IMPG2) gene in patients affected by retinal dystrophies. IMPG2 encodes for a proteoglycan synthesized by photoreceptors and secreted in the interphotoreceptor matrix. Little is known about the structure and function of this protein, we thus decided to characterize zebrafish impg2. In zebrafish there are two Impg2 proteins, Impg2a and Impg2b. We generated a phylogenetic tree based on IMPG2 protein sequence similarity among vertebrates, showing a significant similarity between humans and teleosts. The human and zebrafish proteins share conserved domains, as also shown by homology models. Expression analyses of impg2a and impg2b show a continued expression in the photoreceptor layer starting from developmental stages and continuing through adulthood. Between 1 and 6 months post-fertilization, there is a significant shift of Impg2 expression toward the outer segment region, suggesting an increase in secretion. This raises intriguing hypotheses about its possible role(s) during retinal maturation, laying the groundwork for the generation of most needed models for the study of IMPG2-related inherited retinal dystrophies.Photoreceptor outer segments are surrounded by a carbohydrate-rich matrix, the interphotoreceptor matrix, necessary for physiological retinal function. Few roles for molecules characterizing the interphotoreceptor matrix have been clearly defined. Recent studies have found the presence of nonsense mutations in the interphotoreceptor matrix proteoglycan 2 ( In every tissue, extracellular matrix components interact with cells to finely control and regulate cell and tissue functions. In the retina, the extracellular matrix surrounding photoreceptors' outer segments  is highly specialized. It is mainly composed of proteoglycans and glycosaminoglycans, synthesized by the surrounding photoreceptor cells, retinal pigment epithelial (RPE) cells and Müller glia cells (Mieziewska IMPG2) gene are associated with autosomal recessive Retinitis pigmentosa (arRP)  of the University of Trento and Ministero della Salute (Project Number 151/2019-PR) and were raised following standard procedures (Westerfield https://www.ncbi.nlm.nih.gov/) and a multiple protein sequence alignment was performed by using Clustal Omega sequence alignment program, provided by EMBL-EBI . The same sequence analysis tool was used to generate a phylogenetic tree, based on protein sequence similarity.NCBI database was used to find orthologs to the human IMPG2 . After choosing the vertebrate species to be included in the phylogenetic tree, IMPG2 protein sequences of these animals were obtained from NCBI (https://zhanglab.ccmb.med.umich.edu/I-TASSER/)  and EGF -like domains was performed by using the iTasser webserver (® RNA. cDNA was synthesized by Super-Script® VILO™ cDNA Synthesis Kit (Invitrogen). RT-qPCR was performed using KAPA SYBR® FAST Master Mix (KAPA Biosystems) according to the manufacturer’s instructions. Ube2a was used as housekeeping gene  software. Gene primers are listed in Table Total RNAs from pools of 15 embryos at different developmental stages and pools of adult organs  were extracted by Macherey Nagel NucleoSpinTotal proteins from pools of 15 embryos at different developmental stages and pools of 3 adult eyes and 2 adult brains were extracted using RIPA buffer. 10 µg of total extract were resolved by SDS-PAGE, transferred to a nitrocellulose membrane and then incubated with the antibodies reported in Table Embryos at different stages and eyes dissected from adult fishes at different stages were fixed in 4% paraformaldehyde (PFA) at 4 °C overnight, embedded in 30% sucrose at 4 °C for 2–3 h and included in OCT compound. A Leica cryostat was used to obtain 14 µm retina sections. Immunohistochemistry was performed as follows: slides with sections were incubated in blocking solution (0.1% Triton X-100 and 0.5% BSA in 1 × PBS) for 1 h at room temperature (RT) and then incubated in diluted primary antibody in blocking solution (dilution specific for the primary antibody in use), at 4 °C overnight in a humidified chamber. After 3 washes of 10 min in 1 × PBS, 0.1% Triton X-100, slides were incubated in secondary antibody in blocking solution (1:1000), for 2 h at RT in a humidified chamber. Slides were then washed 3 times for 10 min 1 × PBS, 0.1% Triton X-100 and then incubated with nuclear staining dye  in 1 × PBS for 10 min at RT. Following 3 washes of 10 min in 1 × PBS, 0.1% Triton X-100, slides were mounted using Aqua-Poly/Mount coverslipping medium . As negative controls, sections were incubated with secondary antibodies only and revealed no detectable signal (not shown).Primary and secondary antibodies used for immunohistochemistry experiments are reported in Table Measurements of the length of Impg2 signal were done on images of IHC experiments for Impg2 and Zpr-3. Three eyes dissected from three different animals were used, and three sections were considered for each eye. For each section, three different regions of the retina  were considered to have a total number of measurements equal to 27. Measures included: i) the distance between the outer limiting membrane (OLM) and the more external Impg2 signal; ii) the distance between the (OLM) and the outer limit of the rod outer segments (ROS), identified by the outer limit of Zpr-3 staining.Immunohistochemistry images were acquired using a Leica TCS SP8 confocal microscope equipped with an Andor iXon Ultra 888 monochromatic camera. The HC PL APO 40x/1.30 Oil CS2 (Leica Microsystems) objective was used for the acquisition. All figures were analyzed with Fiji and assembled in GIMP.All data are reported as mean ± SEM. Statistical analysis was performed using the GraphPad Software. Data groups from RT-qPCR experiments and those from measurements of outer limiting membrane (OLM)-Impg2 distance and OLM-ROS distance were compared by one-way ANOVA followed by Tukey’s test for multiple comparisons. The statistical significance level was set at p < 0.05. Values levels of statistical significance are described by asterisks .www.uniprot.org/uniprot/Q9BZV3).Human IMPG2 is a 1241 residues protein with four topologically distinct regions: a 22 aa long signal peptide, an extracellular topological domain (residues 23 to 1099), a helical transmembrane domain (1100 to 1120), and a cytoplasmic topological domain (1121 to 1241). The extracellular region contains two SEA domains and two EGF-like tandem repeats, together with five hyaluronan-binding motifs. The protein is also a target for glycosylation and phosphorylation at different sites  using as query the human IMPG2 sequence. We found statistically significant similarities only among jawed vertebrates (data not shown). We then proceeded to generate a phylogenetic tree by aligning the protein sequences of chosen vertebrate species, retrieving the different IMPG2 protein sequences from the NCBI database. We selected some of the most common species of various vertebrate groups to include in our analysis. We then performed a multiple sequence alignment and generated a phylogenetic tree , have only the Impg2a paralogue, possibly due to the loss of the other paralogue after the whole-genome duplication event. In contrast, the spotted gar (which does not have any genome duplication) only has an Impg2b-like protein.We first investigated IMPG2 conservation during evolution by performing a protein–protein alignment  are conserved in both zebrafish paralogues  modelling to investigate the putative conformations of SEA and EGF-like conserved domains from their amino acid sequences. The following sequences of the human protein were submitted to the iTasser webserver: 239–390 (SEA1), 896–1012 (SEA2), and 1012–1098 (EGF-like tandem repeat). Domain identifications were obtained by checking Pfam  of adult fish. Results reveal very low expression of impg2b at 2.5 days post fertilization (dpf) whilst impg2a mRNA is barely detected  . As described in the above paragraph, this domain is conserved in both zebrafish paralogues, and thus is the epitope. Besides epitope sequence analysis, validation for usage in zebrafish was also performed by antibody dilution series, and incorporation of adequate negative controls in all experiments. We performed western blot experiments on pools of embryos at different developmental stages and pools of dissected brains and eyes of adult zebrafish. The expression of the two proteins becomes detectable at 3 dpf, when rhodopsin also starts being expressed Fig. a. This oTo study their localization within the retina, we performed immunohistochemistry (IHC) experiments on sections of WT embryos at different developmental stages and adult dissected eyes, using the same anti-IMPG2 antibody used in western blots, which recognizes both zebrafish proteins. Thus, we will refer to Impg2 localization in general Figs. a-e’’’’. To understand the localization of Impg2 signal in more detail, we compared it to that of Zpr-3, which labels outer segments (OS) of both rods and short double cones Fig. a-e’’’’. The dynamic distribution of the dotted Impg2 signal observed in retinas of different ages might be due to an effective rearrangement in its localization or to its adjustment to photoreceptor’s elongation during retinal growth and maturation. To discriminate between these possibilities, we measured the distance between the OLM and the outer limit of the Impg2 staining and compared it to the distance between the OLM and the outer limit of ROS identified by the external boundary of Zpr-3 staining. Results Fig. f suggestExtracellular matrix plays a key role in retinal function and disease , which diverged from teleost ancestors before the genome duplication, has only one impg2 gene. Moreover, not all the teleosts that we analyzed have two paralogues. For example, the killifish (Notobranchius furzeri) and the blind cavefish (Astyanax mexicanus) have only one impg2 gene. This is consistent with the idea that, after the genome wide duplication, the two different paralogues can have different fates in different species, and that the most likely outcome is the loss of functionalization of one of the two  , IMPG2 is considered to be a transmembrane or extracellular protein (Salido and Ramamurthy IPM structural integrity is fundamental for its function. According to the data presented in this work and the published literature regarding Impg2 in other species (Salido and Ramamurthy impg2a and impg2b mRNAs and proteins in zebrafish embryos and adults, providing insights into the biology of these disease-related genes. Our data allow a better understanding of the physiology of the IPM and the interactions among its components, laying the basis for further studies on the molecular mechanisms of retinal diseases, in turn leading to innovative therapeutic approaches.This work combines structural analysis of the conserved domains of human IMPG2 and zebrafish Impg2a and Impg2b, and expression analysis of


