Shearing along subduction zones, laboratory experiments on analogue faults, and sliding along glacier beds are all associated with aseismic and co-seismic slip. In this study, an ocean-bottom seismometer is deployed near the terminus of a Greenlandic tidewater glacier, effectively insulating the signal from the extremely noisy surface seismic wavefield. Continuous, tide-modulated tremor related to ice speed is recorded at the bed of the glacier. When noise interference  is low, the tremor is also confirmed via analysis of seismic waveforms from surface stations. The signal resembles the tectonic tremor commonly observed during slow-earthquake events in subduction zones. We propose that the glacier sliding velocity can be retrieved from the observed seismic noise. Our approach may open new opportunities for monitoring calving-front processes in one of the most difficult-to-access cryospheric environments. Anomalously slow earthquakes play a critical role in the earthquake cycle and fault sliding. Here, the authors detect continuous seismic radiation from a glacier sliding over its bed and show persistent coastal shaking to represent an addition to the family of slow earthquakes. Analyses of weeks-long episodic tremor1, which could resemble storm-generated noise3, have now been surpassed by machine learning techniques that yield continuous, years-long seismic chatter along a subduction zone7. These findings raise the possibility of similar phenomena under polar glaciers9, which slide at rates of up to four orders of magnitude higher than tectonic faults11. To date, only low-amplitude, discrete basal icequakes have been reported beneath the Greenland Ice Sheet (GrIS) and Antarctic ice streams16. Researchers have documented the teleseismically detectable Mw 7 stick–slip of an Antarctic ice stream17 and its associated tremor bursts19. However, such events have not been documented in Greenland, as MS50 4.6–5.1 events, which were initially termed ‘glacial earthquakes’21 because they were believed to emanate from the downhill sliding of the glacier trunk, have now been recognized as iceberg capsizing22. Further investigations of the seismic fingerprints of glacier sliding are motivated by (1) the possibility of estimating the frictional state of a dry laboratory fault from seismic signals23, and (2) the results of field and cold-laboratory analyses, which support the use of basal seismicity to assess the coupling at the ice–bed interface26, especially when the parameterization of glacier basal sliding remains the key uncertainty in predictions of ice mass loss and sea-level rise28.Recent discoveries of anomalously slow earthquakes and associated nonvolcanic tremor (commonly termed ‘tectonic tremor’) have revolutionized our understanding of the connection between slow and fast (regular) earthquakes, and their respective roles in fault-zone processes30. Consequently, GrIS was considered an ideal site to monitor the underground nuclear explosions conducted by the Soviet Union31. However, ice-breaking noise forced temporal termination of seismic monitoring in certain GrIS locations, such as coastal Thule32. Indeed, the Blue Trek seismic-noise survey across Northwest Greenland in August 1967 , which is the subject of this study and could be seen as the endpoint of the Blue Trek survey , station drift due to ice flow (~40 m per month at Bowdoin Glacier), and the risk of instrument destruction due to crevassing and calving36. On-rock seismic stations are often installed on the lateral side of the glacier to avoid the latter issues8. However, while such a configuration is located away from the region of the fastest glacier flow and avoid the associated ice-generated noise, the seismic waves from the target area  must travel along a complex signal path and attenuation structure that consists of a multi-layered interface between ice, sediments, and bedrock, and, in the case of Bowdoin Glacier, a lateral river that may serve as a potential fluvial seismic source , and its findings were reproduced using the oldest seismo-geodesic records collected in 2015 at Bowdoin Glacier35. Therefore, the analysis results are of value due to a combination of both submarine and surface instruments and their unique proximity to the calving front. By ‘eavesdropping’ directly at the sliding base of this tidewater glacier, the OBS data reveal that the glacier continuously radiates seismic energy. A tremor-like signal is generated that lasts at least 2 weeks and resembles the Cascadia subduction zone tremor6, but with strong diurnal and semi-diurnal periodicities. A comparison