then used to calculate the amount of lactobacilli culture needed to make specific ratios of uropathogen: lactobacilli and that amount of lactobacilli culture was added to their respective tubes. These tubes were then spun in a centrifuge at 3230 rcf for 10 minutes to form a cell pellet and the supernatant was poured off. The pellets were then resuspended in 10 mL of MRS broth and incubated at 37°C. Measurements of uropathogen density were taken via serial dilutions and colony counting and samples were taken at four time points after beginning incubation: 0 hours, 1 hour, 6 hours, and 24 hours. At each of these time points, 100 μL was taken from each tube and used to make serial dilutions ranging from undiluted to a dilution of 10-7. From each of these, 2 μL was taken and plated in rows on LB agar plates which were then incubated for several hours at 37°C. The number of colonies in least diluted but still countable samples was then used to calculate the CFU/mL of E. coli in each tube.As in the solid agar inhibition assays, lactobacilli strains were grown for two nights in 10 mL of MRS broth and uropathogenic strains were grown overnight in 4 mL of LB broth. The optical density as measured at 600 nm  and L. delbrueckii 5010-2. These two strains come from healthy, postmenopausal women and one with a history of rUTI, respectively. Preliminary genome analysis showed the presence of several bacteriocin genes. In light of these preliminary findings, we sought to assess whether major urinary pathogens can be inhibited by these urinary lactobacilli. We used model UTI strains of three major uropathogens: E. coli, K. pneumoniae, and E. faecalis  and four strains of E. faecalis  were used  is needed for inhibition to occur. Taken together, this seems to indicate that the mechanism of inhibition in the cell-free filtrate is highly dependent on pH and is likely a result of the acidification of their environment by lactobacilli species.The cell-free filtrates from both data not shown).One possible explanation for cell-dependent inhibition is the presence of biosurfactants. Several reports showed that phosphate-buffers can elute such surfactants into solution . To testL. crispatus, L. gasseri and L. jensenii  are from the abundant and frequently identified species in the female urinary tract: jensenii . While lE. coli and K. pneumoniae, including model strains and drug-resistant clinical isolates. Many of these same strains, however, either showed modest levels of inhibition or did not inhibit the growth of the gram-positive E. faecalis. L. gasseri 5006-2 and L. delbrueckii 5010-2 inhibited the growth of uropathogenic E. coli both on solid agar and in liquid media  suggest that the underlying inhibition mechanisms are complex. At least for L. gasseri 5006-2, inhibition has both pH-dependent and cell-dependent components  and UAH Startup funds to TS.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Especially in patients after pulmonary valve replacement (PVR), the PV was more fully visualized by ICE. The assessment of PR by TTE underestimated the severity of PR in comparison to cardiac magnetic resonance imaging (MRI) , while there was no discrepancy between the results of ICE and MRI . In comparison to TTE, ICE can safely provide better visualization of the PV and PR in adults with TOF, especially in patients who have undergone PVR.Pulmonary regurgitation (PR) is a risk factor for sudden cardiac death in adult patients with repaired tetralogy of Fallot (TOF). However, transthoracic echocardiography (TTE) cannot fully visualize the pulmonary valve (PV) and PR. We investigated whether intracardiac echocardiography (ICE) could visualize the PV and PR better than TTE. Thirty adult patients with TOF (mean age 33 ± 15 years) scheduled for cardiac catheterization underwent ICE. The visualization of PV and the severity of PR were classified into three grades. ICE depicted the PV better than TTE (ICE vs. TTE: not visualized, partially visualized, and fully visualized:  The number of adult patients with congenital heart disease is increasing worldwide . SignifiIntracardiac echocardiography (ICE) is usually used to guide invasive procedures, such as myocardial biopsy , device The aim of the present study is to investigate whether ICE could visualize the PV and PR better than TTE in adults with TOF.Adult patients with TOF or pulmonary atresia with a ventricular septal defect (PA/VSD) who were scheduled for diagnostic cardiac catheterization were enrolled in Kyushu University Hospital between July 2013 and September 2017. Patients who were scheduled for cardiac intervention were excluded from the study.In all cases TTE was performed using an iE33/EPIQ 7  or Vivid 7 . The PV morphology was assessed on the parasternal short axis view. The severity of PR was evaluated according to the recent American Society of Echocardiography (ASE) guidelines .TM Ultrasound Catheter  or the 9-Fr ViewFlexTM Xtra ICE Catheter  were used. Both of them were only 2D-capable. After inserting the ICE catheter into the RA, it was advanced to the RV under fluoroscopy in a similar manner to the insertion of a Swan-Ganz catheter. With anterior deflection at the height of the tricuspid valve, the ICE catheter could easily enter into the RV. The PV was visualized from the right ventricle without difficulty  and hydroxyzine (12.5 to 25 mg). All patients underwent right heart catheterization from the internal jugular vein. They received 3000 units intravenous heparin after sheath insertion. The ICE catheter was inserted from the internal jugular vein using a 9-Fr or 10-Fr 11 cm sheath. An 8-Fr AcuNavfficulty . All theMorphological assessments of the PV using TTE and ICE were performed by two cardiologists (S.U. and K.H.) on the same day. The visualization of the PV in TTE and ICE was classified into three