Ocean conditions near the grounding zones of Antarctica’s ice shelves play a key role in controlling the outflow and mass balance of the ice sheet. However, ocean observations in these regions are largely absent. Here, we present a detailed spatial survey collected with an underwater vehicle in a basal crevasse located in the ocean cavity at the Ross Ice Shelf grounding zone. The observations depict fine-scale variability in ocean forcing that drives asymmetric melting along the lower crevasse sidewalls and freezing in the upper reaches of the crevasse. Freshwater release from melting at depth and salt rejection from freezing above drives an overturning circulation. This vertical circulation pattern overlays a dominant throughflow jet, which funnels water parallel to the coastline, orthogonal to the direction of tidal currents. Importantly, these data reveal that basal crevasses influence ocean circulation and mixing at ice shelf grounding zones to an extent previously unknown. In situ observations reveal how ocean physical properties drive ice melting and freezing within an ice shelf basal crevasse. Note that hereafter we simply refer to conservative temperature as temperature and absolute salinity as salinity.The Filchner-Ronne, Ross, and Amery ice shelves have been stable over the modern satellite observational record  an effective temperature of −92.50°C (f< 0°C) in the water column that forms saline marine ice with a temperature characteristic of the salinity- and pressure-dependent freezing point Icefin . These features extended upward from a relatively flat ice shelf base with concave features separating the crevasses . The ocean circulation within the crevasse exhibited a three-dimensional structure that varied considerably with horizontal and vertical position in the feature. The strongest velocities flowed southward  through the crevasse at a moderate rate of v = −6 cm s−1 in a jet concentrated along the western sidewall below 490-m depth  less than 0 s−2 (−5 s−2> N2 ≤ −1 × 10−5 s−2) occurred around 485- and 492-m depth. The shallower instabilities were sampled toward the lower limit of the observed downwelling plume . Visidewall . Interlesidewall . These dan 0 s−2 . On the ng plume  where thng plume  and visung plume  located ng plume . We attr−5 s−2> N2 ≤ 2 × 10−5 s−2) over the 490- to 508-m-depth range of the throughflow jet, which exhibited broad upwelling  section of the western and eastern crevasse walls beneath 476- and 494-m depth, respectively . Scallopd) between 13 and 22 cm (table S1). Symmetrical scallops also occurred on the western sidewall keel at 508-m depth, with λ = 48 to 63 cm and d = 10 to 13 cm. Scallops along the western sidewall above this depth (between 482 and 506 m) were instead asymmetric, with an elongated vertical axis of λ = 88 to 157 cm and a horizontal axis of λ = 43 to 78 cm. These asymmetric scallops also exhibited greater incision depths, ranging from 15 to 32 cm.Scallops exhibited quasi-symmetrical axes along the eastern sidewall, with axes oriented in the horizontal and vertical directions of wavelength (λ) ranging from 45 to 78 cm and incision depth (v), with crests perpendicular to the throughflow jet’s horizontal southward flow  tidally driven currents in the underlying ocean cavity (fig. S1). Scallops along the eastern sidewall followed a similar transition from a more random orientation along the crevasse to keel at 506-m depth to a generally more organized north-south horizontal axis pattern upward in the crevasse to 494-m depth  to meteoric ice .The runnels along the eastern sidewall initiated as 60- to 120-cm-wide and 30-cm-deep features carved into the marine ice above the supercooling horizon (within sensor uncertainty), located at 471-m depth at the time of sampling. They deepened and widened below this depth, etching into the meteoric ice in the above-freezing portion of the crevasse to a maximum of Our observations of a vigorous sinking plume adjacent to the eastern runnels and their progressively deeper incision into the ice with depth in the crevasse agree with a localized melting mechanism. However, the presence of scallops superimposed on the eastern wall runnels below 494-m depth and negligible observed vertical ocean currents indicate that horizontal circulation dominated from this depth to the keel. This is additionally supported by negligible further incision of the runnels despite a twofold widening between 494-m depth and the crevasse keel. We also interpret that the western runnels’ small vertical range results from the primarily horizontal throughflow jet dominating melting along most of the above-freezing portion of this crevasse sidewall.<0.10 cm , which is the square root of the kinematic shear stress exerted by a surface on a flow . At the time of sampling, the throughflow jet adjacent to the western wall exhibited a dominant southward horizontal and secondary vertical circulation pattern from 487-m depth to below the crevasse keel (u∗ = 0.32 cm s−1 on the ocean (table S1). Relating u∗ to the scallops’ horizontal λ produced a dimensionless drag coefficient (CD) between 2.70 × 10−3 and 9.20 × 10−3, with a mean of 5.90 × 10−3 . The point of no flow occurred at a roughness length (z0) of 3.53 cm from the ice surface or d.The immobile crevasse sidewalls exerted drag on ocean flow through the crevasse, which induced velocity shear as it transitioned from free-stream to no flow across the boundary layer. This phenomenon can be represented by the friction velocity . The z0 was smaller at 2.21 cm with a narrower range of d. Despite a much smaller sample number (N = 70) used to derive these parameters along the eastern sidewall than the western sidewall (N = 839), the scallops throughout the crevasse exerted a similar frictional drag on the adjacent oceanic flow.Scallops along the eastern sidewall from 483- to 506-m depth modified the weaker and less-organized horizontal flow on this half of the crevasse to exert an average u∗ = 0.21 cm s−1 and z0 = 4.16 cm. This translated to 1 × 10−3 ≥ CD ≤ 5.50 × 10−3 (mean = 2.30 × 10−3) based on the runnels’ horizontal λ and d. Note that while the analysis yielded a satisfactory fit (table S1), the lack of data located further away from the wall and the localized nature of runnel melt features cause uncertainty regarding the shape of the boundary layer velocity profile. We suspect that d may relate to u∗ and CD in these vertically oriented features more appropriately than λ. Nonetheless, the quantification of frictional forces exerted by various ice morphology on the ocean reveals that they induce shear to increase turbulent kinetic energy, which potentially enhances melting throughout the crevasse.The downwelling plume adjoined vertically oriented runnels along the eastern sidewall over an observed depth range of 471 to 483 m. Implementing the same analysis as above yielded Considering phase changes occurring both directly at the sidewall ice-ocean interface  where shear induced by the throughflow jet efficiently mixed the sparse ocean heat into the ice face. Melt rates along the eastern wall were lower at a maximum around 0.17 ± 0.04 m year−1, due to reduced current speeds on this side of the crevasse. Melting decreased with height in the crevasse to near zero at 486-m depth, as current speeds reached a minimum and pressure release caused ocean thermal driving to approach 0°C. The elevated melt rates along the lower western crevasse sidewall relative to the eastern sidewall are consistent with its shallower slope. This suggests that the throughflow jet is a persistent feature that enhances localized melting and erodes this section of the crevasse to produce its shallower slope.The transition from above to below freezing ocean temperatures paired with highly variable current speeds  affected−1, with frazil crystal formation . We convert our transfer velocities to average values of these parameters  so that they may be considered in future studies that are unable to directly calculate CD. Transfer velocities . A portion of this dive extending upward to 492-m depth in the crevasse located around 4100 m from the GL .The visual onset of marine ice on the eastern sidewall coincided with the depth that neighboring ocean temperatures transitioned to supercooled . The tuned rising and sinking plume model reflects this, with simulated total neap tide freeze rates increasing from ∼65% greater than spring melt rates near the western supercooling horizon to ∼500% greater near the crevasse roof .Predicted spring tide melting persists in the upper portion of the crevasse that was supercooled during neap tide at low rates of 0.03 to 0.12 m yearOur observations expose in unprecedented detail the ice morphology, three-dimensional ocean circulation, and thermodynamics within an ice shelf basal crevasse. We identify a dominant jet flowing through the crevasse and an overturning circulation driven by freshwater release from melting on the lower sidewalls and salt rejection from freezing above . This ovThe dominant horizontal throughflow jet intensifies estimated melting by four times along the lower western sidewall, when compared to rates driven solely by the overturning crevasse circulation. The higher velocities in this jet more efficiently entrain ambient water from the center of the crevasse, which dilutes cold and fresh GMW released during melting and preserves the upwelling plume’s positive thermal driving. This process raises the supercooling horizon along the western half of the crevasse to the depth predicted solely by pressure release, thus largely negating the cooling and freshening from melting. However, we posit that GMW accumulation from the integrated melting driven by the throughflow jet along the crevasse sidewall will lower thermal driving and the supercooling horizon downstream (south at the time of sampling) of our study site, which would in turn influence crevasse geometry.Icefin survey conducted outside of the crevasse identified quasi-two-dimensional ripple formations in the ice base with crests oriented orthogonal to the northeast-southwest (perpendicular to GL) tidal currents that dominated circulation to first order in the underlying ocean cavity  and faster current speeds that drove melt rates on the order of 100 m year−1 showed that ice morphological features can form in under 12 hours (The portion of the 12 hours  (20), thThe crevasse width is an order of magnitude lower than the internal deformation radius given the observed ocean conditions, and recorded maximum north-south velocity shear across the crevasse exceeds predicted geostrophic velocities by six times . This signifies that Earth’s rotation does not alter the flow direction as it feels the friction of the crevasse wall  to a 110-m2 area to convert area fluxes  to volume fluxes . Meltwater only comprised ∼0.40% of the jet’s volume transport due to the low GMW concentrations from relatively weak ice shelf melting. Nevertheless, the nontrivial total volume