of the OBS signal with the horizontal displacement rate of the glacier demonstrates that the background noise may potentially serve as a proxy for the ice speed and ultimately the physical state of the ice–bed interface.Here, we overcome the aforementioned challenges by the deployment of an ocean-bottom seismometer (OBS) near or under the calving front of Bowdoin Glacier Fig. b–d. This23. Furthermore, since we are assuming the overall similarity between a fault and the sliding glacier bed, we note that while the continuous tremor of the Cascadia subduction zone exists down to ~1 Hz, the signal in the 8–13 Hz band is nonetheless strong6. We first deconvolve the instrument response and compute the power spectral density–probability density functions  to determine the statistical features of the continuous seismic data that best correlate with glacier dynamics while also keeping the signal processing as simple as possible. The representative velocity spectrum of the OBS data highlights that the horizontal components carry more power (see the “Methods” section for details), with an elevated energy level between approximately 0.6 and 14 Hz, and peaks at 0.7–2.0 and 7–14 Hz . We investigate the seismic noise levels by computing the PSD for each 30-s segment of the OBS data and integrating the power in several frequency bands of interest that were chosen based on the PSD–PDFs analysis . We then present the temporal evolution of the normalized power in each band as density plots and extract the lowest noise level in 30-min-long sliding windows  with the global positioning system (GPS) displacement rates of the glacier that were measured ~880 m away  + b) has an uncertainty range of ±0.16 m d−1, which is equivalent to ~12% of the mean ice speed. We note that using the horizontal components does not lead to a significant improvement in the results.The broad-band seismic signal (3.5–14.0 Hz) correlates well , moulins, and flow in crevasses are very local acoustic sources38 that are unlikely to converge into a coherent, widely observed tremor.We discuss below the ‘basal-sliding-seismicity’ hypothesis over competing hypotheses. Furthermore, in support of our arguments, we use surface stations on ice and rock located at different distances from the fastest moving part of the glacier in the same and previous years. Among the alternative sources of noise fluctuations, our key competing hypotheses are surface crevassing36. First, the correlation between the strain rate, R = 0.45), even if the peaks and troughs in the longitudinal extension that cause microseismic activity occur close to the peaks and troughs in ice speed . Furthermore, such a double-hump  feature of tidal modulation is generally difficult to associate with subglacial discharge45. In this regard, a separate line of evidence suggesting that the subglacial discharge under Bowdoin Glacier is unlikely to dominate the observed broad-band tremor is provided by the first direct and continuous monitoring of a subglacial discharge plume near the centre of the calving front40. Specifically, even during such extreme hydraulic events as subglacial drainage of an ice-dammed lake, the associated seismic tremor remained primarily below ~1 Hz40.There are two key differences between the observed continuous seismic signal and strain-rate variations that drive the intense microseismicity due to surface crevassinger R = 0.5, even ier R = 0.5, even ier R = 0.5, even imentclass2pt{minimFor the period of distinctly strong tidal modulation of the ice speed , correspond to relatively short interference that masks the tremor signal. Fortunately, these calving events can be distinguished by their seismic signature having a particularly low characteristic frequency , 20 July , and 23 July , yields similar patterns and correlation results , which do not overlap with an earlier proposed subglacial-discharge band  being relatively stable and corresponding to cold (−1. 