How lake temperatures across large geographic regions are responding to widespread alterations in ice phenology  remains unclear. Here, we analyse satellite data and global-scale simulations to investigate the contribution of long-term variations in the seasonality of lake ice to surface water temperature trends across the Northern Hemisphere. Our analysis suggests a widespread excess lake surface warming during the months of ice-off which is, on average, 1.4 times that calculated during the open-water season. This excess warming is influenced predominantly by an 8-day advancement in the average timing of ice break-up from 1979 to 2020. Until the permanent loss of lake ice in the future, excess lake warming may be further amplified due to projected future alterations in lake ice phenology. Excess lake warming will likely alter within-lake physical and biogeochemical processes with numerous implications for lake ecosystems. How lake temperatures are responding to widespread changes in lake ice remains unclear. Here the authors show the excess lake warming during the ice-off and ice-on month due to earlier ice loss and later ice formation across the Northern Hemisphere. When lakes freeze, ice acts as a barrier shielding the water surface from atmospheric forcing, notably the incoming radiation, and also acts to increase the lake’s albedo3. Ice phenology (the timing of ice formation and loss), in turn, regulates the seasonal surface energy and thermal regimes of many lakes worldwide5. Yet, lake ice is vulnerable to climate change7. Within a warming world, many lakes have experienced a substantial change in ice phenology, with a later onset and an earlier break-up of ice cover and, subsequently, a longer duration of the ice-free season9. A change in ice phenology can influence the seasonal and inter-annual variability of lake surface water temperature11, with knock-on effects on other physical as well as biogeochemical processes in lakes14.More than 90% of the world’s lakes are situated north of 30 °N ref. , and man17. Warmer conditions during the ice-off month can also lead to an earlier onset of thermal stratification18 and to warmer surface water temperature during the open-water season11. The extra heat absorbed by lakes during the open-water season can also contribute to the delay of ice formation in the following winter, with drastic consequences for socioeconomic and cultural ecosystem services20. Later ice-on dates can, subsequently, lead to more incoming radiation into lakes by extending the open-water season during the ice-on month, but also lead to greater heat loss due to increased evaporation rates22.Historical trends in ice phenology can influence lake warming rates during the months of ice-off and ice-on . During the ice-off month , an earlier break-up of ice cover results in an increase in the amount of incoming short-wave radiation due to reduced surface albedo and an extended period of open-water. This likewise results in an increase in net surface heating, and thus warmer water temperature, with knock-on impacts on lake ecology. For example, warmer conditions during the ice-off month can facilitate the growth of phytoplankton earlier in the year and, in turn, bring forward the onset of the spring bloom with implications for water quality23, solar radiation24 and wind speed25, the magnitude of change in lake surface temperature during the months of ice-off and ice-on remain uncertain. Some in situ observations and model simulations have suggested that earlier ice-off and/or later ice-on in some lakes can lead to excess lake surface warming at these times of the year28 .Compared to the warming rates often reported during summer, which are influenced by changes in, among other things, air temperaturer28 Fig. . Howeverr28 Fig. ; and  or ice-on (LSWTION) to the computed trend during the open-water season . With the ice-off month from March to July, the average RIOFF of lakes in the Northern Hemisphere increases from 1.2 ± 0.9 to 1.4 ± 1.3 (± standard deviation across Northern Hemisphere lakes). Our data suggest that 51% of lakes have a RIOFF > 1, 34% > 1.5, and 13% > 3  is much larger than in North America (0.7 ± 1.1) and Europe  than in Asia (0.6 ± 0.5) and North America  in 43% (29%) of lakes , implying the important role of lake ice phenology on excess lake warming at this time of year.As potential drivers of lake surface temperature, climatic variables including surface air temperatureIOFF  with a delay in ice-off over the study period . We hypothesize that lake depth could explain the large variation in the trend of LSWTIOFF across lakes that experience similar changes to the timing of ice-off shown in Fig. IOFF and lake depth  is 0.49 (p < 0.001), suggesting an important role of lake depth in mediating excess lake warming. Given that lake stratification will change the volume of water exposed to direct surface heating35, this hypothesis could be invalid if stratification occurs during the ice-off month. Using the simulated temperature difference between the epilimnion and hypolimnion of lakes , we calculated lake stratification dates across the Northern Hemisphere for 1979–2020 (see Methods section). We found that only 16% of lakes experienced stratification during the ice-off month  and lake depth (24%) compared to the three climate variables (<11%), thus suggesting a dominant contribution of lake ice phenology and lake properties on excess lake warming during the ice-off month , but the downward long-wave radiation  and surface air temperature  at this time show a higher correlation with the trend in LSWTION than lake depth  or a one-day delay of ice-on . The average SIOFF across the North Hemisphere is estimated at 0.14 °C day−1 . Our data suggest that without an advancement to the timing of ice break-up, the LSWTIOFF warming rate would be similar to that calculated during the open water season (1.4 ± 0.7 °C). The average SION in the North Hemisphere (0.06 °C day−1) is much smaller than the SIOFF  than during the ice-off month . This excess warming of LSWTION would be offset by the “dimming” of downward short-wave radiation during the ice-on month, compared to “brightening” during open-water season  with increasing lake depth  compared to the SIOFF  at least once during the study period (see Methods section). The extended stratification period thereby could contribute to excess lake warming in spring and summer.The enhancement of lake warming due to earlier ice break-up can last about three months . Water clarity can influence both lake surface and bottom water temperature, as darker lakes typically absorb more incoming radiation at the surface, which can lead to (1) an increase in surface water temperature in summer but result in faster cooling in autumn77, and (2) a decrease in summer bottom temperature due to thermal shielding79. In addition, as there is no water balance equation in FLake, lake depth and surface area are constant in time, which can be an additional source of uncertainty, particularly in some shallow lakes where lake levels change dramatically at seasonal and inter-annual timescales. Despite these limitations, we stress that this model has been used previously to successfully estimate the thermal environment of lakes globally, and our results provide an important step forward in understanding changes in lake thermal conditions within a warming world.To correspond with the lake-mean satellite observations described above, we elected to investigate simulated water temperatures from a one-dimensional (1D) lake model. Compared to the more comprehensive, but computationally expensive, 2D or 3D models, a 1D model assumes a simple bathymetry for the simulated lakes, thus introducing an additional source of uncertainty in our projections22. Changes in lake ice phenology, notably a later onset and earlier break-up of ice cover, have been suggested to occur in lakes worldwide and are expected to continue with future warming81. In this study, we calculate excess lake warming during the months of ice-off and ice-on due to an earlier loss of ice cover and later ice formation throughout the Northern Hemisphere. Meanwhile, as a result of higher incoming solar radiation during the ice-off months, the sensitivity of excess warming to the timing of ice loss is quantitatively higher than during the month of ice-on. Our results highlight the excess warming during these transitional months between the ice and open-water seasons using historical datasets. Projecting future lake warming due to changes in ice phenology is important for understanding lake thermal regimes, lake ecological processes and the provision of lake ecosystem services within a warming world.Both ice phenology and lake surface temperature are essential physical lake variables, which are particularly vulnerable to climate change85, which is coupled into the Hydrology Tiled ECMWF Scheme for Surface Exchanges over Land (HTESSL) of integrated forecast system (IFS). ERA5 lake surface temperatures have been validated against observations in previous studies86, and have been used to simulate lake thermal responses to climate change86. The lake grid cells in ERA5 are identified with a >0% lake cover fraction. For lake warming attribution, hourly surface solar radiation downwards, surface thermal radiation downward, surface 2-metre air temperature, lake bottom temperature, and static lake depth with a spatial resolution of 0.25° × 0.25° were also downloaded from the ERA5 reanalysis datasets for 1979–2020. All hourly climate and lake temperature data were aggregated into daily or monthly averages for further analyses.To calculate monthly lake warming rates across the Northern Hemisphere (1979–2020), we used hourly lake surface water temperature (notably the temperature of the upper mixed layer) simulations from European Centre for Medium Range Weather Forecasts (ECMWF) ERA5, which has a spatial resolution of 0.25° × 0.25° refs. ,82. Lakewww.laketemp.net)87 to calculate warming rates across the Northern Hemisphere lakes. ARC-Lake provides daily lake-wide mean surface temperature in 1628 lakes from June 1995 to April 2012, produced by ATSR-2 and the Advanced ATSR. Lake-mean satellite observations for 963 lakes north of 30 °N are used in this study to average across the intra-lake heterogeneity of lake thermal responses to climate change65, and to be comparable to the lake mean model used. Lake warming rates show good agreement between ERA5 and ARC-Lake  Reprocessing for Climate: Lake Surface Water Temperature and Ice Cover (ARC-Lake) dataset (http://cci.esa.int/lakes)88 was also used to validate the results from ERA5  Lakes project  or individual lake (ARC-Lake), we followed Layden et al.87 and identified ice-cover periods when lake surface temperature is <1 °C. This method has been used in previous studies66. The ice-on date was defined as the first day when ice-cover periods last for 10 consecutive days after 1 July while the ice-off date was the first day when ice-free periods last for 10 consecutive days. This 10-day threshold was used to account for periods of intermittent ice cover during the transitional periods of ice cover and open-water. We only selected lake grid cells (ERA5) or individual lakes (ARC-Lake) with at least 1-year ice phenology data for further analysis. Meanwhile, we excluded lakes with an ice-cover duration <30 days to prevent lakes from experiencing break-up and freeze-up in the same month (this included <0.2% of lakes in ARC-Lake and <0.3% of lake grid