funneled through the crevasse in this jet at the time of sampling argues that the crevasse extends laterally across RIS for a considerable distance.The dominant jet flowing through the crevasse transported a volume of water southward at 5.54 m3 s−1 over a 550-m2 area at the time of sampling. The small area that this water funneled through obscures the potential larger influence of this transport. If the volume transport rate through the crevasses is standardized by the flux gate area, then it becomes 28% of the inflow through one of the main pathways that translates the source ocean water mass (HSSW) beneath RIS that feeds our study region  onboard features  and a prIcefin: a Neil Brown Ocean Sensors conductivity-temperature (CT) sensor and a Valeport ultraP pressure (P) sensor. The manufacturer-stated accuracies are C: ± 0.010 mS cm−1, T: ± 0.005°C, and P: 0.100 dbar. The C, T, and P accuracies translate to uncertainties in SA of ±0.008 g kg−1 and Θ of ±0.018°C. All sensors were factory calibrated before entering the field in 2019, and the CT sensor was field calibrated through a cross comparison of vertical profile data from beneath the borehole at RIS with data obtained independently with an RBR Concerto3 CTD profiler. This resulted in offsets/root mean square residuals of C: 0.249 mS cm−1/0.003 mS cm−1 and T: 0.018°C/0.002°C. The fact that these residuals are lower than the manufacturer-stated accuracies lends confidence to the quality of data after application. The CT sensor recorded at a frequency of 5 Hz and the P sensor recorded at 1 Hz. Pressure measurements were interpolated to match the 5-Hz CT data to derive hydrographic variables.We follow the processing reported in  Remove background atmospheric pressure reading;C and T (excluding borehole data);2) Remove outliers ±2 standard deviations (SDs) from the mean for C, T, and P;3) Apply three-point weighted mean filter to C and T measurements with time lag ;4) Align C and T data when Icefin moved at speeds less than 5 cm s−1;5) Remove SA, density (ρ), etc., using the Thermodynamic Equation of Seawater 2010 (6) Derive hydrographic variables such as Θ, 7) Average hydrographic variables into 1-m bins with a ±1 SD range.SA and Θ were 0.002 g kg−1 and 0.002°C for the west profile and 0.002 g kg−1 and 0.003°C for the east profile. These ranges are well below the initial uncertainties derived from manufacturer-stated C, T, and P accuracies, which supports our analysis and interpretation of small-scale variability in these properties within the crevasse.After postprocessing, a mean of 77 and 73 samples were averaged into each 1-m bin of the associated hydrographic variable for the west and east crevasse profiles, respectively. The average ±1 SD range for each 1-m bin of z direction discussed here. Three-dimensional ocean current velocities come from the DVL on board Icefin, which doubles as an acoustic Doppler current profiler (ADCP). As the DVL retrieves vehicle motion from an immobile surface, the ADCP function monitors water column velocities in 2-m bins at a variable start distance from the vehicle. The minimum nominal altitude from a surface for the ADCP to operate is 10 m, and gradients in vehicle pitch, roll, heading, and velocity control the distance of the first bin from the vehicle and the sampling frequency. Here, we subsample ADCP velocities to a fixed rate of 1 Hz; they can reach a maximum of 5 Hz. Manufacturer-stated accuracies for our ADCP are 1% of the DVL-recorded vehicle velocity in that direction. Icefin typically travels at ≤50 cm s−1 in the x direction, making uncertainty in ADCP × velocities 0.50 cm s−1. Vehicle motions in y and z are typically substantially slower, so the associated ADCP y and z uncertainties are also lower.We follow the processing reported in  Remove measurements if bin depth is below seafloor depth;2) Recalculate bin depth and horizontal location considering vehicle pitch and roll;−1 or unadjusted (before differencing vehicle velocity) value is 32,767 m s−1 ;3) Remove measurements if exactly 0 m s4) Remove measurements if absolute value of vehicle pitch or roll is greater than 30°;x and y velocities);5) Recalculate measurements considering vehicle pitch and roll (excluding z velocities);6) Convert measurements from vehicle reference frame to geographic reference frame (excluding 7) Apply 30-s rolling mean filter;8) Filter measurements for gradients greater than 1 SD from the mean in vehicle speed, pitch, roll, and individual bin velocity; and9) Collate bins into 1-m vertical bins and then remove measurements if they exceed 1 SD of the mean for that 1-m depth range.x, y, and z velocity components reflect ocean currents in the eastward (u), northward (v), and vertical (w) directions.After postprocessing, the ADCP Icefin. The altimeter manufacturer states an accuracy of up to 1 mm. The DVL takes into account pitch, roll, and heading when producing ranges, and we apply these corrections to the altimeter in postprocessing.We follow the processing reported in (The elevation data from each of these sensors were postprocessed in the following manner:1) Remove data when vehicle pitch or roll exceeds 30°;2) Remove data when the gradient in vehicle pitch, roll, or heading exceeds 1 SD of the respective mean;3) Despike by removing data that exceed 2 SDs of the mean of the gradient in elevation of the surface;4) Investigate elevation profiles and manually remove outliers; and5) Apply a 3-point weighted mean filter to ice elevations and an 11-point weighted mean filter to seafloor elevations.After postprocessing, 90% of the ice elevation measurements were at a horizontal resolution of 63 ± 59 cm (mean ± 1 SD), with a minimum and maximum spacing of 0.06 and 627 cm, respectively. Eighty-six percent of postprocessed seafloor data were at a horizontal resolution of 44 ± 23 cm (mean ± 1 SD), with a minimum and maximum spacing of 18 and 169 cm, respectively.SA, and σΘ differences at each depth bin from 492 to 535 m between dive 1 and dive 3 (fig. S3). Note that we averaged dive 3 east and west profiles into a mean “central” crevasse profile in this exercise, because the dive 1 data were located toward the crevasse’s center. Dive 1 and 3 profiles exhibited very little change in Θ, SA, and σΘ with depth between the upper layer and in the crevasse, large variability across the pycnocline, and contrasting behaviors in the lower layer . The differences between profiles reflected these patterns , which led us to select the upper and crevasse layers for our offsets. We decided this because variability was most consistent between profiles in these layers, and the target of this effort is to investigate ice-ocean thermodynamics in an ice shelf basal crevasse. The Θ, SA, and σΘ differences in the upper and crevasse layers ranged from 0.0091° to 0.0293°C, from −0.0041 to 0.0052 g kg−1, and from −0.0037 to 0.0038 kg m−3, respectively. We selected the mean difference of Θ = 0.0191°C, SA = 0.0014 g kg−1, and σΘ = 0.007 kg m−3, and applied each to the dive 3 profiles as constant offsets to predict hydrographic conditions in the crevasse during spring tide. Note that the AS and σΘ differences are within the uncertainty range of the bin-averaged data and thus not significant.During dive 1, a small subset of data were collected up to 492-m depth in the crevasse located around 4750 m from the GL . These dSA changes reflect the turbulent mixture between three water masses:We estimate the concentrations of water masses in the ocean cavity beneath RIS with a three-point endmember partition  HSSW formed in Ross Sea polynyas during winter  Fresh GMW produced by ocean-driven ice shelf melt (SA = 0 g kg−1.3) Subglacial freshwater (SGW) produced by geothermal and frictional heating of the ice upstream of the GL (−1)−1 determined by the following equation  denotes current speeds  and y intercept (b) of the boundary layer logarithmic profile along sections of each crevasse wall with distinct circulation patterns. We express the respective z0 value as a fraction of the morphological incision depth d of features along the crevasse wall (table S1).We combine ADCP velocities with scaled ice morphologies obtained with  effects , which d effects . The logU ≥ 3 cm s−1) with the quadratic stress formula , practical salinity (BS), and pressure (Bp) at the ice-ocean interface and BT resides at the freezing point dictated by the local salinity and pressure; a = −5.73 × 10−2°C psu−1, b = 9.39 × 10−2, and c = −7.53 × 10−8°C Pa−1 are considered constants. The heat  flux divergence is the difference between the ocean heat  flux toward the ice We proceed with the conservation of heat equations in the following formw and T denote the measured ocean density and temperature along the crevasse walls, pc = 3974 J kg−1°C−1 is once again the specific heat capacity of cold seawater, and In i = 918, ipc= 2009 J kg−1°C−1, and −6 m2 s−1 are the density, specific heat capacity, and thermal diffusivity of ice, respectively. We assume that the temperature gradient through the ice shelf . Several nondimensional parameters commonly used in In B, T, and BS. BT and BS and solve for BT and BS can be calculated.The three unknowns in the melt rate parameterization are We follow a similar method to that described in the above section to estimate freeze rates of frazil ice crystals suspended in the supercooled water column by parameterizing heat and salt transfer at the crystal-ocean interface (Bp). In The conservation of heat and salt is as followsC represents the fraction of frazil crystals suspended in the water column at any time (varied from 1 × 10−9 to 1 × 10−5), and in T and S) are identical to the observations used at each depth bin in In Nu = 1 is the dimensionless Nusselt number, the ratio of convective to conductive heat transfer at a boundary, r is their radius. We choose the respective mean, minimum, and maximum value of r to be 0.60, 0.50, and 0.70 mm, which are within the range of observations , then use it to solve for D over the full crevasse extent along each sidewall. Values of D were initially set to half the width of the crevasse to compute D values. The plume model equations are as followsWe integrate our hydrographic observations of 2 s−1) in the region of the crevasse where we lack data in both the western and eastern halves by setting the upwelling flux wwestD equal to the downwelling flux weast(l − D), where l − D is the total width of the crevasse minus the upwelling plume width. We make the assumption that l − D equals the eastern half-width of the crevasse at our uppermost measurement of weast (471-m depth), effectively setting the downwelling plume width. We then find a value of D at our uppermost measurement of wwest (487-m depth) that after running the model produces a best fit to weast(l − D) at 471-m depth, with a residual of 0.01 m2 s−1 or ∼10% of the observation. The model iterates in 1-m bins to be consistent with our sampling resolution, considering the conservation of mass , and the entrainment constant E0 = 0.07  and the ambient fluid density ρa is the mean of ρwwest and ρweast at each depth bin; ρ0 = 1027 kg m−3 is the reference density.Beginning with  of mass  and 28 a of mass . In Eq. −1, we interpret that this marine ice does not melt away during spring tides. Therefore, we consider a range of initial values for C (1 × 10−9 to 1 × 10−5) in our model and select the lowest initial value of C, which produces an Cinitial = 1 × 10−6 fulfills this requirement for the full range of CD values considered , and a lower layer density of ρ2 = 1027.95 kg m−3 that represents the mean of the water column below the pycnocline (thickness H2 = 12 m); this produces iR = 559 m. We choose to consider the full water column above the pycnocline as our upper layer, because the vertical density gradient was very weak over this depth range.We follow (We follow (gv is directed through the crevasse and is based on an assumed level of no flow at our lowest data point (532-m depth). When the lateral density gradients (gv = −1 cm s−1. This is one-sixth of the maximum observed southward velocities in the throughflow jet.The calculated geostrophic velocity All statistics were performed in MATLAB. The above sections provide the details of these statistical analyses, with the underlying mean and SD functions being native to MATLAB.