8 °C) Polar Water50 during the period of our OBS observations.In contrast to the 2019 on-rock station Fig. , the on-6. Although this may be a coincidence, the band-limited noise is difficult to attribute solely to attenuation because our off-ice stations do not miss high-frequency seismicity  suggest similar physical processes, whereby the observed tremor is generated by slab sliding and friction under high pore-fluid pressure. We suggest that the elastic waves are generated at the ice–bed interface via either entrained-sediment friction24 or protruding clasts, which are dragged by the sliding glacier and either plough/deform the water-saturated till or scratch the hard patches of the bed11. Rock-on-rock friction due to entrained sediments was recently proposed as the most likely cause of subglacial shear seismicity, as inferred from cold-laboratory experiments24. Photographs of calved icebergs  may partly explain this result . Another difference we expect is that glaciers can flow at high velocities without a proportionate increase in seismic noise, as a result of either partial or full decoupling of the glacier from its bed at particularly low effective pressures. Specifically, recent cold-laboratory experiments and theoretical models exploring ice-on-till friction indicate that the rate-weakening friction is a necessary condition for basal seismicity24. Relatively low effective pressures at the sliding interface may correspond to reduced frictional weakening24. For example, such decoupling can occur when there is insufficient development of the subglacial system to remove excess water pressure lifting the ice, or when the western zone of the Bowdoin Glacier transitions from near-floating to floating in the future49. Such a major loss of effective pressure is difficult to imagine within a given subduction zone.Glacier tremor is stronger and more clearly correlated with the GPS data than subduction-zone tremor10. However, this interpretation has been challenged in recent studies28. In fact, as an alternative to traditional power-law basal rheologies relating slip velocity to basal stress, recent studies have proposed numerical ice-sheet modelling based on analogue experiments with glacial till27. Such modelling indicates the validity of using Coulomb friction and shows reduced basal shear stress near the grounding line27. Furthermore, the shear stress is independent of a high slip velocity at low effective pressure because shallow bed deformation is initiated at the failure strength of the bed by particles dragged by the flowing ice11. Therefore, our conclusion that seismic radiation is related to sliding rate does not contradict our current understanding of the underlying physical processes , at least from a conceptual perspective.Finally, our findings are somewhat at odds with the interpretation of velocity-independent basal drag corresponding to basal-friction-independent glacier sliding32 contains a wealth of glaciological information. Furthermore, our approach is valuable in difficult-to-access glacier-fjord settings. This study may motivate long-term interdisciplinary studies employing similar integrated multi-purpose ocean acoustic systems to reveal tremor location, migration, triggering, and termination via external factors1, as well as the presence of slow-earthquake phenomena  in other glacial settings. The use of multiple OBS stations, rather than just one as in this study, would be particularly helpful in locating the tremor, although, considering the cost of OBS instruments and the difficulty of approaching calving fronts, single-station submarine experiments may be more feasible. Such observations may increase the understanding of the coupling between the ice and its subglacial bed, especially as many marine-terminating glaciers continue to thin and approach floatation.In summary, we present a seismic acquisition method  to circumvent the noisiness of calving-front environments while simultaneously harvesting glacioseismic information from continuous seismic data. This work reflects the recent paradigm shift in seismology from discrete to continuous analysis, and demonstrates that inconvenient ‘noise’57. This setup consisted of a velocity seismometer, recorder, and lithium batteries that were sealed in a glass sphere under vacuum, which was protected by a plastic shell and surrounded by autonomous external instrumentation (mainly a Mitsuya anchor unit and Kaiyo Denshi STH-10B acoustic transponder). The sensor consisted of a gimballed three-component geophone with a 4.5-Hz natural frequency . The seismic data were digitised and sampled at 128 Hz, with the internal clock information assigned using a Katsujima HDDR2 recorder with a 16-bit A/D converter. The clock drift and instrument drift from the drop point were both negligible due to the relatively short and shallow deployment48.A pop-up-type OBS station with an acoustic release system (previously used in subduction-zone and other tectonic studies offshore of Japan) was deployed near the glacier calving front58. The temperature at the bottom of the fjord provides valuable information for both understanding the performance of the system and detecting deep intrusions of warm Atlantic water, the latter of which is important for subaqueous ice melt and has previously been observed in Bowdoin Fjord50.The system also included a radio beacon , and a compact hydrophone integrated with a recorder  that sampled at 96 kHz and an internal thermometer (one sample per minute) that measured a mean water temperature of −1.8 ± 0. 