cells in ERA5). To validate the annual ice phenology data derived from daily LSWT from ERA5 (1979–2020) and ARC-Lake (1995–2012), we also used a dataset of lake ice phenology from Advanced Microwave Scanning Radiometer for EOS and Advanced Microwave Scanning Radiometer 2 (AMSR-E/2) sensors91. This dataset contains daily ice phenology time series for 76,671 lake pixels with a spatial resolution of 5 km × 5 km across the Northern Hemisphere from 2002 to 2015, derived from brightness temperature data of AMSR-E/2 sensors. We selected lake pixels with temporal coverage >85% of the period 2002–2015. Annual ice-off and ice-on dates derived from ERA5, ARC-Lake and AMSR-E/2 show a good agreement (R > 0.73) in lake grid cells across the Northern Hemisphere  to indicate excess warming in the ice-off and ice-on month across the Northern Hemisphere, respectively. To indicate the sensitivity of lake warming during the ice-off and ice-on months to changes in ice phenology, we calculated the ratio of the trend in LSWTIOFF to the trend in ice-off dates (SIOFF), and the ratio of the trend in LSWTION to the trend in ice-on dates (SION). The trends in LSWTIOFF and LSWTION are trends of monthly lake surface water temperature for the months of ice-off and ice-on from 1979 to 2020, respectively. In this way, the open-water season of each lake grid cell was defined as the months after the ice-off month and before the ice-on month. The ice-off (ice-on) month for each lake was defined as the month of mean ice-off (ice-on) date during the period 1979–2020, not varying between years. Given that when ice-off occurs at the end of a month the ice-off has a limited impact on lake surface water temperature in that month but a larger impact during the following month, we also tested another definition for ice-off and ice-on month. When the mean ice-off date is later than the 25th day of the month or the mean ice-on date is earlier than the 5th day of the month, then the following month is defined as the ice-off month and the previous month is the ice-on month. With this alternative definition, we found similar results that lake ice phenology is the dominant factor of excess lake warming in both ice-on and ice-off month (Supplementary Fig. We used the ratio of the trend in LSWT92, we used the difference between lake surface and bottom temperature from ERA5 to calculate the lake stratification during 1979–2020. The onset of stratification is defined as the first day when the temperature difference between the epilimnion and the hypolimnion is >1 °C and lasts for 3 consecutive days (Supplementary Fig. 43.We used correlation analysis to explain the impacts of climate variables, lake ice phenology and lake depth on the excess lake warming during the ice-on and ice-off month. In addition, we also consider the role of lake stratification after ice-off on lake warming. On the basis of the methods described by Woolway et al.Supplementary InformationPeer Review File


Background: Chronic spontaneous urticaria (CSU) is defined as the recurrence of unprovoked transient wheals and itch for more than 6 weeks. Currently, there is an unmet need concerning response prediction in CSU. The present study investigated biomarkers of type I and type IIb autoimmunity as potential predictors of response to omalizumab in CSU. Materials and methods: Differences in levels of IgG and IgE autoantibodies targeting the high- and low-affinity IgE receptors , as well as spontaneous and specifically triggered leukotriene C (LTC)4 release by basophils from the investigated subjects, were evaluated in 18 consecutive, prospectively enrolled CSU patients and 18 age- and sex-matched, healthy non-atopic controls. Results: The patients with CSU had higher levels of anti-FcεRI IgE (542 (386.25–776.5) vs. 375 (355–418), optical density (OD), p = 0.008), and IgG (297 (214.5–431.25) vs. 193.5 (118–275) OD, p = 0.004) autoantibodies relative to the controls. Simultaneous anti-FcεRI IgG and IgE positivity  was recorded only in late- and non-responders . Discussion: Significantly higher anti-FcεRI IgE autoantibody levels were found in the CSU patients as compared to the controls, supporting FcεRI as an autoallergic target of IgE  in the complex pathophysiological scenario of CSU. The co-occurrence of anti-FcεRI IgG and IgE autoantibodies was documented only in late- and non-responders, but not in early ones, crediting the co-existence of autoimmune and autoallergic mechanisms as a driver of late/poor response to omalizumab. Chronic spontaneous urticaria (CSU) is defined as the recurrence of unprovoked transient wheals and itch for more than 6 weeks . CSU treIn addition to IgG autoantibodies directed against the high-affinity IgE receptor (FcεRI), whose involvement in CSU pathogenesis is well-recognized, IgE directed against a variety of autoallergic targets  ,5 have bThe present study investigated biomarkers of type I and type IIb autoimmunity  as potenPatients with severe CSU, i.e., Urticaria Activity Score (UAS)7 > 25, underwent subcutaneous omalizumab treatment at a dose of 300 mg every 4 weeks, following the indications of the European Academy of Allergy and Clinical Immunology (EAACI) guidelines . The patAnti-FcεRI, anti-FcεRII IgE, and IgG autoantibodies were measured by a sandwich enzyme-linked immunosorbent assay at baseline, as described in detail elsewhere . For cutrho. p-values below 0.05, two-sided, were considered statistically significant .The results are expressed as the medians and interquartile ranges (IQR). The differences in terms of autoantibody levels and LTC4 release were evaluated by means of the Mann–Whitney U test between the CSU patients and the controls, and among the patients according to their response to the omalizumab. The correlations between the autoantibody levels and the LTC4 release were assessed by means of Spearman’s The demographics, clinical, and laboratory features of both groups are summarized in p = 0.008), and IgG (297 (214.5–431.25) vs. 193.5 (118–275) OD, p = 0.004) autoantibodies relative to the controls (The patients with CSU had higher levels of anti-FcεRI IgE (542 (386.25–776.5) vs. 375 (355–418), optical density (OD), controls a,b.p = 0.029) (p = 0.183), albeit without reaching statistical significance.Interestingly, the basophils from the CSU patients released significantly more LTC4 upon stimulation with anti-FcεRII antibodies than the basophils of the controls (2130.15 (535.325–3383.425) vs. 543.6 (304.5–854.5) pg/mL, = 0.029) c. The bap = 0.010) (p = 0.06).Among the CSU patients, a statistically significant inverse correlation was documented between anti-FcεRII IgE autoantibody levels and LTC4 released from the patients’ basophils after in vitro stimulation with anti-FcεRII antibodies  d. MoreovSimultaneous anti-FcεRI IgG and IgE positivity  was documented in the late- and non-responders , but not in the early responders to omalizumab.Herein, we found significantly higher anti-FcεRI IgE autoantibody levels in the CSU patients as compared to the controls. This seems to confirm FcεRI as an autoallergen in the complex pathophysiological scenario of CSU , with IgOur data on the inverse correlations between LTC4 release in vitro from patients’ basophils and autoantibody levels may support the concept that autoantibodies may desensitize basophils to further activation. Future studies on the release of histamine, calcium, and β-hexosaminidase from basophils may be of help to confirm this view. In line with what we have observed, MacGlashan et al. demonstrated that the presence of autoantibodies to IgE or FcεRI is highly correlated with the non-responder phenotype of the basophil in CSU . Althougmilieu of co-existing type I and type IIb autoimmunity in the pathophysiology of CSU, underscore the challenges in finding reliable response predictors to omalizumab.In conclusion, our findings, while confirming a


HTT gene, for which no disease modifying therapies are currently available. Much of the recent research has focused on developing therapies to directly lower HTT expression, and while promising, these therapies have presented several challenges regarding administration and efficacy. Another promising therapeutic approach is the modulation of HTT post-translational modifications (PTMs) that are dysregulated in disease and have shown to play a key role in HTT toxicity. Among all PTMs, modulation of HTT phosphorylation has been proposed as an attractive therapeutic option due to the possibility of orally administering specific kinase effectors. One of the kinases described to participate in HTT phosphorylation is Protein Kinase CK2. CK2 has recently emerged as a target for the treatment of several neurological and psychiatric disorders, although its role in HD remains controversial. While pharmacological studies in vitro inhibiting CK2 resulted in reduced HTT phosphorylation and increased toxicity, genetic approaches in mouse models of HD have provided beneficial effects. In this review we discuss potential therapeutic approaches related to the manipulation of HTT-PTMs with special emphasis on the role of CK2 as a therapeutic target in HD.Huntington’s Disease (HD) is a devastating neurodegenerative disorder caused by a CAG trinucleotide repeat expansion in the  HTT), resulting in a mutated poly-glutamine (polyQ) expanded HTT protein (mtHTT) [Huntington’s disease (HD) is an inherited autosomal dominant neurodegenerative disease that affects approximately 1 in 10,000 individuals worldwide. HD is caused by a CAG trinucleotide repeat expansion in exon 1 of the huntingtin gene ( (mtHTT) . In addi (mtHTT) . Anxiety (mtHTT) . Unfortu (mtHTT) .Despite decades of research since the causal mutation of HD was identified by the Huntington’s disease Collaborative research group in 1993 ,6. AlterRecent reports have demonstrated that mtHTT levels and toxicity can be regulated by both posttranscriptional and posttranslational events. mtHTT-RNA undergoes an aberrant and incomplete splicing that generates HTT-exon1 transcripts and contributes to mtHTT toxicity ,33. ReceCurrently, no disease modifying therapies are available for this devastating disease that results in death most commonly between 15 to 20 years after diagnosis . The incOne of the most promising therapies against HD has been the development of antisense oligonucleotides (ASOs) against HTT. From a drug-discovery perspective, one major challenge regarding targeting HTT with small-molecule therapeutics has been due to the large size of HTT (>300 KDa) and that it lacks a characterized enzymatic function . ASOs caGiven that the current strategies for lowering HTT, while promising, have not yet achieved the desired success in clinical trials, a need for alternative therapies is imminent. With increasing evidence for the role of HTT PTMs in regulating mtHTT toxicity, abundance, and localization, therapies targeting the molecules that modify them have promise in becoming more successful therapeutic strategies.HTT PTMs have been shown to affect mtHTT toxicity and HD progression. PTMs do occur in wild-type HTT, but can become dysregulated in disease affecting aggregation, protein localization, mitochondrial function, protein interactions, axonal transport and other cellular functions that contribute to the toxicity of mtHTT and disease progression ,74,75,76HTT phosphorylation is one of the most heavily studied PTM in HD, and thus there is an abundance of literature and information on identified phosphorylations. A summary of all identified HTT phosphorylations