Over the past few decades, drug therapy problems (DTPs) have become a significant public health concern worldwide. DTPs in patients with diabetes are responsible for uncontrolled glycemia, disease worsening, early development of complications, high healthcare expenses, prolonged and recurrent hospitalizations, and mortality.This study aimed to identify the prevalence of drug therapy problems and determine the associated factors among patients with type II Diabetes Mellitus at a University Teaching Hospital in Southwest Ethiopia.Hospital-based cross-sectional study was conducted between September and October 2022. Data were collected through medical record reviews and interviewer-administered structured questionnaires, which were then analyzed using SPSS version 26. Cipolle’s method was adapted for classification of DTPs. Bivariate followed by multivariate logistic regression analysis was used to assess the association between predictor variables and the outcome variable. P-value ≤ 0.05 was employed as a cut-off point to determine statistical significance.Among 117 participants, 172 drug therapy problems (DTPs) were identified, with an average of 1.47 DTPs per patient, and83 (70.9%) participants had at least one type of drug therapy problem. Of the seven DTPs identified, need additional drug therapy was the most common, 50 (42.7%), followed by non-compliance with medication, 45 (38.5%) and ineffective drug therapy, 25 (21.4%). Occupational status and comorbidity were factors that associated with the occurrence of DTPs. Farmers were approximately four times more likely to develop DTPs than housewives were (adjusted odds ratio (AOR) = 3.56, 95% CI: 1.12–11.38, P = 0.03). The odds of drug therapy problems were twice as high in those with four comorbid conditions than in those without comorbidities .In the current study, the proportion of type 2 diabetes patients with drug therapy problems was high. This potentially lead to uncontrolled glycemia and early development of comorbid conditions, increasing morbidity and mortality rates. This could be attributed to the failure to effectively integrate clinical pharmacy services in different hospital wards, which is the case in virtually all hospitals in Ethiopia. Diabetes Mellitus (DM) is a common metabolic disease characterized by chronic hyperglycemia resulting from defects in insulin secretion, action or both. Type 1 DM which accounts for 5–10% of all diabetic cases is characterized by absolute insulin deficiency. Whereas, Type 2 diabetes is mainly due to insulin resistance with a relative insulin deficiency, and accounts for 90–95% of all diabetes cases. Gestational diabetes mellitus and other specific type (monogenic diabetes) are other forms of diabetes mellitus Reviewers' comments:Reviewer's Responses to Questions  Comments to the Author1. Is the manuscript technically sound, and do the data support the conclusions?  The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: YesReviewer #2: Yes**********  2. Has the statistical analysis been performed appropriately and rigorously?   Reviewer #1: YesReviewer #2: Yes**********  3. Have the authors made all data underlying the findings in their manuscript fully available?PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.  The Reviewer #1: YesReviewer #2: Yes**********  4. Is the manuscript presented in an intelligible fashion and written in standard English?  PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.Reviewer #1: YesReviewer #2: Yes**********  5. Review Comments to the Author  Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. Reviewer #1: Dear Author,1) It is mentioned that “severely ill patients were excluded”. What do you want to mean as severely ill patients? What is its coverage? Could you define clearly?2) Who and how many people collect the data? Who did provide training to data collectors? Can you define the concept of this training?Reviewer #2:  Dear Authors,I enjoyed reading your manuscript. It is well-written and flows smoothly. However, I have a few minor concerns that, if addressed, would greatly enrich your manuscript. Please find my specific comments and suggestions below:1. Please consider using the phrase "Patients with type II diabetes mellitus" instead of "Type 2 Diabetic Patients."In the study abstract:2. I recommend modifying your objectives as follows:Objectives: This study aimed to identify the incidence of drug therapy problems and determine the associated factors among patients with type II Diabetes Mellitus at a University Teaching Hospital in Southwest Ethiopia.Regarding the Results section of the study abstract:3. Please write "adjusted odds ratio" in full before using the abbreviation.4. When reporting numerical values, limit the decimal places to two digits.5. In your study results, you mentioned the following finding: "Farmers were about four times more likely to develop DTPs than housewives ." Could you please elaborate on the clinical significance of this finding? Is it related to non-compliance with home medications?In the Methodology section:6. Provide further details about the study design, including the process of recruiting your study sample and how participants were selected and approached.7. Specify the ambulatory setting where you conducted your sample selection during follow-up visits. Did you target endocrinology or family medicine clinics? I suggest describing the ambulatory setting of the hospital and the specialties involved.8. When providing operational definitions, make sure to cite all the references used after each definition.Regarding the Data Collection Instrument:9. You mentioned using "A structured questionnaire prepared from previous studies with minor modifications." Please cite the articles you used to prepare your questionnaire and describe the specific modifications made.10. Instead of describing the process of data collection, provide a detailed description of the questionnaire used.11. Create a separate section for the data collection process.12. Clarify how training of data collectors took place. I have a few additional questions regarding the data collection process:13. I didn't fully understand the reason for interviewing patients to detect DRPs. Could you please describe the specific components of information retrieved directly from the patients' medical files and those obtained during the interviews?14. How did the interviews take place? Were they conducted in the clinic with the attending physician or prior to entering the clinic?15. In case the interviewers detected any DRPs, how did they respond? Did they inform the prescriber or intervene to resolve these drug-related problems?In the Results section:16. Avoid starting a new sentence with numbers.17. Please ensure consistency when reporting frequencies and percentages. For example, write "27 (23.1%) of the study subjects developed microvascular complications, with nephropathy accounting for the highest proportion." Remember to add a space between the frequency and the percentage.18. Add a footer to Table 4.Lastly, it would be beneficial to mention any study limitations other than the issue of causality. (e.g. selection bias as you only selected patients during a follow-up visits) Overall, your manuscript is well-structured and engaging. By addressing these minor concerns and incorporating the suggested revisions, your work will be even more comprehensive and accessible to the readers.**********  6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.If you choose “no”, your identity will remain anonymous but your review may still be made public.Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.  Reviewer #1: NoYes: Rania ItaniReviewer #2: **********https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool,  16 Jun 2023Response to the editor1. Journal requirementsThank you. All the journal related issues raised  are addressed in the revised manuscript.2. Abstract- Conclusion• Consider removing any values for the study results from this part. This also applied for the conclusion part at the end of the manuscript.Thank you for your comment. The figures/values are now removed from the conclusion parts (Please see line numbers 32-36 and 292-300 of the revised manuscript).3. Page 3, Line 48: Consider writing (20 and 79 years old).Thank you. The correction has been made (please see line number 47 of the revised manuscript)4. Page 3, Line 56-57: ‘pharmacologic management of hypertension and hyperlipidemia’Consider writing ‘pharmacologic management of comorbid disease conditions, such as hypertension and hyperlipidemia’.Thank you for your comment. The correction has been made (please see line numbers 55 & 56 of the revised manuscript)5. Discussion• Page 15, Line 231/232: The findings do not match the order of the referencesThank you very much. The references are now correctly reordered (please see line numbers 232 & 233 of the revised manuscript).6. Page 15, Line 243-245: Consider providing a reference for the sentence.Thank you for your suggestion. References are now added (please see line numbers 240-246 of the revised manuscript).7. Limitations of the study. • More limitations are required to be reported for this study.Thank you. Limitations of the study section is now revised (please see line numbers 286-291 of the revised manuscript)Response to Reviewer #1Dear Reviewer #1, thank you for your constructive questions.1. It is mentioned that “severely ill patients were excluded”. What do you want to mean as severely ill patients? What is its coverage? Could you define clearly?Thank you for your concern. Severely ill patients refer those who were unable to respond to the questions due to their medical condition/s. It is now clearly stated in the revised manuscript (please see line numbers 96-97 of the revised manuscript).2. Who and how many people collected the data? Who did provide training to data collectors? Can you define the concept of this training?Thank you. Two pharmacists collected the data, and the principal investigator gave the training. Because the data collectors are experienced in data collection, the training mainly focused on explaining the components of the questionnaire. (Please see line number 148-149 & 153-154 of the revised manuscript)Response to Reviewer #2Dear Reviewer #2, thank you for your invaluable comments and suggestions.1. Please consider using the phrase "Patients with type II diabetes mellitus" instead of "Type 2 Diabetic Patients."Thank you for your suggestion. Amendments are now made in the revised manuscript.2. In the study abstract: I recommend modifying your objectives as follows:Objectives: This study aimed to identify the incidence of drug therapy problems and determine the associated factors among patients with type II Diabetes Mellitus at a University Teaching Hospital in Southwest Ethiopia.Thank you very much for your recommendation. The correction has been made in the revised manuscript (please see line 14-16 of the revised manuscript)Regarding the Results section of the study abstract:3. Please write "adjusted odds ratio" in full before using the abbreviation.Thank you. The correction is now made. (Please see line number 29 of the revised manuscript). 