grades; not visible, partially visible, and fully visible . Cases iTo evaluate the size and function of the RV and left ventricle (LV), cardiac MRI was performed before cardiac catheterization. All patients underwent 3-Tesla MRI , which was equipped with dual-source parallel radiofrequency transmission, 32-channel phased-array torso coils used for radiofrequency reception and a four-lead vector cardiogram used for cardiac gating. RV volumes were measured with axial cine MRI images, as reported in the past . Phase-cp values of <0.05 were considered statistically significant. All statistical analyses were performed using JMP Pro 14 .We used Fisher’s exact test to analyze differences in categorical variables between the two groups. Cohen’s kappa coefficient was used to measure the agreement between the two observers. n = 6; SJM Epic, n = 1; Hancock II, n = 1). Twenty-seven patients (90%) were in sinus rhythm during TTE and ICE. One patient had undergone implantation of a permanent pacemaker.A total of 30 adult patients with TOF or PA/VSD were included in the present study . The meaThe hemodynamic data obtained by cardiac catheterization are shown in The conventional assessment by TTE is shown in Cardiac MRI was performed in 29 patients . There were 18 patients (62%) with severe PR and 5 patients (17%) with moderate PR. The pulmonary regurgitation fraction (PRF) was 37 ± 21%. The right ventricle was dilated and the right ventricular ejection fraction was mildly impaired .n = 1 [3%], n = 13 [43%], and n = 16 [53%] vs. n = 14 [47%], n = 13 [43%], and n = 3 [10%], p < 0.001) (p = 0.006). Cohen’s kappa coefficient showed very good agreement between the assessments of the two observers .ICE depicted the PV better than TTE  . ICE wasp = 0.004). On the other hand, the number of patients who were diagnosed as having severe PR was not significantly different between ICE and MRI .The severity of the PR was assessed in 29 patients who underwent cardiac MRI . TTE undWe performed right heart cardiac catheterization and ICE from the internal jugular vein in all cases. The average time required to perform ICE assessment was 5 min. No procedure-related adverse events  were observed in the present study. Premature ventricular contraction and its short-run were occasionally observed during the manipulation of ICE. However, there were no cases that experienced sustained ventricular tachycardia or hemodynamic deterioration.This is the first study to demonstrate the superiority of ICE in the assessment of PV morphology and the severity of PR in comparison to TTE. The native PV is relatively thin and has highly pliable leaflets, which makes its entire visualization by TTE difficult . In adulSeveral invasive and non-invasive modalities are available for assessing the anatomy of the PV. Cardiac MRI is useful for volume and flow measurement, while it is not the best option for morphological assessment of the PV. CT provides very good spatial resolution. However, the time resolution is inferior to TTE and ICE. The artifact caused by the sewing ring after PVR is also an obstacle to the clear depiction of the valve. 3D TEE is superior to TTE in the assessment of the pulmonary valve. However, the PV is the most anteriorly and superiorly located valve, and it is often challenging to visualize the PV because of interference from other structures, and its far-field location. It is important to choose the best modality for the assessment of the PV function and ICE could be a reliable diagnostic tool, especially when the patient is scheduled for cardiac catheterization. Although we have not performed a quantitative evaluation of PR with ICE, multiple Doppler parameters may improve the quality of the evaluation of PR. Surgical PVR for severe PR has been performed in recent years . HoweverICE is a relatively invasive modality. However, we could assess the PV and PR with ICE from the internal jugular vein without any complications in our patients. The RV is dilated and/or hypertrophied in many patients with repaired TOF. In addition, the RV outflow tract is often repaired with an outflow patch and the possibility of severe complications  is expected to be relatively low. On the other hand, previous reports on the performance of ICE from the internal jugular vein are scarce . It has The present study was associated with several limitations. First, our study enrolled a relatively small number of adult patients with TOF. The usefulness and safety of ICE should be examined in larger study populations and in patients with heart disease other than TOF. The utility and safety for pediatric patients should also be investigated. Second, we did not perform 3D TEE, that is one of the possible modalities to visualize the pulmonary valve. In addition, we did not use quantitative assessment using PISA, which may provide a more precise evaluation of the severity of PR. Further studies using these modalities and techniques will provide useful information to choose the best imaging option in these patients. Third, ICE was performed by a single operator. The safety and quality of images are highly dependent on the experience of the operator. Although overall risks related to ICE are reported to be low, ICE is known to potentially cause transient arrhythmias and vascular-access related complications. Using biplane right ventriculography as a reference guide is helpful to minimize the risks associated with ICE manipulation. Finally, ICE is not feasible in cases after mechanical tricuspid valve replacement, cases with RA/RV thrombus, and intracardiac tumors. Other modalities should be considered in these patients.ICE can safely provide better visualization of the PV and PR than TTE in adults with TOF. It is especially useful in patients after PVR, in whom the information provided by TTE can be quite limited.