and their effects on HTT-mediated toxicity can be found in Mass spectrometry analyses in cell models transfected with HTT exon 1 fragments have shown that T3 phosphorylation (pT3) is one of the most prevalent phosphorylations present in exon 1 . Interesbona fide phosphorylations by introducing site-specific phosphorylations on the N17 HTT region. In this context, phosphorylation at S13 or S16, but not pseudo phosphorylation, results in a significant retardation of mtHTT exon 1 aggregation [bona fide phosphorylations have revealed beneficial effects of site-specific induced phosphorylation including decreased mtHTT aggregation in response to T3, S13, and S16 phosphorylation [Phosphorylations on S13/S16 have also been shown to improve HD-like phenotypes in mouse models that have a HTT-S13/16 phosphomimetic mutation . Howeverregation . Additiorylation ,98,110.Phosphorylations outside of the N17 region also influence HTT abundance, aggregation, and toxicity properties ,105,107.All this evidence demonstrates that increasing the phosphorylation of HTT could be a potential venue to ameliorate mtHTT toxicity and improve HD-like phenotypes. This therapeutic potential could be achieved by strategies targeting phosphatases to prevent HTT hypophosphorylation or by targeting key kinases to modulate HTT phosphorylation. Since knowledge on HTT-specific phosphatases is very limited ,112, kinHTT is subjected to several other PTMs besides phosphorylation and a brief summary of other PTMs is described below:Acetylations of multiple lysine residues in the HTT protein have been identified, including lysines 6, 9, and 15 in the N17 region  and lysiPalmitoylation is the covalent attachment of fatty acids, such as palmitic acid, to Cys and less efficiently to Ser and Thr, catalyzed by a family of palmitoyl acyltransferases (PATs). HTT is palmitoylated at Cys214 by the huntingtin-interacting protein 14 (HIP14) and 14-like (HIP14L) acyltransferases ,67,114. SUMOylation consists of the addition of small ubiquitin-like modifier (SUMO) polypeptides to internal Lys residues of target proteins. HTT SUMOylation has been reported to by dysregulated in HD  and seemMethylation consists of the addition of methyl groups to the nitrogen-containing sidechains of Arg and Lys and it is mediated by methyltransferases. Arginine methylations are mediated via arginine methyltransferase (PRTMs) 4 and 6 (PRTM4 and PRTM6) which have been identified as mediators of identified methylations of HTT ,70. MethUnderstanding the specific kinases that regulate HTT phosphorylation could result in the identification of effective therapeutic targets to treat HD. Kinases that have been implicated in HTT phosphorylation include, but are not limited to: Tank-binding kinase 1 (TBK1), Inhibitor of nuclear factor kappa-B kinase subunit beta (IKKβ), Cyclin-dependent kinase 5 (CDK5), Nemo-like kinase (NLK), and Protein kinase 2  ,105,107.Hdh cells resulted in more phospho-N17 HTT and enhanced HTT nuclear accumulation and aggregation but did not affect cellular toxicity [IKKβ belongs to the IKK family of kinases, and it has been implicated in mediating HTT phosphorylation at S13, S16, and T3 ,96,100. toxicity . FurtherC. elegans models of HD [TBK1 also belongs to the IKK-kinase family and was identified in a kinase-based screening assay for its role in phosphorylating HTT, specifically at S13 . TBK1-mels of HD . When IKls of HD . NeverthCdk5 displayed improved cognitive function and motor behavior [Cdk5 haploinsufficiency in HTT phosphorylation, the study suggests that other CDK5 targets, such as Src kinase, which impacts the localization of the NMDA receptor subunit NR2B, may play a more important role in disease progression than HTT phosphorylation.CDK5 is a proline-directed serine/threonine kinase that belongs to the family of cyclin-dependent kinases. CDK5-mediates phosphorylation of HTT at S434, S1181, and S1201 which prevent aggregation and toxicity of mtHTT ,107. CDKbehavior , which ibehavior ,107. WhiHdh Q111 cells which is thought to contribute to mtHTT toxicity [NLK is a proline-directed serine/threonine MAP kinase . A largetoxicity . Overexptoxicity . Given ttoxicity . While tHdh Q111 cells, and in striatal tissues from patients with HD [CK2 is a serine/threonine protein kinase extensively studied in the context of cancer and inflammation ,133 and  with HD ,134,135. with HD . More re with HD .S13LKS16FQQQ) lacks a CK2 consensus sequence (SxxE/D) [Hdh-Q111 cells using siRNA against different CK2 subunits showed that silencing the catalytic subunit CK2α’, but not CK2α or CK2β, improved cellular toxicity [(+/−)) provided ample benefits in ameliorating several HD-like phenotypes suggesting that CK2α’ plays a detrimental role in HD [It is important to mention that the studies where the protective role of CK2 in HD was investigated were conducted almost exclusively using treatment by benzimidazole derivatives (DRB and DMAT), which not only inhibit CK2 but also potently inhibit several additional kinases, RNA Pol II transcription, and DNA replication ,138,139.(SxxE/D) ,141. Alt(SxxE/D) , genetic(SxxE/D) ,133. Rectoxicity . Similarle in HD ,135. Unfle in HD ,142, itsAs previously discussed, CK2 holoenzyme is composed of two different catalytic subunits, CK2α and CK2α’ ,133. In Previous studies reported that mtHTT impairs the ability of cells to activate the Heat Shock Response (HSR), a molecular pathway governed by Heat Shock Factor 1 (HSF1) that is essential to induce molecular chaperones  that facilitate protein homeostasis and prevent protein aggregation . Severa(+/−)) decreased S303 and S307 phosphorylation, increased HSF1 protein levels and HSPs expression, and subsequently decreased mtHTT aggregation [Upregulation of CK2α’ in HD is involved in the pathological degradation of HSF1 . CK2α’ dregation . CK2α’ hregation ,135, thuHdh-Q111 cells treated with siCK2α’ and in zQ175 mice with CK2α’ haploinsufficiency [(+/−) mice showed decreased levels of proinflammatory cytokines compared to zQ175 mice including TNF-α, IL-27, IL-7 and others, as well as decreased astrocyte pathology in the striatum [CK2 regulates signaling pathways involved in inflammation ,161,162.ficiency . Knock-dstriatum . This stSlc30a3  selective for glutamate synapses), Grm2 (Glutamate Metabotropic Receptor 2), Slc17a7 , and Nrp2 (Neuropilin 2). Restored expression of these genes in zQ175:CK2α’(+/−) mice also correlated with an improved frequency of striatal mEPSCs (miniature excitatory postsynaptic currents) observed by reducing CK2α’ levels [Excitotoxic damage from dysregulation of glutamatergic signaling is hypothesized to contribute to HD pathogenesis . RNA-seq’ levels . Since d(+/−) mice are warranted to shed light on whether this particular subunit is involved in NMDAR regulation.In addition to the transcriptional regulation of glutamate-signaling related genes, CK2 also controls the function and localization of other genes involved in glutamatergic signaling such as NMDARs. CK2 directly phosphorylates S1480 of the NR2B subunit and such phosphorylation limits surface NMDAR expression by disrupting interactions between the receptor and the postsynaptic scaffolding protein PSD-95 . In addiAlongside the toxicity mediated by mtHTT protein and its aggregates is its pathogenic interactions with other aggregation-prone proteins such as alpha-Synuclein (αSyn) ,174,175,(+/−) mice, pS129-αSyn levels are decreased compared to zQ175 mice, and this is associated with the restoration of glutamate-related synaptic genes dysregulated in zQ175 mice, as well as improved motor behavior and synaptic function [CK2α’ promotes αSyn phosphorylation at S129 (pS129-αSyn) in HD . This PTfunction . All thiCK2 has been implicated in a range of neurodegenerative disorders including Alzheimer’s disease (AD), Parkinson’s disease (PD), Amyotrophic lateral sclerosis (ALS) and Spinocerebellar ataxia type 3 (SCA3) and has emerged as a potential therapeutic target across multiple NDs  Figure . Since tIncreased levels of CK2 have been detected in affected brain regions in patients with AD  and was A kinase screening in a N2a cell model of tauopathy looking at potential kinases involved in tau aggregation revealed CK2 as one of the kinases with the most profound effects in modulating tau aggregation . The rolContrary to what was proposed in HD, CK2 contributes to NR2B phosphorylation and to a detrimental imbalance in NR2B synaptic/extrasynaptic ratio in AD, which is also associated with tau accumulation . In addiAs discussed above, phosphorylation of αSyn at S129 (pS129-αSyn) is a hallmark of PD and other synucleinopathies ,180. SevCK2, specifically CK2α, has also been associated with L-DOPA-induced dyskinesia (LID) in PD. L-DOPA is one of the most effective pharmacological treatments in PD but its long-term use generates a loss in efficacy and LID . ConditiCK2 phosphorylates Trans-activation Response DNA-binding Protein-43 (TDP-43), a protein associated with ALS and other NDs ,203. In Ataxin-3, the protein responsible for spinocerebellar ataxia 3 (SCA3), has multiple phosphorylation sites and specific phosphorylation patterns can decrease or increase its aggregation. Ataxin-3 phosphorylation mediated by CK2 increases protein accumulation in the nucleus, and inhibition of CK2 by DMAT or TBB in SCA3 cell models results in lower nuclear inclusions ,206. ThiBased on the evidence provided above, it is clear that upregulation of CK2 and the mediated effects could represent a convergent mechanism of neurodegeneration across multiple NDs and therefore targeting CK2 has therapeutic potential for the treatment of several NDs. It also highlights that different CK2 catalytic subunits may play different roles in the pathogenesis of those NDs and therefore having the ability to selectively inhibit CK2α and CK2α’ is critical for the development of effective therapeutic strategies to target CK2 in neurodegeneration, and more specifically in HD.50 1 nM) to be safely administered in vivo and currently is in human clinical trials for various forms of cancer [In recent years, more potent and specific CK2 inhibitors to those derived from carboxyl acids, and discussed above, have been developed. f cancer ,213,214.f cancer . Althougf cancer ,217 theyf cancer ,147,210.f cancer . AlthougCK2 inhibition holds promise as a potential therapy, not only for HD, but for other neurodegenerative disorders. However, there are still gaps in knowledge and conflicting reports that need resolution to better inform the effectiveness of CK2 inhibition in HD. These include: (1) use of more genetic approaches to exam the effects of selectively targeting CK2α or CK2α’ in vivo, (2) evaluation of the impact of CK2 depletion in several HD mouse models, and (3) development and optimization of selective CK2α’ and CK2α inhibitors to test their efficacy in modulating mtHTT toxicity. As discussed in this review, there is a large amount of evidence indicating that CK2α’ mediates detrimental effects in HD at the level of protein homeostasis, inflammation, synaptic gene expression, synucleinopathy and motor behavior. Although pharmacological inhibition of CK2 has provided detrimental effects in HTT phosphorylation, the current research has not resolved whether there is a differential contribution of the different CK2 catalytic subunits to this effect. We therefore suggest CK2α’ specific inhibition as a potential therapeutic strategy that could provide long-lasting beneficial effects in ameliorating several HD-related phenotypes.