4. When reporting numerical values, limit the decimal places to two digits.Thank you. The decimal places of all numerical values are limited to two digits. Please see the revised manuscript.5. In your study results, you mentioned the following finding: "Farmers were about four times more likely to develop DTPs than housewives ." Could you please elaborate on the clinical significance of this finding? Is it related to non-compliance with home medications?Thank you. Yes, it is attributed to non-compliance to prescribed medications. It is discussed in the discussion section, page 17, line 278-284 of the revised manuscript).In the Methodology section:6. Provide further details about the study design, including the process of recruiting your study sample and how participants were selected and approached.Thank you. Hospital-based cross sectional study design was used (line 83), and the participants were selected using a simple random sampling technique (line 117). Details on the process of approaching and recruiting the study subjects is explained in the ‘data collection method’ section (line 137)7. Specify the ambulatory setting where you conducted your sample selection during follow-up visits. Did you target endocrinology or family medicine clinics? I suggest describing the ambulatory setting of the hospital and the specialties involved.Thank you. The ambulatory setting of MTUTH where diabetes patients receive a follow-up care is called diabetes outpatient clinic, and there are no specialties involved. 8. When providing operational definitions, make sure to cite all the references used after each definition.Thank you for your suggestion. All operational definitions are now are cited. (Please see line numbers 119-136 of the revised manuscript).Regarding the Data Collection Instrument:9. You mentioned using "A structured questionnaire prepared from previous studies with minor modifications." Please cite the articles you used to prepare your questionnaire and describe the specific modifications made.Thank you for your constructive comment. A comprehensive questionnaire was prepared, and the minor modifications are on socio-demographic characteristics of the study subjects, in which some essential variables like social drug use and BMI (body mass index) were added. The references used are now cited. (Please see line 14 and 142-143 of the revised manuscript) 10. Instead of describing the process of data collection, provide a detailed description of the questionnaire used.Thank you. The contents of the questionnaire are sufficiently described (line numbers 139-152) and the tool is attached as a supplementary material.11. Create a separate section for the data collection process.Thank you. All relevant information on the data collection process is stated in the ‘data collection tool and process’ section. (Please see line numbers 139-152 of the revised manuscript)12. Clarify how training of data collectors took place. Thank you. Training of the data collectors was carried out before initiation of the study. Because the data collectors are experienced in data collection, the training mainly focused on explaining the components of the questionnaire/data collection tool. Please see line numbers 155-156 of the revised manuscript. I have a few additional questions regarding the data collection process:13. I didn't fully understand the reason for interviewing patients to detect DRPs. Could you please describe the specific components of information retrieved directly from the patients' medical files and those obtained during the interviews?Thank you. Patient interview focused on collecting sociodemographic characteristics of the study participants and assessing unreported adverse drug reaction, while clinical and medication-related information were collected retrospectively from the patients’ medical records. (Please see line numbers 144-146 of the revised manuscript)14. How did the interviews take place? Were they conducted in the clinic with the attending physician or prior to entering the clinic?Thank you. The study participants were interviewed before they enter into the diabetes outpatient clinic. 15. In case the interviewers detected any DRPs, how did they respond? Did they inform the prescriber or intervene to resolve these drug-related problems?Thank you. DRPs were detected mainly by reviewing their medical record. The study did not address/intervene the identified DRPs, which is included in the limitation of the study.In the Results section:16. Avoid starting a new sentence with numbers.Thank you, and your comment is considered in the revised manuscript.17. Please ensure consistency when reporting frequencies and percentages. For example, write "27 (23.1%) of the study subjects developed microvascular complications, with nephropathy accounting for the highest proportion." Remember to add a space between the frequency and the percentage.Thank you very much for the comment. Consistencies are now ensured in the revised manuscript.18. Add a footer to Table 4.Thank you, and a footer in now added to Table 4 of the revised manuscript  .Thank you. ‘Limitations of the study’ section is now revised (please see line numbers 286-291 of the revised manuscript).AttachmentResponse to Reviewers.docxSubmitted filename: Click here for additional data file. 19 Jun 2023Pharmacotherapy Problems and Associated Factors among Type 2 Adult Diabetic Patients on Follow up at Mizan-Tepi University Teaching Hospital, Southwest EthiopiaPONE-D-23-09993R1  Welday Kahssay  Dear Dr. We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.Within one week, you’ll receive an e-mail detailing the required amendments. 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For more information, please contact Kind regards,Anmar Al-Taie, Ph.D.Academic EditorPLOS ONEAdditional Editor Comments :All comments have been addressedReviewers' comments:Reviewer's Responses to Questions  Comments to the Author  1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.Reviewer #1: All comments have been addressedReviewer #2: All comments have been addressed**********  2. Is the manuscript technically sound, and do the data support the conclusions?  The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: YesReviewer #2: Yes**********  3. Has the statistical analysis been performed appropriately and rigorously?   Reviewer #1: YesReviewer #2: Yes**********  4. Have the authors made all data underlying the findings in their manuscript fully available?PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.  The Reviewer #1: YesReviewer #2: Yes**********  5. Is the manuscript presented in an intelligible fashion and written in standard English?  PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.Reviewer #1: YesReviewer #2: Yes**********  6. Review Comments to the Author  Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. Reviewer #1: The manuscript is technically sound, and the data supports the conclusions. Statistical analysis has been performed appropriately. I have no additional comment.Reviewer #2: (No Response)**********  7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.If you choose “no”, your identity will remain anonymous but your review may still be made public.Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.  Reviewer #1: NoYes: Rania ItaniReviewer #2: ********** 26 Jul 2023PONE-D-23-09993R1 Pharmacotherapy Problems and Associated Factors among Type 2 Adult Diabetic Patients on Follow up at Mizan-Tepi University Teaching Hospital, Southwest Ethiopia Dear Dr. Welday Kahssay:I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. onepress@plos.org.If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact plosone@plos.org. If we can help with anything else, please email us at Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staffon behalf ofDr. Anmar Al-Taie Academic EditorPLOS ONE


Induction of general anaesthesia is often accompanied by hypotension. Standard haemodynamic monitoring during anaesthesia relies on intermittent blood pressure and heart rate. Continuous monitoring systemic blood pressure requires invasive or advanced modalities creating a barrier for obtaining important information of the circulation. The Peripheral Perfusion Index (PPI) is obtained non-invasively and continuously by standard photoplethysmography. We hypothesized that different patterns of changes in systemic haemodynamics during induction of general anaesthesia would be reflected in the PPI. Continuous values of PPI, stroke volume (SV), cardiac output (CO), and mean arterial pressure (MAP) were evaluated in 107 patients by either minimally invasive or non-invasive means in a mixed population of surgical patients. 2 min after induction of general anaesthesia relative changes of SV, CO, and MAP was compared to the relative changes of PPI. After induction total cohort mean(± st.dev.) MAP, SV, and CO decreased to 65(± 16)%, 74(± 18)%, and 63(± 16)% of baseline values. In the 38 patients where PPI decreased MAP was 57(± 14)%, SV was 63(± 18)%, and CO was 55(± 18)% of baseline values 2 min after induction. In the 69 patients where PPI increased the corresponding values were MAP 70(± 15)%, SV 80(± 16)%, and CO 68(± 17)% . During induction of general anaesthesia changes in PPI discriminated between the degrees of reduction in blood pressure and algorithm derived cardiac stroke volume and -output. As such, the PPI has potential to be a simple and non-invasive indicator of the degree of post-induction haemodynamic changes. Haemodynamic monitoring during general anaesthesia (GA) traditionally relies on simple and readily available parameters such as blood pressure and heart rate. Most patients will only have intermittent non-invasive monitoring of blood pressure. During the induction phase an abrupt reduction in blood pressure is often seen, with possible deleterious consequences for organ perfusion , 2. The The peripheral perfusion index (PPI) is obtained non-invasively by photoplethysmography – ubiquitously present in the perioperative setting. The PPI is a simple ratio describing the proportion of pulsatile to non-pulsatile signal attenuation. No specialized equipment is needed as the PPI is inherent to photoplethysmography, although not all pulse-oximeters are by default set up to display PPI. The PPI in awake patients is right skewed and dominated by sympathetic tone , 4. ConvThe present study was designed to explore the clinical utility of the non-invasively obtained peripheral perfusion index to detect cardiovascular compromise during induction of general anaesthesia. We hypothesized that during induction of GA changes in systemic haemodynamics in the form of mean arterial pressure (MAP), cardiac stroke volume (SV) and -output (CO) would be reflected in the PPI, albeit in a complex manner. Thus, a predominant vasodilatory response might be differentiated from a response dominated by decreased CO.To examine the ability to of the PPI to describe different haemodynamic responses during induction of GA, we investigated, in a prospective mixed cohort of patients undergoing surgery under GA, associations between acute changes in PPI, MAP, SV, and CO during the first minutes of induction.Furthermore, we wanted to investigate whether baseline PPI could predict haemodynamic instability during induction, as well as offer baseline data of PPI in a mixed cohort of surgical patients.For this prospective observational study we included 107 patients scheduled for acute or elective surgery at our institution. Patients were included when one of the investigators were available and logistics permitted. If no vasopressor or inotrope is administered a mean decrease in MAP 25% after induction is likely , 7. HoweAll patients were monitored with continuous blood pressure. Either by arterial catheter (n = 49) dependent on local protocol or by continuous non-invasive blood pressure monitoring by a CNAP module . Calculations of SV and CO from raw BP data were performed by a LiDCO Unity monitor . A Masimo Radical 7 pulse oxymeter probe  was placed on the third finger of the right hand and shielded from ambient light, providing continuous recordings of PPI.After 5 min of baseline recording induction of anaesthesia was performed in the supine position using propofol and remifentanil. Elective or rapid sequence induction, airway management, and drug dosage was at the discretion of the attending anaesthesiologist. All measurements were recorded at 2 min post-induction, after which the study was considered terminated. No vasopressor or inotropic agent was administered routinely during induction but could be used if deemed necessary for patient safety before the 2 min post induction measurements, which was the case in three patients. This setup was chosen to mimic the clinical reality of intermittent non-invasive blood pressure measurements by brachial cuff for the vast majority of patients.Changes in MAP, SV, and CO stratified by changes in PPI, both by quartiles and dichotomized by PPI decreasing vs. increasing.Baseline correlations between the investigated variables. Predictive power of baseline PPI for post induction haemodynamic instability.Data from the LiDCO monitor  was exported and analyzed using LiDCOviewPRO V1 . Data from the MASIMO monitor was exported to excel spreadsheet. The sampling periods were 30 s one minute before induction and 30 s 2 min after induction. The mean values of the variables during the sampling periods were calculated. Relative changes for SV, CO, MAP, and PPI were calculated.Distribution of outcome data were tested with the Kolmogorow-Smirnov test for normality and inspected graphically with qq-plots. Normal distributed data  are reported as mean(± st.dev.) and data with non-normal distribution (PPI) as median[IQR].Correlation between variables was evaluated by the Pearson correlation test or the Spearman Rank-order correlation where appropriate. Differences of means between groups were evaluated by T-test.Analyses were performed using SPSS version 25 statistical software  and a 2-sided p-value < 0,05 was considered statistically significant.Basic characteristics of the cohort are presented in Table At baseline PPI was 1,7%, MAP was 99(± 19)mmHg, SV was 99(± 34)mL, and CO was 7,3L/min.Pre-induction PPI correlated weakly and negatively with MAP , p < 0,05. No correlation with CO or SV was found. No correlation with age was found. PPI did not differ between patients with known hypertension and those without. No significant effect of treatment with ACE-inhibitors or ACE receptor blockers was found.No correlation between baseline PPI and hemodynamic response to induction was found.2 min after induction PPI was 2,9%, MAP was 65(± 17)mmHg, SV was 74(± 36)mL, and CO was 4,6L/min. These values correspond to relative changes of + 36[-21–105]%, -35(± 16)%, -26(± 18)%, and − 37(± 19)%.To better describe the breadth of circulatory changes within the cohort we stratified the patients based on PPI response to induction, where the first quartile was the largest decrease in PPI at 2 min, whereas the fourth was the largest increase in PPI at 2 min. Doing this we found a gradual increase in cardiovascular compromise, the largest in the 1st quartile (PPI range 0–78% of baseline) with MAP 54(± 13)%, SV 59(± 16)%, and CO 49(± 16)% of baseline values, and the least in the 4th quartile (PPI range 206–720% of baseline) with MAP 75(± 16)%, SV 86(± 16)%, and CO 71(± 18)% of baseline values  MAP was 57(± 14)%, SV 63(± 18)%, and CO 55(± 18)% of baseline values. In the group with PPI stable or increasing (n = 69) MAP was 70(± 15)%, SV 80(± 16)%, and CO 68(± 17)% of baseline values. All responses differed significantly between the 2 groups, p < 0,001.During induction of general anaesthesia a range of changes to the circulation happens in a very short span of time, with possible deleterious consequences for organ perfusion : Loss ofThe peripheral perfusion index (PPI) is obtained non-invasively by photoplethysmography, ubiquitously present during GA. The PPI in awake patients is predictive of the complication rate in major surgery , is a prThe plethysmographic signal consists of a pulsatile “AC” and non-pulsatile component “DC” with PPI = AC/DC. Sympathetic tone is the major determinant of PPI in the awake person. Indeed, sympathetic block demonstrated by increasing PPI has been described as determinant of successful neuraxial  or peripClearly the PPI during the transitional period of induction of anaesthesia is subject to multiple changes from both sympatholysis and alterations of global circulatory parameters. Still, we hypothesized the possibility of.describing two different patterns of sympatholysis: One dominated by vasodilatation with preserved stroke volume and flow (≈ PPI increasing) and one with decreased cardiac stroke volume dominating (≈ PPI decreasing).The LiDCO Unity monitor is an uncalibrated device calculating SV from the arterial pulse waveform. The algorithm tracks changes in SV/CO with good reliability , 34, 35.In this cohort of mixed surgical patients, we found a baseline PPI of 1,71,0–3,8]% (median and quartiles), not unlike 1,4% previously reported in healthy volunteers [,8%  to be predictive of post-induction instability as a low PPI might suggest a circulation dependent on high sympathetic tone. Most likely our cohort consisted of “too healthy” patients to show this effect, indeed only 4 patients had a very low baseline PPI < 0,5%. This may be due to patients being included only when the investigators were present with sufficient time to setup the equipment, but also due to the fact that acutely ill patients may not be able to give informed consent for a number of reasons. Furthermore, anxiety or pain, may also lead to a low PPI, but not a higher likelihood of post-induction instability. Thus, the negative correlation found between baseline MAP and PPI reflects the effect of sympathetic tone on PPI in the awake patient [On average for the total cohort 2 min after induction PPI increased by 36%, whereas MAP, SV, and CO decreased by 35%, 26%, and 37%, respectively. This is consistent with a recently published study describing an inverse correlation between ∆MAP and ∆PPI during induction [These changes likely reflect different degrees of reduction in cardiac function due to induction of anaesthesia. The highest increase PPI quartile thus have decreased arterial vasomotor tone but largely intact stroke volume contrasting the low PPI quartile with compromised cardiac function – due to sympathetic dependent cardiac function, reduced cardiac filling by venous dilatation, or both. As noted, the major determinant of PPI in the awake patient (i.e. pre-induction/baseline) is sympathetic tone. Upon induction, all things being equal, the PPI will increase as vascular tone decreases. However, as sympathetic tone is decreased changes in SV becomes the major determinant of PPI; with a large post-induction decrease in cardiac function leading to net decrease in PPI, whereas a cardiac function only mildly affected by induction of anaesthesia leads to a net increase in PPI .A recent study using non-invasive monitoring during induction found a stable SV and decreased MAP after induction . The patTo increase the clinical usefulness of our results, we dichotomized our data into PPI decreasing vs. PPI stable or increasing Fig. . With PPWe must point out that algorithm-based SV measurements may be inaccurate during the induction period; this may hold even more true when the pulse waveform is derived from CNAP instead of arterial cannula. However, due to the high temporal resolution of this methodology it enables to obtain some insight, where more accurate but slower methods cannot be used. Thus, these results point a direction for further research into this evolving field of interest.In summary, in a mixed cohort of surgical patients undergoing general anaesthesia using minimally- or non-invasive monitoring of MAP, SV, and CO we demonstrated the expected effects of induction of anaesthesia with reduction of haemodynamic parameters. When stratifying the patients according to the effect of induction upon PPI it was possible to elicit a plausible physiological background to the observations and to discriminate between trivial and more severe degrees of cardiovascular compromise.