Myriad hypotheses have been suggested, but few experiments have helped narrow the field of possible explanations. In addition, the complex visual features of real zebra pelage and the natural range of stripe widths have been largely left out of experimental designs. In paired-choice field experiments in a Kenyan savannah, we found that hungry Stomoxys flies released in an enclosure strongly preferred to land on uniform tan impala pelts over striped zebra pelts but exhibited no preference between the pelts of the zebra species with the widest stripes and the narrowest stripes. Our findings confirm that zebra stripes repel biting flies under naturalistic conditions and do so at close range  but indicate that interspecific variation in stripe width is associated with selection pressures other than biting flies.The best-supported hypothesis for why zebras have stripes is that stripes repel biting flies. While this effect is well-established, the  Equus quagga, males have stripes closer to true black than females). Stripes are clearly not aposematic warning signals, nor do they provide camouflage in either the woodland or savannah habitats common across zebra ranges2. So, striping presents an ideal evolutionary puzzle: a trait so refined it seems it must be “for” something, but one that confers no clear advantage upon its bearers and imposes apparent costs (conspicuousness) that cannot be explained in Zahavian terms.The evolutionary origins of zebra stripes have been investigated—and debated—for centuries. The trait is rare, conspicuous, and intensely expressed, and thus appears to beg an adaptationist explanation. However, the utility of a complete coat of densely packed, starkly contrasting black-and-white stripes is not immediately apparent. Unlike many conspicuous visual traits, striped pelage is expressed with comparable intensity in both sexes and is thus unlikely to have arisen through sexual selection alone . The hypotheses suggest various ways in which stripes may provide a social function , a temperature-regulation benefit6, an anti-predator effect8, or an anti-parasite effect10. There is continued debate over both the merits of individual hypotheses and the likelihood of stripes having arisen via a single driver vs. a confluence or alternation of multiple selective pressures11.Scientists have proposed and investigated several possible explanations for the evolution of zebra stripes (reviewed in12). Zebras, like most ungulates, are harassed by tabanid, glossinid and Stomoxys species of biting flies, which can inflict significant blood loss, transmit disease, and weaken hosts when fly-avoidance behaviors reduce the host’s feeding rate14. Yet zebras are attacked far less than sympatric ungulates across their African range16, and also less than other equids17. Zebras also produce odors that may augment their anti-fly defenses18, but so do other sympatric ungulate species19, and a host of observations and experiments have demonstrated that black-and-white stripes alone are unattractive, or actively repellent to tabanid, glossinid, and Stomoxys flies23.The present study addresses the hypothesis that has thus far received the most empirical support: the anti-parasite hypothesis  at which they would likely operate:afar: stripes might make it harder for flies to locate and distinguish zebras from background vegetation, perhaps by breaking up their outline9 or varying the way they polarize or reflect light31 especially from distances at which composite eyes support only low-resolution vision and cannot resolve zebra stripes as clear bands of alternating color on a single host .From close range (estimates range from 0.5 to 4.0 m26): stripes might interfere with orientation or landing behavior via any of several disruptive or ‘dazzle’-related visual effects27. For example, stripes might affect ‘optic flow’, or the fly’s perceived relative motion to its target as it approaches, by creating an illusion of false direction or speed of motion . Alternatively, relative motion to a striped pattern within the visual field may create the perception of self-rotation, inducing the fly’s involuntary ‘optomotor response’ and resulting in an avoidance turn in an effort to stay on a straight course29.At transition between long- and short-distance orientation. If zebras appear as blurred gray from a distance and then, at closer range, suddenly resolve into a sequence of floating black and white bars, this abrupt ‘visual transformation’26 might disrupt the behavioral sequence that facilitates landing.Finally, stripes might cause confusion in the Though the effect of stripes on flies is well-established, the source of the effect remains unexplained. Since Waage’s foundational studies in the 1970s and 1980s6. Importantly, few experiments have controlled the distance from which flies are first able to view potential landing sites (but see23). While growing evidence supports a mechanism operating at close range26, failing to restrict the starting distance of the fly means that the full set of possible mechanisms outlined above all remain plausible contributors to most previous results.Within these categories, hypotheses have proliferated faster than experimental tests of many of the proposed mechanisms. The very active literature on this question has grown in somewhat haphazard fashion, as curious researchers test new possibilities without eliminating old ones17, cloth (Experiment 2 in22), horse blankets or sheets26, and paint on live animals30. These have all clearly demonstrated a broadly replicable visual effect: stripes, and some other juxtapositions of black and white , repel flies. However, insofar as specific features of zebra pelage factor into proposed mechanisms of fly repellence—the reflective properties of “smooth, shiny” coats31; the orientation of the stripes32; the light-polarizing effects of black and white hair vs. background vegetation25; and the complex structure of hair25—there is a need for more experiments that present natural targets to wild flies (but see33). Similarly, most experiments have compared landing preferences between black-and-white striped, solid black, solid white, and occasionally solid grey substrates, which have served as important controls for determining that light polarization, rather than a combination of polarization and brightness, is sufficient to induce the effect of stripe avoidance17. However, it is now time to refocus on the original question by presenting flies with more realistic choices. Since biting flies seeking a bloodmeal on the African savannah seldom encounter solid black hosts, and even more rarely solid white hosts, landing choices should be compared between zebra stripes and common coat colors of sympatric mammals, namely various shades of brown. Further, tabanid, glossinid, and Stomoxys flies all avoid landing on stripes that are the same width or narrower than the widest zebra stripes 23, and there is some evidence that narrower stripes are even more repellent to tabanids17. This pattern is potentially significant in the application of the anti-parasite hypothesis to an adaptive explanation for the striking variation in stripe width across zebra species and between the different areas of the body on individual zebras22, but must first be confirmed with experiments using real zebra pelage.Additionally, while many studies have, appropriately, used artificial stimuli to isolate basic effects of color, pattern, brightness, and light polarization of  test surfaces, possible contributions of several aspects of natural zebra pelage remain untested. Controlled experiments have used various landing substrates, including striped and solid oil tray traps, sticky plastic, smooth plasticAepyceros melampus), mimicking the actual choices made by flies cruising the African savannah. Using pelts over live animals allowed us to remove potentially interacting effects of fly-deterrent behavior  that are less relevant to specific questions about the evolution of zebra stripes. We further isolated visual properties of the pelts by salt-curing the hides, a well-established gentle preservation method that (a) minimally impacts the specific luminance, reflectance, and pigment saturation, as well as the depth, density, and structure of animal hair, while (b) minimizing the persistence of volatile olfactory compounds that might be independently aversive to flies34. To further minimize the potential confound of odor, we chose impala skin as the alternative landing substrate to zebra pelts; like zebras, impala are consistently unattractive hosts for biting flies36. Finally, we tested pelts from the zebra species with the widest stripes overall  and the narrowest stripes  under the same conditions, to investigate whether cross-species variation in stripe width could represent differences in their abilities to repel flies.Here, we present a simple experiment designed to address each of these gaps in the literature on the anti-fly benefits of zebra stripes. In this field experiment, the landing choices of flies were tested entirely within the range at which all estimates agree flies should be able to perceive the presented stripes (< 1.0 m). This restriction enabled us to determine whether hypothesized mechanisms that act at close range are sufficient to produce the fly-repulsion effect, potentially revealing other mechanisms to be unnecessary . Our paired-choice experiments used real animal pelts, both striped (zebra spp.) and solid tan . However, there was no difference in preference between the two zebra species .The flies had a strong preference for impala skin over zebra skins of either species . This effect was remarkably similar between the two zebra species (187 landings on black PZ stripes and 177 on black GZ stripes vs. 59 landings on white PZ stripes and 60 on white GZ stripes).Across subtrials , most flies landed in the approach zones on either side of the arena , followed by the ‘indifferent’ zone near the center . The fly count on impala skin was highest after that (N = 1155) while landings on zebra skins occurred at roughly one quarter of this frequency . In each subtrial, very few flies tended to choose to land on zebra skins, but relatively few flies landed near the center (‘indifferent’ zone) as well, suggesting that there was motivation to move towards one of the ends of the arena , and all interactions occurred within that range. The strong preference flies showed for impala pelt over zebra pelts suggests that a mechanism operating within the distance at which flies perceive stripes is sufficient to deter them from landing. Visual effects occurring at long distances —or sudden ‘visual transformations’ as flies approach and stripes come into view—may still occur. They are simply not necessary to repel flies from landing. Some prior studies have observed apparent close-range effects, but did not constrain the starting distance of approaching fliesOur results also begin to address an imbalance in the literature, between hypotheses that invoke fine-grained details of natural zebra-fly interactions and experiments that test simplified, artificial visual targets. While artificial targets might amplify the visual effects of coloration by isolating and intensifying the visual signal, our results demonstrate a strong effect using targets that retain the structural complexity of natural pelage, while still controlling for other features of live-animal interactions in the wild—e.g., odor and anti-fly behaviors—that can also confound tests of a visual phenomenon. While it is possible for some residual differences in odor to persist after salt-curing the hides, our finding that flies landed equally frequently in the approach zones close to zebra and impala skins indicates that a visual effect acting at very close range is more likely than odor to regulate landing preferences on the skins. The jagged boundaries between black and white stripes on natural pelage, their curved shape, the intricate patterns on some areas of natural pelts, and the light-scattering effects of hair, all distinguish real zebra pelts from the artificial stimuli tested in most experiments to date. Any or all of these natural features might have altered the visual effects of stripes that have been previously demonstrated in experiments with clear, straight-banded artificial stimuli. Instead, our results match those of past experimental work, thereby lending biological validity to the full body of experimental evidence for the fly-repellent effects of zebra stripes.17). Our comparison of striped and brown targets is more relevant to the actual choices flies must make in the wild. Solid white animals are very rare on the African savannah, but tan, brown, and dun-colored coats are common. We confirm that, given a choice between natural brown pelts and zebra pelts, biting flies show