The origin of these modes, not observed for protons in bulk water, is attributed to protons with reduced coordination number in confinement based on Density Functional Theory calculations. This study therefore demonstrates a useful tool for the characterization of chemical species under 2D confinement.The hydration structure of protons has been studied for decades in bulk water and protonated clusters due to its importance but has remained elusive in planar confined environments. Two-dimensional (2D) transition metal carbides known as MXenes show extreme capacitance in protic electrolytes, which has attracted attention in the energy storage field. We report here that discrete vibrational modes related to protons intercalated in the 2D slits between Ti 3C2Tx MXene interlayer during electrochemical cycling have a different hydration structure than protons in bulk water.Intercalation of protons in 2D materials plays a major role for several applications in energy storage and conversion. Here, the authors show that protons intercalated in Ti The solvation structure of a proton is usually described as the Eigen cation3, where a hydronium ion is solvated by three water molecules, or the Zundel cation4, where the excess proton is equally solvated by two flanking water molecules. Recent theoretical and experimental studies are increasingly pointing to a distorted structure, or a structure in between these two limiting cases5. Due to the dynamic hydrogen-bonding (H-bonding) patterns of water leading to rapidly interconverting proton hydration structures, protons in bulk solution display a broad vibrational signature that spans from ca. 1000 cm−1 to beyond 3000 cm−16. This so-called acid continuum absorption makes it difficult to assign features to any particular structure. However, the infrared (IR) spectra of protonated water clusters present sharper bands compared to the spectra of bulk solutions, which let to breakthroughs in the understanding of proton hydration9. Recently, ultrafast two-dimensional (2D) IR spectroscopy measurements have demonstrated that protons are mostly solvated as Zundel complexes in bulk solution11. In contrast to clusters, which are limited to ca. 21 water molecules6, and bulk water, the vibrational modes of hydrated protons in 2D confinement remain largely unexplored.The hydrated excess proton is of fundamental importance in diverse systems ranging from biological processes12, exotic phases13 and high proton conduction14. Few reports detailing the spectroscopic characterization of such confined water layers are available to date and are limited to neutral water18. The hydration structure of protons under confinement requires further attention. Confined protons are indeed expected to play a major role in the pseudocapacitive electrochemical charging of 2D materials such as MXenes in acidic environment20. MXenes are a large family of 2D transition metal carbides, nitrides and carbonitrides which are most commonly prepared by the selective etching and removal of the A layer from MAX phases21. The resulting MXenes are conductive with hydrophilic surfaces terminated by -O, -OH, and other functional groups represented by Tx in the chemical formula. Over the last years, Ti3C2Tx MXene has shown remarkable electrochemical properties in acidic electrolytes, putting this material at the forefront of pseudocapacitive energy storage materials22. Nevertheless, experimental evidence of the proton intercalation mechanisms in MXene remains elusive so far. Confined water in Ti3C2Tx MXene, the most studied MXene so far, has been characterized by NMR23 and inelastic neutron scattering24; however, an effective method of directly characterizing the hydrated protons confined within the layers of MXenes has yet to be demonstrated.Water in 2D confinement presents bulk-like water in the planar dimension, while being limited to thicknesses of 1-3 layers of water, thus constituting an ideal platform to bridge the knowledge gap between clusters and bulk water. 2D confinement affects the H-bonding structure of water, leading to anomalous properties such as low dielectric constantoperando Fourier Transform Infrared (FTIR) spectroscopy to probe the water molecules and hydrated protons confined within the interlayer spacing of Ti3C2Tx during potential-induced proton intercalation. FTIR spectroscopy is ideally suited for the study of water due to the strong IR absorption of O–H bonds but has not yet been utilized to probe intercalation and solvation phenomena in MXenes. By combining FTIR spectroscopy and theoretical modelling, we have been able to show that the H-bonding structure of hydrated protons electrochemically intercalated into 2D slits between Ti3C2Tx MXene layers is fundamentally different from bulk solution.In this work, we utilized operando FTIR system is shown schematically in Fig. operando spectroscopy to emphasize the fact that infrared spectroscopic characterization of the Ti3C2Tx MXene film was acquired during the CV measurement, without stopping the potential at any fixed value. Each spectrum is averaged over a scanning range of 60 mV (see SI for details). The measurements were made in the attenuated total reflectance (ATR) mode using microstructured Si substrates as the internal reflection element (IRE). All IR-active vibrational modes of Ti3C2Tx MXene itself, except for the terminal O–H stretch, are calculated to appear below the wavenumber range accessible with this setup25. The ATR mode helps to remove much of the bulk electrolyte signal since the probing depth is less than the thickness of the Ti3C2Tx film . Successful etching of the parent MAX phase is demonstrated by the X-ray diffraction (XRD) pattern, where only the (00 l) reflections are present  and 3400 cm−1 (stretching mode). The sharp band at 3640 cm−1 is attributed to dangling O–H bonds of water molecules28. Exposing the dry Ti3C2Tx film to an acidic solution replaces the residual Li+ ions, which were present in the Ti3C2Tx interlayer spaces as a result of the MXene synthesis and delamination process, with protons, leading to a significant decrease in the water bands and the appearance of new bands at 1820 cm−1, 2040 cm−1, 2560 cm−1 and 3090 cm−1, which are attributed to confined hydrated protons , 2 (~2080 cm−1), 3 (~2380 cm−1) and 4 (~2840 cm−1) are attributed to hydrated protons and are not observed when the same measurement is performed using a neutral electrolyte  are also detected, assigned primarily to vibrations from water molecules located beyond the first excess proton hydration shell. A positive band that shifts from 3300 cm−1 to 3220 cm−1 (peak 5) is accompanied by a distinct negative band at 3530 cm−1 (peak 6). These two bands together show that neutral water is eliminated within the Ti3C2Tx interlayer spacing due to proton intercalation, and that the remaining water molecules experience stronger H-bonding. This observation, also performed on water trapped in graphene nanobubbles at high temperature31, has previously been assigned to an increase of the water density in the confined environment32. The increase of the band at 3630 cm−1 (peak 7) from dangling O–H bonds is also observed in a neutral electrolyte and therefore not specific to proton intercalation  water reordering toward stronger H-bonding due to polarization at the MXene-electrolyte interface and (ii) proton intercalation and/or surface redox reactions. Process (i) is independent of proton concentration and is related to changes in water stretching modes as evidenced by peaks 4 and 5, the areas of which are nearly identical at both acid concentrations. Also, peak 7, attributed to water molecules with a dangling OH bond28 appears at almost the same frequency and intensity in both, acidic and neutral electrolytes  is dependent on the proton concentration and gives rise to peaks 1-3 and loss of peak 6. Peaks 1-3 are associated with the diffusion of protons within the MXene interlayers, which replaces the weakly bonded neutral water (peak 5).The spectral evolution is similar in both concentrations, with some notable exceptions Fig. . Peaks 13C2 surface and how that affects the way protons are hydrated in the interlayer space. As can be observed by visual inspection of the CV curve in Fig. 3C2 electrode deviates from the ideal, where the anodic and the cathodic peak potentials would be symmetric. The Ti3C2 film was optimized for IR study and the dropcasting method produced a film with a higher resistance compared to conventional supercapacitor electrode films.Some peaks exhibit considerable hysteresis in their integrated areas, which may be related to the state of protonation of the Ti2SO4, respectively  calculations were performed to clarify the vibrational structure of the new bands in the region 1800-2400 cmits Fig. . The calSO4 Fig. . The norSO4 Fig. , confirm33. Asymmetrically hydrated Eigen cations (one water molecule removed from or one water molecule added to H9O4+) exhibit redshifts in the Eigen core frequency and present bands close to peaks 1 and 2 observed here7. Similarly, hydrated protons confined inside reverse micelles were found to exist predominantly in asymmetric Eigen-like hydration structures, with one weaker H-bond and two stronger H-bonds formed by the central hydronium ion5. Moreover, vibrational sum frequency spectroscopy measurements that are sensitive to interfacial modes show that hydrated protons at a charged liquid-vapour interface have a preferred orientation and strongly favour an Eigen-like configuration34. We do not observe a peak at 1750 cm−1, which is prominent in difference spectra of bulk acid solutions and widely attributed to the bending mode of a Zundel cation35, further supporting our DFT results and the assignment of peaks 1-3 to Eigen modes. The assignment of the experimental features based on these DFT calculations is summarized in Table The classification of proton hydration structures as Eigen or Zundel is often based on the proton-sharing parameter, δ, which describes the difference in the distance of the shared excess proton from the O atoms of the two nearest water molecules (for more details see Methods). δ = 0 Å for an ideal Zundel cation, whereas configurations with δ > 0.1 Å are classified as Eigen states. The simulated snapshots show practically no Zundel character for any normal modes contributing to peaks 1 and 2; instead, these peaks arise predominantly from Eigen modes. For peaks 1-3, both δ and peak frequency decrease in the order 3 > 2 > 1 , may be more appropriate to resolve the Ti-OH contribution. X-ray absorption spectroscopy has proved useful in revealing the change in the Ti chemical bonding environment induced by hydroxylation and redox reaction of titanium carbide MXenes38.However, we are currently unable to definitively assign a particular absorption band to the surface hydroxyl groups. DFT calculations predict the stretching mode of the surface -OH to appear at ca. 3730 cmD), and the oxygen in the hydronium accepts one H-bond from a neighbouring water molecule (labelled OA). These interactions are highlighted in the inset of Fig. 39, the proton coordination number drops from 3.5 in the bulk to 3.0 in the confined interlayer space at both concentrations. This decrease of the hydronium coordination number matches well with the main normal modes of the confined protons, which all involved three water molecules. Recent theoretical and experimental studies have found that the protonated water cluster H+(H2O)21, referred to as a magic number cluster due to its stability, exhibits IR bands in the 1800-2400 cm−1 region40. In the most stable conformer of the H+(H2O)21 cluster, the excess proton is found in an Eigen configuration with three donated H-bonds but no accepted H-bond, similar to the H-bonding structure found in our DFT calculations. The loss of the accepted H-bond is therefore the main change in the hydration of the protons which is observed when the protons are confined within a two-dimensional environment in MXene, as represented schematically in Fig. To find out the structural origin responsible for the vibrational modes of hydrated protons in confinement, we compared the H-bonding network of the hydrated proton in confinement with that of a hydrated proton in bulk water. For a fully solvated hydronium ion, the three hydrogen atoms each donate a H-bond to a water molecule . Li2SO4 and H2SO4 (Sigma-Aldrich) were used as received. All electrolytes were deoxygenated by bubbling with nitrogen for 30 min prior to experiments. A Ti3C2Tx stock suspension was diluted to 1 mg/ml, 125 µl was pipetted onto a graphene-covered Si wafer (vide infra) and the droplet was allowed to dry at room temperature. Some manual manipulation of the drying droplet was necessary to ensure that the infrared beam probed an area fully covered by MXene. The resulting film was ca. 600 nm thick. Measurements were also made with thinner films by reducing the amount of Ti3C2Tx MXene suspension pipetted onto the wafer.All electrolytes and Ti3C2Tx working electrode was made through the graphene film, although measurements were also performed without the graphene layer with very similar electrochemical results. A coiled Pt wire and an Ag wire were used as the counter and quasi-reference electrodes, respectively. Prior to use, the Ag wire was anodized at 50 mV vs. OCP in 0.1 M HCl. The potential was controlled with a Bio-Logic SP-200 potentiostat running EC-Lab software. With all electrodes mounted, the cell was filled with electrolyte, sealed, mounted into the infrared spectrometer and the spectrometer was evacuated until the pressure in the sample chamber reached 0.8 mbar . After this equilibration time, the open-circuit potential (OCP) of the system was recorded and used as the starting potential for the CV. Each CV was recorded between 0 V and −0.6 V with a scan rate of 2 mV/s. Several cycles were performed to verify the stability of the MXene electrode.Electrochemical measurements were performed in a three-electrode spectroelectrochemical cell designed and built in-house  was a microstructured Si wafer (Irubis) covered by a monolayer film of graphene (Graphenea) transferred as per instructions. A fresh graphene-covered wafer was prepared for each measurement. The graphene layer improved adhesion between the MXene film and the IRE, leading to a higher sensitivity to interlayered water layers and more reversible spectra during electrochemical cycling. The optical accessory was designed and built in-house to accommodate the microstructured Si wafer as the IRE and provides an angle of incidence of 28.74°. Each spectrum consisted of 128 scans and took ~30 s to record, with a 20-s waiting time between spectra resulting in one spectrum every 50 s. With a cyclic voltammetry scan rate of 2 mV/s, this equates to one spectrum per 0.1 V. The timing was manually synchronized with the cyclic voltammetry such that the midpoint of each spectrum collection coincided with potential = 0.1n V, where n = integer. For example, an FTIR spectrum, the collection of which started when the potential was −0.17 V and finished when the potential was −0.23 V, is designated as the spectrum at −0.2 V. A linear or cubic baseline was subtracted from spectra.The measurements were carried out at the IRIS beamline at the BESSY II electron storage ring operated by the Helmholtz-Zentrum Berlin für Materialen und Energie43. The nuclei-electron interaction was treated by projector augmented wave (PAW) potential44. The Perdew-Burke-Ernzerhof (PBE) version of generalized gradient approximation (GGA) was adopted for exchange-correlation functional45. The energy cutoff of the plane wave basis sets was set to 500 eV. The optB88-vdW functional was adopted to account for the van der Waals interaction46. Supercells containing 12 formula units of Ti3C2O2 were used to model MXene. 17 water molecules with different numbers of protons  were randomly placed in between MXene layers. The convergence criteria were set to 10−5 eV in energy and 0.01 eV/Å in force.All the density functional theory (DFT) calculations were performed by Vienna Ab initio Simulation Package (VASP)47. From each trajectory, snapshots at every 0.5 ps from 2 ps to 10 ps were then selected for vibrational analysis. For each snapshot, density functional perturbation theory (DFPT) was applied to obtain the normal modes 48To sample the configurations of intercalated water/proton, first principles molecular dynamics simulations (FPMD) were performed at 300 K for 10 ps with a timestep of 0.5 fs, in the canonical ensemble (NVT) using the Nose-thermostat−1. The final IR spectrum was then obtained by averaging over the selected snapshots. The frequency axis was scaled by a factor of 1.017 and 1.039 such that the water bending mode was centered at 1635 cm−1 and 1665 cm−1 in the bulk and confined spectra, respectively. To mimic the heterogeneity of proton concentration in experimental measurement, the simulated spectrum in Fig. 3C2O2·1.42H2O·0.08H and Ti3C2O2·1.42H2O·0.25H.To simulate the whole IR spectrum, the peak associated with each normal mode was then broadened by a Gaussian function with a width of 60 cmD―H—OA hydrogen bond are referred to as the special pair, and OD (OA) refers to the oxygen from the H-bond donor (acceptor) molecule. The classification of hydration structures is defined through the proton-sharing parameter δ:r is the distance between Oi and H. Special pair configurations where δ < 0.1 Å are classified as Zundel and δ > 0.1 Å as Eigen33. It is important to note that this cutoff value is arbitrary; other classification methods can be found in the literature49. A perfectly symmetric Zundel cation would have δ = 0 Å and a perfectly symmetric Eigen cation would have δ = 0.53 Å41.The hydrated proton is often described as either a Zundel or Eigen cation. In the Zundel cation, one excess proton is equally shared by two water molecules with a further four water molecules in the first hydration shell, whereas in the Eigen cation the excess proton is more closely associated with one water molecule, surrounded by a hydration shell of three additional water molecules. The two water molecules sharing the excess proton through an OSupplementary InformationPeer Review File