a strong preference to land on the former. When they do land on striped substrates, flies have been found to prefer to land on black stripes over white stripes17, and we confirm that this is also true for natural zebra skins and for the full range of stripe widths seen across zebra species.Many experiments using artificial targets have tested fly landing behavior on striped surfaces compared to solid black or solid white surfaces. The resulting effect sizes have varied widely, and some have even shown a lower preference for solid white surfaces than for striped ones , illustrating the wide variation in zebra striping across environments. If, as our results indicate, stripes that span the range of interspecific stripe width variation repel flies to the same extent, adaptive explanations for stripe width variation would have to pertain to some other selective pressure. The native range of the Grevy’s zebra in the Horn of Africa is more arid than that of the plains zebra38, and the Grevy’s zebra forms loose bonds in unstable groups while plains zebras form stable family groups40. This may implicate some of the other hypotheses for the evolution of zebra stripes  to explain how this trait may have been refined within the zebra clade, as opposed to how it initially arose. It is also possible that the costs of starkly contrasting stripes  weigh differently on the different zebra species in their native habitats and that stripe width variation reflects the mitigation of these costs to different degrees.Why the Grevy’s zebra evolved the narrowest stripes of all zebras, and the plains zebra the widest stripes, remains an open question. The third extant zebra species—the mountain zebra, 41. This has been suggested to align with the anti-parasite function of zebra stripes, as flies will often take flight from vegetation and cruise close to the ground, encountering the legs or face of a grazing zebra before other parts of the body43. But if narrower stripes are not better at deflecting biting flies, why are stripes narrowest in these regions of the body? There may be an adaptive reason for this, or it may simply be a matter of ontogeny, such as an artifact of differences in the spacing of differentiated melanocytes between more proximal parts of the body and the extremities46.Our finding that stripe width did not alter fly repulsion also addresses proposed explanations for the variation in stripe width between different parts of the zebra body. In all zebra species, stripes are the narrowest on the face and legsOur experiments were intended to facilitate future investigations into the origins of zebra striping in two ways: by rendering some categories of mechanistic hypotheses unnecessary and by reintroducing the natural structural complexity of real animal pelts into a body of evidence that has largely focused on visually simplified, artificial stimuli and conditions. We believe our results strongly implicate a visual mechanism that operates at distances within the range at which dipteran eyes can resolve stripes, and one in which all stripes within the natural range of stripe width expressed by zebras have an equally strong effect on fly landing preferences. However, we remain in the dark as to precisely what visual, perceptual or cognitive mechanism drives the avoidance of striped pelage as flies make landing choices. Clearly, there is still a need for creative investigations of stripes from a fly’s-eye view to uncover what makes them visually aversive or challenging for flies to approach. Furthermore, if variation in the width of stripes on natural pelage does not compromise their anti-fly properties, it seems that researchers will have to look to other sources of selection pressure for adaptive explanations for cross-species variation in zebra stripes.2 piece from the side of the rump from one plains zebra and one Grevy’s zebra (SI1), and one section from the flank of an impala , all found dead in the field. Each of these squares was scraped free of any attached muscle and fat, cured with salt, and sun-dried at the research center.Sections of zebra and impala skin were extracted from carcasses found at Mpala Research Centre (MRC), Laikipia District, Kenya. We took a ~ 400 cm47 to collect local biting flies next to a camel corral close to the research center, baited with a bucket of a mixture of fresh camel urine and milk at the center of the trap. Traps were set for 6–24 h until a sufficient number of flies was collected for an experiment, and flies were harvested from traps 12 h before the morning experiments. To ensure that all flies were hungry, they were kept in fly boxes outside overnight . Researchers from the International Centre for Insect Physiology and Ecology (ICIPE) were on site to verify that the flies were Stomoxys species.We set Nzi trapsThe experimental arena comprised a four-sided rectangular Plexiglas box (120 cm × 30 cm × 30 cm) with a removable square piece of wood wedged in at each end to serve as the background for an animal skin , the position of each was noted as being within one of six zones in the arena  and in the afternoons (15:00–16:30), as these are the times of day when 0–10:00 avs. a plains zebra skin, a plains zebra skin vs. an impala skin, and a Grevy’s zebra skin vs. an impala skin . Landing counts on each skin in each subtrial were combined into averages per trial to avoid pseudoreplication due to the non-independence of a fly’s position between subtrials. These averages were compared between skins for every choice combination using paired Wilcoxon Rank Sum tests . Note that in a Wilcoxon Rank Sum test, a W = 0 (or V = 0 in the case of a paired test) result indicates that all observations from one group rank above all observations of the other group (a perfect difference in ranks). The skins were alternated between trials such that each skin was tested at both ends of the arena for each choice combination to enable the statistical control for a bias for which side of the arena was chosen by the flies  (The paired choice combinations tested comprised a Grevy’s zebra skin ing fly) .Supplementary Information.


Background: Pedicle screw fixation has become the workhorse for the stabilization of the thoracolumbar spine. Since accurate pedicle screw placement is necessary for a successful surgery, three-dimensional navigation has become a mainstay for placing pedicle screws. However, the published studies have an overrepresentation of lumbar screws despite the prevalence of thoracic fractures. Furthermore, no robotic-assisted pedicle screw study has focused solely on traumatic fractures. The goal of this study was to address whether (1) robot-assisted pedicle screw placement had comparable accuracy in the thoracic and thoracolumbar region and (2) robot-assisted spine surgery was feasible in an acute, traumatic setting.Methods: We performed 14 consecutive, thoracolumbar spinal stabilization procedures in which 126 pedicle screws were placed using the Globus ExcelsiusGPS® spine robot in an acute, traumatic setting. Operative times were measured, and the accuracy of pedicle screws was assessed with the Gertzbein and Robbins classification system by two board-certified neuroradiologists.Results: A total of 60-thoracic (T3-T11), the 24-thoracolumbar junction (T12-L1), 40-lumbar (L2-L5), and two-sacral pedicle screws were placed. Pedicle screw placement was accurate with a < 1% (1/126) pedicle breach rate. Thoracolumbar robotic spine surgery in an acute, traumatic setting was demonstrated to have a good safety profile with only one minor neurological deficit which was related to positioning. Furthermore, surgical times were inversely related to the case number.Conclusions: These results together suggest that robot-assisted spine surgery is accurate in the thoracic spine. Furthermore, placement of thoracolumbar screws in an acute trauma is non-inferior to other methods when based on accuracy. Over 160,000 spine traumas occur yearly in North America with over 90% of those being localized to the thoracolumbar spine . FactorsThoracic spine pedicle fixation can be particularly difficult due to the kyphosis in the thoracic spine producing vertebra-specific optimal trajectories . Robot-aAdditionally, despite spinal trauma accounting for a significant portion of potential spinal procedures, robot-assisted surgery remains largely untested in acute, spinal trauma. In a sample of studies with a large number of robot-assisted pedicle screws, trauma only represented a small number of the cases representing 0% up to 18% of cases ,11. ThisDespite the evidence emphasizing the accuracy of robot-assisted spine surgery, the adoption of these techniques has been slow. Reasons for not using a robot include lack of accessibility to a robot, familiarity with older techniques, and increased setup time. However, these disadvantages need to be balanced against the advantages of robot-assisted spine surgery including dual point registration/verification, a rigid arm to help support, and guide tools, which translate to less deformity on the unstable spine, and pre-operative planning with intraoperative plan modification. These factors have led robotic-assisted spine surgery to be more accurate than other methods outside of acute spine traumas  and may Given the advantages of navigated, robot-assisted surgery, we performed a case series to determine the feasibility of robot utilization for stabilization of acute, thoracolumbar spine injuries. We investigated the accuracy of a robotic system, the amount of blood loss, the length of hospital stays, and the duration of surgery to address the feasibility of robot-assisted spine surgery in a thoracic-biased setting of an acute, traumatic setting. We hypothesized that robot-assisted pedicle screw fixation would be non-inferior to other pedicle screw fixation techniques.This study is a case series of acute, thoracolumbar spinal fracture patients collected from May of 2020 to February of 2022 at a single institution. Each patient was evaluated by a neurosurgeon to determine the individual’s pathology as well as the best operative course. Patients were taken to the operating room within 24 hours of presentation. All surgeries were performed by a single neurosurgeon with resident assistance. All thoracolumbar pedicle screws were planned and placed using a robotic navigational system . Chart reviews were conducted for demographics, operative reports, and perioperative complications. Post-operative CT scans were obtained either intraoperative or post-operatively. The post-pedicle screw placement scans were evaluated in the axial and sagittal cuts for screw breaches in Centricity imaging software by two board-certified neuroradiologists. Any difference of opinion on pedicle screw breach was discussed among the radiologists until a consensus was reached independently of the surgeons. The cut with the largest deviation was measured by the neuroradiologists. The Gertzbein and Robbins system (GRS) was utilized for grading the pedicle screw breaches. A screw was considered accurate if the screw had an acceptable trajectory (in the pedicle or with an in-out-in trajectory) without a neurological deficit. Operative times were defined as when the time of the initial incision to the closure of the skin. Blood loss was estimated for each procedure by the attending neurosurgeon. This study was approved via the University of Oklahoma Health Sciences Center Institutional Review Board (2/2020/IRB #11641).WorkflowAfter a thin-cut (0.65-mm) CT scan was obtained, screw trajectories were planned in all three planes and loaded into the ExcelsiusGPS® system. Patients were sedated, intubated by the anesthesiologist, and positioned on an open Jackson table. The patients were prepped and draped. The area of surgery was identified via fluoroscopy. The dissection of the bony elements surrounding the corresponding level of injury was performed. The surveillance frame was clamped to a spinous process. A second surveillance point was placed into the PSIS through a stab incision. Anterior/posterior and lateral images were obtained using a special attachment with a C-arm x-ray machine. Close attention was paid to the stability of the clamp to optimize navigation and the merging of pre-operative and intraoperative images. A breath hold was utilized during imaging based on the utility of the breath hold in unpublished experience. The CT-planned images were merged with the pre-operative radiographs. The ghost construct was verified by the surgeon ensuring the screw trajectories were merged appropriately with the fluoroscopic images using the merge scores. Landmarks were verified before proceeding. The robot was draped in a sterile fashion and brought into the field. The robot arm was aligned with the potential screw hole. The hole was drilled and tapped with concurrent breath holds. The corresponding pedicle screw was placed. Intraoperative neuromonitoring was conducted on all surgeries. After the screws were placed, the corresponding fusion was performed as well as any additional procedures.There were 14-acute, spinal column trauma patients who were included in this single-center study for a total of 126 pedicle screws , 24-thoracolumbar junction (T12-L1), 40-lumbar, and two-sacral pedicle screws were placed. The most common indication for surgery was a burst fracture. The demographics for the patient population are summarized in Table -T11, 24-Twenty-six percent 4/15) of patients had pre-operative deficits with three out of four improving after surgery (Table 5 of patiPedicle screw placement was accurate with a <1% 1/126) pedicle breach rate when discounting the pre-planned in-out-in trajectories. Pedicle screw breaches which occurred due to an in-out-in trajectory were noted in Table 6 pedicleNone of the screws required replacement. The average anterior vertebral body screw breach was 2 mm. The only true pedicle breach (when discounting IOI trajectories) was 2 mm Figure  and was This case series demonstrates the feasibility of utilizing robotic assistance for thoracolumbar pedicle screw placement in acute, traumatic spine surgery. In this paper, after placing 126 pedicle screws in 14 patients we demonstrated the feasibility and safety of robot-assisted spinal fixation. Of the 126 pedicle screws placed, 84 screws were placed from T1-L1 with only a single breach (1.2% breach rate). Our breach rate was comparable to previous studies utilizing O-arm navigation demonstrating the non-inferiority of the robot-assisted pedicle screws in the thoracic spine.Our secondary findings of this case series include the feasibility of robotic assistance in an acute, traumatic setting. We demonstrated a good safety profile with a single new neurological deficit unrelated to robot use. In fact, three of the four pre-operative neurological deficits improved with surgery. Additionally, blood loss was low (191 mL), and operative times were decreased with a case number.Pedicle screw placement accuracy is critically important for not only improved fusion rates/strength of the fusion construct but also for patient safety. Previous work has demonstrated that intraoperative navigation improves pedicle screw placement accuracy from 68% to 95% . The addAdditionally, our study is unique as the cases occurred in an acute, traumatic setting. Acute, spine surgery provides unique challenges which are nonmodifiable including increased likelihood of hypotension from shock, increased neurological complications, increased operative hemorrhages, polytraumas, and operating in non-ideal conditions despite operative methods . UnderstThe robotic arm is especially useful in an acute, traumatic setting due to the rigidity and additional support provided to instruments. In unstable spines, touching the instruments to a fractured vertebra may displace the bony elements and lead to undesirable trajectories. However, with additional aids such as the robotic arm, such outcomes are less likely. Furthermore, swelling can lead to distortion of landmarks, which inhibits independent verification of landmarks, which can be partially mitigated by dual point verification in robotic spine surgery. However, despite robot-assisted spine surgery having many advantages, no work has demonstrated the feasibility of robotic-assisted surgery with a focus on either the thoracic spine or acute, traumatic surgery. A review of previous studies for robot-assisted pedicle screw placement is heavily biased in two ways: (1) to L4-S1 and (2) to elective cases. A study by Keric et al., one of the largest robotic pedicle screw placement studies with 2,067 pedicle screws, had 321/406 79%) cases focused on the lumbar/lumbosacral or lumbosacral area % cases f. MeanwhiThe accuracy of our pedicle screw placement can be traced to our meticulous attention to merge scores and stabilization of instruments via the robotic arm so as not to deform the spine while placing screws. Specifically, we found the instability of the spine can lead to motion of the spinal column even during breathing if the fracture is severe. Spinal instability would lower the accuracy of our pre-operative and interoperative image merge. To help address a proper merge, we have found breath holds were crucial. For improved screw accuracy, we found apnea before initial drilling and screw placement, stabilization of instrumentation by a robotic arm, and minimizing pressure on unstable areas when drilling as methods to maximize accuracy. Using these steps, we have managed to have accurate screw placements in an acute, traumatic setting. Also, it is important to note the dual verification required the placement of a second array in the pelvis during surgery. When performing navigated screws, the distance from the array can lead to increased variability in infrared detection. However, the dual array utilized in this study had no issues.One major critique of this paper may be the increased operative time associated with utilizing a robot. However, we do not discuss additional procedures being performed such as laminectomies which were included in our operative time. Furthermore, we demonstrated that operative times are continuing to improve as no plateau has been met Figure . These fThe second critique of this paper may be the planned IOI trajectories. Intuitively, if one is placing pedicle screws with robotic assistance, each screw should be wholly within the pedicle. However, nuances in anatomy make this difficult. When placing pedicle screws in the thoracic spine, some pedicles are smaller than the standard smallest screw diameter. Furthermore, when placing screws optimally, not all screws can be wholly in the pedicle and allow for attachment to the rod despite the polyaxial heads. The surgical goal in regard to pedicle screw trajectory was to optimize the trajectory while enabling feasible direct attachment to the rods. Therefore, after taking into account both pedicle size and rod attachment, IOI trajectories were at times a necessity.Another critique of this paper would be the blood loss. Previous work has demonstrated decreased blood loss for robot-assisted spinal surgery as opposed to freehand techniques . HoweverThis study does have caveats. This study had a low number of cases. Therefore, as with any technology, we cannot state that robotic-assisted spine surgery would be appropriate in all acute, spinal trauma cases and can only claim noninferiority versus other methods. However, given that our goal was proof of concept, we believe that despite our low number of cases, it provides valuable insight for thoracic spine surgery during the acute, traumatic setting.A second caveat may be that spine robots are expensive and cost-prohibitive for a number of hospitals . HoweverRobot-assisted pedicle screw placement is a feasible alternative to other pedicle screw placement techniques in thoracic and lumbar spine fixation surgery during an acute, traumatic setting with many benefits. These benefits include multiple redundancy checks for screw accuracy, improved stability of instruments, and less deformity of fractured vertebrae due to the rigid robot arm supporting the tools. Additionally, we demonstrated the operative times decrease as the surgeon gains experience. Furthermore, we demonstrate safe pedicle screw placement in both the lumbar and thoracic spine. We hope through the adoption of robot-assisted spine surgery in the thoracic and lumbar region during the acute, traumatic setting, patients will have the safest surgeries possible.


The Northeast Greenland shelf (NEGS) is a recipient of Polar Water (PW) from the Arctic Ocean, Greenland Ice Sheet melt, and Atlantic Water (AW). Here, we compile hydrographical measurements to quantify long-term changes in fjords and coastal waters. We find a profound change in the vertical distribution of water masses, with AW shoaling >60 m and PW thinning >50 m since early 2000’s. The properties of these waters have also changed. AW is now 1 °C warmer and the salinity of surface waters and PW are 1.8 and 0.68 lower, respectively. The AW changes have substantially weakened stratification south of ~74°N, indicating increased accessibility of heat and potentially nutrients associated with AW. The Atlantification earlier reported for the eastern Fram Strait and Barents Sea region has also propagated to the NEGS. The increased presence of AW, is an important driver for regional change leading to a likely shift in ecosystem structure and function. A comprehensive database of summer data reveals long-term changes in the vertical distribution of water masses on the Northeast Greenland Shelf, which will be important for driving ecosystem change through altered stratification and nutrient supply. The supply of freshwater to the NEGS is changing due to acceleration of mass loss from the Greenland Ice Sheet (GrIS)4, increased central Arctic Ocean freshwater content5, increased Arctic sea ice melt, and changes in freshwater6 and sea ice export, in the western Fram Strait8. Similarly, the air-sea exchange of heat just off the NEGS has greatly increased due to the westward retreat of the sea ice edge9.Advection of low salinity water and sea ice from the Arctic Ocean is a key characteristic of the Northeast Greenland Shelf (NEGS). Dominance of this low saline layer over much of the area enforces strong upper water column stratification10. The upper 150–200 m of the EGC consist of Polar Water (PW) formed within the central Arctic Ocean11. Warm waters of Atlantic origin (AW) are found below12, and are originally carried northwards with the Norwegian Atlantic Current (NAC) and West Spitsbergen Current 13, thereafter merging with the EGC to return southwards12 14. The properties of these waters can provide a record for observing ongoing Arctic climate change. Greenland fjords serve two important roles in this context: Firstly, they are the main gateways through which GrIS melt enters the ocean; and secondly, their bathymetry and circulation regulate the propagation of warm oceanic waters to marine-terminating glaciers, contributing to mass loss through submarine melting and undercutting15. Whilst the mechanisms by which AW reaches the head of fjords are not yet fully understood16, variability in coastal shelf waters is thought to be central in setting hydrographic conditions in the fjords17. In summer, the Northeast Greenland (NEG) fjords with deep sills that allows entrance of AW therefore have a three-layer structure similar to that found on the shelf: cold , fresh  PW advected from the Arctic Ocean comprises the intermediate layer and is capped by a thin, fresh local surface layer from summer melt and terrestrial runoff18. Finally, warm (θ > 0 °C), saline (Sp > 34.4) AW fills the deepest parts of the fjord. However, for fjords with shallow sills at the entrance, AW is inhibited from entering. The deep waters in these systems are therefore typically local modifications of the intermediate PW19. Note, that the detailed bathymetry of many NEG fjords and nearby shelf is still poorly known due to a dearth of observations.As opposed to the dynamic environment at the shelf-break and wider shelf area where advection and eddy activity can cause rapid modification of hydrographic conditions, shelf signals can be preserved within subsurface waters of fjords with long residence times20. Change has also occurred at depth where increased supply of heat from the passage of warmer AW to the 79N glacier has been documented22 resulting in increased ocean induced melting of marine-terminating glaciers24.Due to its sea ice coverage, the NEGS is relatively under sampled. There are two sustained long-term (>10 years) observation programs in the Young Sound fjord system (~74°N) and the western Fram Strait (~79°N). Results from the Young Sound monitoring program have shown a freshening in subsurface fjord and adjacent coastal waters over the 2003–2015 period25. Increased advection of AW or increased vertical mixing could directly increase the nitrate available to sustain phytoplankton production with potential cascading effects through the food web. Because PW and AW have different origin and temperature, the two water masses also contain species with different biogeographic affinity. Increased abundance of boreal species on the East and West Greenland shelf have been linked to changes in water mass distribution28. The importance of changes in water masses for ecosystems is very apparent in the Barents Sea, where a distinct increase in AW presence has reduced sea ice cover, changed species distributions, and transformed ecosystem structure29. Since AW is also important for the NEGS we aim to answer if the Atlantification observed elsewhere has propagated to the NEGS. For this purpose, we consolidate previously disparate summer measurements of oceanographic conditions over the period 1923 to 2019 from the NEGS to reveal recent trends in selected fjords and place these in the context of hydrographic changes of the adjacent NEGS waters. Our analysis, which is restricted to waters up to 100 km from the coast, finds significant changes in core PW and AW properties and their vertical distribution during the last two decades. There are, however, distinct latitudinal differences of which the impact on upper ocean stratification is quantified and potential implications of these changes for the NEGS ecosystem are discussed.The distribution of PW and AW has important consequences for the shelf ecosystem across the Greenland shelf. Regions more influenced by AW generally show higher productivity due to the higher concentration of nitrate, while especially the NEGS has low overall productivity due to limited nitrate in PW and delayed spring blooms due to sea ice coveragep < 34.4 and θ < 0 °C) and AW (Sp > 34.4 and θ > 0 °C) characteristics are reflected in the hydrography of the studied fjords  over the 2003–2014 period. However, recently this trend reversed, and salinities have increased slightly  temperatures are variable Fig. , there iThe properties of AW in the region have also changed with core temperatures increasing ubiquitously over the near-shore NEGS for the past four decades Fig. . CurrentIn addition to pronounced PW freshening and AW warming the vertical distribution of these waters have changed. The record of PW thickness shows that the PW layer is typically between 100 m and 200 m thick on the shelf but has been thinning from the 2000’s and onwards Fig. . This th30. AW is particularly important in this context as it acts as a marine heat source for ice melt as well as an important source of nutrients for phytoplankton in the photic zone. APE is quantified from the surface to the depth of the upper AW boundary and is used to document changes in the accessibility of heat and nutrients in AW to surface waters over the NEGS. Changes in APE are dependent on density changes as well as interface depth changes (see Methods)31. As such, APE is a measure of the integrated effects of changes in stratification in the upper water column.The combined impact of the hydrographic changes on stratification can be evaluated through the concept of available potential energy (APE), which represents the energy required to break down vertical density gradients and bring a given water column into mixed state−3) and has decreased 14% to 1130 J m−3 in recent years , a central region from 77°N to 74°N, and a southern region between 72°N and 69°N where stratification is comparatively weak Fig. . We notears Fig. . In the  AW Fig. , and to  AW Fig. . Similar AW Fig. . The latIn contrast to the northern and southern reaches of the shelf, the central region experiences no significant change in APE between the two periods Fig.  due to t32, acceleration and thinning at the ice sheet margins has since then, accelerated mass loss to the coastal environment33. The total freshwater flux from Northeast Greenland has nearly doubled from 125 ± 9 km3 y−1 between 1992 and 2010, to 240 km3 y−1 from 2007 to 201635. Stable isotope and dissolved organic matter measurements have shown that freshening of NEGS waters due to GrIS melt is largely limited to surface waters with salinities less than 31.536. So it is possible that GrIS melt has contributed to the decline in surface water salinities, but not the decline in PW salinities.There is evidence for a significant summertime freshening of the surface and PW layer over much of NEGS. Potential sources are GrIS melt water and freshwater exported from the central Arctic Ocean via Fram Strait in either liquid or solid state. Although the Northeast GrIS appears to have been in equilibrium up until the early 2000’s37. Whilst the composition in terms of source waters of the Fram Strait outflow is highly variable, evidence for increased positive sea ice melt water contribution to the total freshwater content since 2009 has been reported38. Freshwater transport within the shelf-break EGC showed no trend between 1997 and 2009 (~1300 km3 yr−1 39) but increased between 2010 and 2015, amounting to an additional freshwater volume of 3106 km3 40. While this may have caused some freshening on the NEGS, the bulk has likely followed the shelf-break EGC southward to Denmark Strait as the fresh anomaly was also observed here with little delay. However, since the freshwater transport with the PSW Jet on the 300 km wide northern NEGS is not monitored year-round, a liquid FW contribution from the Arctic Ocean through a pathway on the shelf to the observed freshening on the shelf cannot be ruled out. Recent findings from further east on the NEGS report a reduction in Polar freshwater export through the Fram Strait since 2015, show a thinning of PW layer and a westward movement of AW6, which supports the observations reported from the coastal waters.Liquid freshwater export from the central Arctic Ocean via Fram Strait is composed of contributions from sea ice melt, river discharge, precipitation and less saline ocean water entering through the Bering Strait7. Supplementary Fig. 7 to the late summer (July–Aug–Sept) sea ice volume estimate on the NEGS, assuming a one-month transit time from the Fram Strait into the NEGS. The late summer (JAS) sea ice volume in the region has reduced from approximately 265 km3 to 135 km3 from the 1990s to the 2000s while at the same time, the sea ice volume transport from the central Arctic for the summer months shows no statistically significant trend , as well as freshwater from the GrIS, however, may outweigh the local sea ice melt in the future. The importance of these sources are expected to increase in the coming decades as mass loss of the Northeast GrIS accelerates4 and liquid freshwater export through Fram Strait may increase41.Decline in sea ice extent and thickness within the NEGS as well as sea ice exported from the central Arctic Ocean and melted locally can also contribute to the observed freshening. Arctic sea ice volume export through Fram Strait has been declining since the early 1990’s and particularly since the early 2000’s mostly due to thinning of sea ice43 and the warming of AW entering Fram Strait since 1997 and warming of RAW which recirculates in Fram Strait before merging with the EGC and returning southward along the shelf break44. The increase in AW core temperature derived in our analysis  increased heat supply for ice melt, (b) change in stratification and nutrient supply, and (c) increased biological connectivity to the Atlantic Fig. . Closer 48. Ice coverage over the NEGS limits the extent of mixing and surface waters entrain nutrients from PW, which has a lower nitrate content and nitrate to phosphate ratio, than AW38. Over the summer months the surface mixed layer is established, ice coverage retreats, and phytoplankton become nitrate limited49  resulting in greater light attenuation51. Freshening and thinning of PW will dilute and reduce the contribution of CDOM to vertical light penetration. This combined with a low contribution to light attenuation by phytoplankton in surface and PW waters due to nutrient limitation, can result in increased light penetration (photic depth). Decreased sea ice cover both increases light availability and increases energy available for vertical mixing, potentially resulting in increased summer phytoplankton productivity on the NEGS  Oceanographic Toolbox built for MATLAB60. Subsequent analysis and computation of derived variables were done using potential temperature and practical salinity, from here on referred to as “temperature” and “salinity” unless otherwise specified.Prior to 1990, the majority of profiles were sampled using reversing thermometers and bottles to measure temperature and salinity at standard levels. Modern profiles were primarily sampled with CTD instrumentation, typically with an accuracy between 0.001 °C and 0.005 °C for temperature, 0.003 to 0.01 for practical salinity S61. The northern extent of the study domain is set at 81.3°N north of which the EGC has different dynamics62, and the southern extent is set at 69°N corresponding to the approximate latitude at which the EGC bifurcates and circulation dynamics take on a new character63. While the geographical coverage of the dataset is fair, there are seasonal and spatial biases , is characterized by temperature >0 °C and salinity >34.4. Polar Water (PW) is defined by temperature <0 °C and salinity <34.4. Hydrographic changes of PW and AW are characterized by the core temperature and salinity properties of the two water masses as well as by their vertical positioning and extent. Each metric is described in the following.66). A final concern regarding the ability of historical data with coarse vertical resolution, to accurately estimate core properties was addressed with a small experiment. In this experiment the procedure was applied to high-resolution profiles sub-sampled at typical bottle depths . Core properties identified using the sub-sampled and full resolution profiles were then compared. The sub-sampled profiles captured the core properties well  and differences in temperature and salinity properties of both PW and AW were found to be small. However, the experiment indicated that depth of the AW core requires higher vertical resolution to be reliably estimated.PW and AW cores are identified by the temperature minimum and maximum within the two water masses, respectively. These features are identified for each profile after linear interpolation onto a 1 m spaced grid from 0 m to 600 m. Only profiles with depths larger than 100 m were considered, thereby excluding shallow banks. In the absence of a pronounced temperature minimum, the mean properties between 40 m and 60 m were used to characterize the profile. This was particularly relevant for shallow sill fjord systems such as Young Sound. Identifying the AW core can likewise be problematic if the sampling depth does not penetrate adequately into the AW layer. To this end, temperature from the 1 m binned data of the 3 depth-levels nearest the identified temperature maximum were compared. If the temperature deviated by more than 0.01 °C between subsequent depth-levels, the core property estimates were deemed unreliable and discarded as we cannot be confident that the true AW core was sampled  are presented.To limit across shelf discontinuity in water mass properties, a spatial constraint is adopted for time series construction. This constraint limits the spatial extent of the study region to a 100 km band from the shoreline unless the shelf is narrower in which case the 500 m isobaths is used , z1 and z2 are the depths between which the metric is computed, h is the difference between z2 and 1, z is depth measured positively upward from z2, ρ is potential density (kg m−3) and z2. APE is determined by two quantities, namely density and layer thickness. Change in APE between the 1980–2004 and 2014–2019 periods, is segmented into the relative contribution of these components . This metric represents the work required to erode vertical density gradients and bring a given water column into a mixed state. This criterion provides information on changes occurring between the considered depths and can therefore be considered a bulk measure of stratification. APE is calculated from the 1 m binned data with units of J m8, was averaged over June–July–August from 1992 to 2018. The NEGS sea ice volume is estimated from monthly mean sea ice extent (defined as the area with more than 15% sea ice concentration) in the region 70°N–82°N, 20°W–15°E from monthly sea ice concentrations at 25 × 25 km resolution from the National Snow and Ice Data Center  multiplied with the monthly mean effective sea ice thickness estimate across the Fram Strait between 13°W and 0°W8 at a northern limit and the assumption that the sea ice thickness between the Fram Strait and 70°N goes linearly to zero in the summer months.Monthly mean sea ice volume transport dataSupplementary InformationPeer Review File


A total of 42 (1.5%) complications directly related to TSC: 0.9% in the ICE group and 3.1% in the no ICE group (p < 0.001). The independent predictors of all complications were age , TSC with the use of ICE  and AF ablation . The independent predictors for TSC complications were age  and TSC with the use of ICE . Conclusions: ICE reduced periprocedural and TSC-related complications during electrophysiological procedures for ablation of left atrial arrhythmias.Aims: To explore the impact of the use of intracardiac echocardiography (ICE) in the ablation of supraventricular arrhythmias requiring transseptal catheterization (TSC), whilst analyzing the reduction in periprocedural complications and complications specifically related to TSC. Methods: A retrospective multicenter study collecting data from consecutive atrial fibrillation (AF) and supraventricular ablation procedures that required TSC was performed in five Italian centers. Based on physician discretion, TSC was performed with or without ICE. Periprocedural complications, separating all complications from complications directly related to TSC, were collected. Independent predictors of periprocedural complications and TSC-related complications were investigated. Results: A total of 2181 TSCs were performed on 1862 patients at five Italian centers from 2006 to 2021, in 76% of cases by AF ablation and in 24% by ablation of other arrhythmias with a circuit in the left atrium. Overall, 1134 (52%) procedures were performed with ICE support and 1047 (48%) without ICE. A total of 67 (3.1%) complications were detected, 19 (1.7%) in the ICE group and 48 (4.6%) in the no ICE group,  During the last ten years, the use of transseptal catheterization (TSC) in the electrophysiology laboratory has increased, especially in its use in the ablation of the substrate of atrial fibrillation (AF). This technique, although highly effective, is still burdened by complications of around 1–6% ,3,4,5. TIn this retrospective multicenter study, we collected data from consecutive AF and supraventricular ablation procedures that required TSC, performed at the various participating centers  and patients who underwent the TSC with ICE (ICE group).Baseline characteristics of patients, such as age, gender, presence of cardiac disease, left ventricular ejection fraction, left atrial diameter and periprocedural anticoagulation regimen, were collected. We considered as “uninterrupted anticoagulant” the patients in whom no dose  or only morning dose  was withdrawn before the procedure. Regarding patients taking vitamin K antagonist, uninterrupted therapy meant patients therapeutically anticoagulated at the time of the procedure without needing heparin bridge the days before. All acute periprocedural complications during in-hospital follow-ups were recorded. All acute complications related to TSC were recorded. Independent predictors of periprocedural complications and TSC-related complications were investigated. We used the simplified technique described by De Ponti et al. : two catWith ICE, the TSC technique is the same as above with the addition of ICE to confirm engagement of tip of the needle and sheath of fossa ovalis.® Catheter, Biosense Webster or ViewFlexTM Catheter, St. Jude Medical-Abbott) was used only in selected difficult cases at physician discretion. Subsequently, from 2016 onwards, systematic use of the ICE was made in most of the TSC procedures. The effectiveness of TSC was also considered as the ability to complete it with adequate positioning of the catheters in the left atrium. All TSCs were performed directly or under the supervision of an experienced operator, defined by the previous performance of >50 procedures. In the case of double TSC during ablation of atrial fibrillation, a single puncture was considered. TSC through patent foramen ovalis was excluded. The majority of procedures were performed under sedation with midazolam plus morphine/fentanyl while general anesthesia was used in a small minority of selected cases. In terms of learning curve, procedures were divided in the whole cohort in chronological order between 1–100, 101–200 and >200.The 5 participating centers shared a similar experience, up to 2015 ICE , acute aortic root puncture (with or without requiring pericardiocentesis or cardiac surgery), atrial wall injury , pericarditis and/or pericardial effusion, thromboembolism , acute coronary syndrome, hemidiaphragm paresis and death.Periprocedural TSC complications included: acute aortic root puncture (with or without requiring pericardiocentesis or cardiac surgery), atrial wall injury , pericardial effusion, thromboembolism (transient ischemic attack/stroke), acute coronary syndrome and death.The primary endpoint was the occurrence of periprocedural complications. The secondary endpoint was the occurrence of TSC complications.p < 0.05). Statistical analyses were performed with SPSS Statistics version 25.0 (IBM Corporation).Statistical analysis was performed per procedure. The data analysis was descriptive. Continuous variables with normal distribution were expressed as mean ± standard deviation. Categorical variables were expressed as an absolute number and percentage (%). The comparison between continuous variables was performed by t-test. Comparison of proportions was performed by Fisher’s test or chi-square as appropriate. In stepwise logistic regression, the dependent variable was represented by the periprocedural complications and complications related to TSC and the independent variables correspond to the significant clinical characteristics  complications directly related to TSC: 0.9% in the ICE group and 3.1% in the no ICE group (p < 0.001)  patients, 0 (0%) in the ICE group and 14 (1.3%) in the no ICE group, p < 0.001; none of these patients received cardiac surgery but 4 (29%) required pericardiocentesis. The other periprocedural complications were not significantly different between the two groups  complications were detected, 19 (1.7%) in the ICE group and 48 (4.6%) in the no ICE group, < 0.001) . In parto groups .p = 0.10) and TSC complications . However, a trend toward lower complications was observed when the number of procedures was increasing . Considering these 41 procedures, a total of 18 procedures produced complications, 1 in the ICE group and 17 in the no ICE group (p < 0.001), and 23 were forced to stop the procedure without further complications, 18 in the ICE group and 5 in the no ICE group (p = 0.02).The procedural effectiveness was 98%. An inability to perform the transseptal puncture and introduce the scaler catheter into the left atrium was observed in 41 procedures: 19 procedures in the ICE group and 22 procedures in the no ICE group (p = 0.036), TSC with the use of ICE  and AF ablation  were independent predictors of complications (p = 0.013) and TSC with the use of ICE  (p < 0.001) and TSC complications  . Further < 0.001 ).The main finding of this study was that the use of ICE significantly reduced periprocedural complications and TSC-related complications, with a relative risk reduction of 70%. Furthermore, the present findings show that the beneficial effect of ICE-guided procedures was independent of the learning curve. The other independent predictors of complications were advanced age and AF ablation. Age is a well-known factor in increasing procedural risk in most cardiac and non-cardiac interventions . AblatioInterestingly, in the last 5 years, supraventricular arrhythmias ablations increased, especially because of an increase in AF ablation procedures. This can be explained by the new recommended guidelines ,12 that The rate of periprocedural complications in the study population was 3.1%, in line with previous studies, and, in particular, was 1.7% in the ICE group and 4.7% in the no ICE group. Recently, Pimentel et al. reported in a series of more than 2000 patients a rate of complications that was 2.9% in the ICE group and 5.8% in the no ICE group, which is in line with our results .In our cohort, the complications rate directly related to TSC  fell to a value of 0.8%, equal to or less than previously published rates ,2,3,4,5.All these results were confirmed by only analyzing AF ablation procedures. Of interest, age in this subgroup of patients was not independently related to complications. This may be due to the range of age being narrow, which may have homogenized the risk. An uninterrupted anticoagulation strategy, currently in class I in the ESC guidelines on AF management, was also associated with a significant reduction in global complications at univariate analysis but not independent in multivariate analysis where the only use of ICE was associated with lower complications. Considering the potential hemorrhagic risk of this strategy, the use of ICE could be of benefit by preventing accidental punctures of fragile structures. Even an infusion of heparin boluses before TSC, which in some studies has been associated with a lower incidence of intra-atrial thrombosis, could also be made safer by the use of ICE.The impact of ICE on the incidence of TSC complications was not significant in a study by Swedish authors on 4690 procedures; however, it was used only in 0.5% of cases . Our resRegarding the learning curve, we observed that there was a trend toward a higher number of periprocedural and TSC complications in the group of the first 100 procedures (1–100) and procedures 101–200 without, however, reaching statistical significance. This showed that experience alone is not enough to prevent complications, because cardiac anatomy is unpredictable. Therefore, the use of ICE is of paramount importance. The procedural efficiency, understood as the ability to complete TSC, was high (around 98%) in both groups of this study, although the complications arose significantly more often in the non-ICE group.A recent meta-analysis  has showIn conclusion, the additional cost of the ICE seems justified by the possibility of significantly reducing the incidence of complications and improving the procedural efficiency of TSC. Added to this is the possibility of highlighting and then treating the formation of any pericardial effusion, to visualize the esophagus during ablation and intra-atrial thrombus. In addition, the formation of microbubbles, visible with ICE, may be a frequent indication of excessive heating of the tissues during radiofrequency delivery and, finally, to select in certain cases the site of puncture of the oval fossa in relation to the type of procedure, with the possible benefit of the maneuverability of catheters . FinallyThe present study is retrospective, thus discounting the intrinsic limitations of this type of analysis. The time span covered is very long, with relative difficulty in completing the data collection. It was not possible, in fact, to evaluate the impact of the ICE on procedural times and on the times of fluoroscopy since such data were not available for all patients in the study. The data collection involved a limited number of centers with medium to high procedural volume, which, however, showed homogeneity in the execution of the TSC method. The use of ICE was not random but at the physician’s discretion and has become systematic from 2016 onwards. Complications not related to hospitalizations such as atrio-esophageal fistula and pulmonary vein stenosis were not reported. However, the present study focused on the role of ICE in reducing acute and TSC complications.In this retrospective multicenter study, ICE significantly reduced periprocedural and TSC-related complications during electrophysiological procedures for the ablation of left atrial arrhythmias.


In the present work, we demonstrate that phosphorylation of the enzyme at Ser505 is an important step for the translocation of the enzyme to PtdInsP2–enriched membranes in human cells. Constructs of eGFP-cPLA2 mutated in Ser505 to Ala (S505A) exhibit a delayed translocation in response to elevated intracellular Ca2+, and also in response to increases in intracellular PtdInsP2 levels. Conversely, translocation of a phosphorylation mimic mutant (S505E) is fully observed in response to cellular increases in PtdInsP2 levels. Collectively, these results suggest that phosphorylation of cPLA2α at Ser505 is necessary for the enzyme to translocate to internal membranes and mobilize arachidonic acid for eicosanoid synthesis.Group IVA cytosolic phospholipase A Afterward, they were washed extensively and overlaid with 0.5 mL of serum-free medium supplemented with 0.5 mg/mL albumin and treated with 5 µM thimerosal for 15 min to block fatty acid reacylation [The cells were labeled with 0.5 µCi/mL [cylation ,39. The cylation . After ecylation . Spots c2α-mediated production of bioactive lipid mediators represents a key event in the execution of physiological and pathophysiological responses to external stimuli. This study has focused on the complex interactions that govern cPLA2α translocation to membranes and the multiple factors that may be involved in its regulation. Specifically, we have described the interconnected role of two of these factors, phosphorylation of the enzyme at Ser505 and cellular PtdInsP2 levels. Both of them seem to work together to promote membrane translocation and activation of cPLA2α under low intracellular Ca2+ levels. Together, the studies described here represent a relevant working model to further understand the intricacies of the cellular regulation of cPLA2α and the molecular mechanisms underlying it.cPLA