1 °C (±shows the standard deviation). The high-frequency hydrophone was included for recording the soundscape of Bowdoin Fjord, which is seasonally visited by narwhalsThe OBS station was manually deployed in the centre of Bowdoin Fjord (243 m water depth) on 21 July 2019 from two small boats, ~640 m from the calving front  suggests a possible presence of soft sediments of up to a few metres thick , due to their close proximity to each other (<100 m), but both are used here to maximize the observed time series (the 22–25 July data from GPS#9 are incorporated into the B1901 time series when necessary for the time-series comparisons). We also note that the surface GPS records closely correspond to basal sliding due to low internal ice deformation near the calving front as commonly assumed in models27 and directly measured in boreholes at Bowdoin Glacier61.GPS stations were installed close to the calving front  and farther upglacier (B1902 at 2 km) by attaching the antennas to stakes that were drilled into the ice. B1901 and B1902 were dual-frequency GEM-1 receivers, whereas GPS#9 was a single-frequency Emlid Reach M+ receiver. The stations near the calving front are described in the previous studies3 datalogger (Omnirecs) that sampled at 400 Hz. A Lennartz LE-3Dlite MkIII 1-s seismometer was installed on the coast near Bowdoin Glacier, ~630 m south of the calving front and 1.9+ km from the GPS stations; the sensor was buried in a shallow pit and connected to a Centaurus datalogger that sampled at 500 Hz.A Lennartz LE-3D/BH 1-s borehole seismometer was deployed in a shallow borehole (~3 m below the ice surface) within 280 m of each GPS station and connected to a DATA-CUBETime-lapse images of the calving front (7377 × 4935 px) were taken every hour using a NIKON D800 camera that was positioned on the east side of Bowdoin Fjord from 2 July until 29 July.36. An overview of considered seismic data is provided as spectrograms in Supplementary Fig. GPS station B1501 was deployed in July 2015 at the same site as B1901 in 2019. A Lennartz LE-3D 5-s seismometer was deployed in a shallow ice pit 70 m from GPS station B1501, which was in approximately the same area as the 2019 on-ice station (ATD). The station was connected to a Guralp CMG-DAS-S6 digitiser that sampled at 500 Hz. A Guralp CMG40T 30-s seismometer was deployed on the coast at the same site as BWG24 of 2019. The station (CFH) was connected to a CMG-DAS-S6 recorder that sampled at 100 Hz. Further details of the 2015 campaign are provided in previous studies47. An AWS was operated at the same site in July 2015. The median wind speeds in 2015 and 2019 were similar (0.9 and 1.0 m s−1). However, the 2015 campaign had remarkably calm weather conditions that were favourable for ambient noise measurements36.An automatic weather station (AWS) was installed to the east of the glacier and operated from 1 to 28 July 2019; the details of the AWS have been reported previouslyr2 = 0.98), and, due to contamination by local calving-generated tsunami signals, the latter is more convenient to process39. Detailed analysis of the tidal role in the Bowdoin Glacier ice speed and strain rate is provided in our previous studies49. In brief, the tide-modulated ice speed varies greatly, usually with two daily peaks corresponding to falling or low tide  and are shown in Fig. 41 using continuous seismic data that were parsed into 360-s-long segments with a 50% overlap. We first computed the PSD for each segment after removing the instrument response over the 0.005–100 Hz range. For H/V analysis, we also differentiated the signal. We then smoothed the PSDs in 1/2-octave averages at 1/8-octave intervals, and collected the corresponding powers in 0.5-dB bins, which served as the basis for computing the PDFs. We present the PSD–PDFs in decibels relative to velocities.We estimated the PSD–PDFsi denotes the time window, and f1 and f2 are the lower and higher frequencies of each band (with a width Δf = f2 − f1). The corresponding power is assigned to the central absolute time of each segment, and normalized by the band-width (×Δf−1) and its average logarithmic levels . We then divided the average of the horizontal power (two components) by the vertical component (one component). This revealed two distinct peaks (0.9 and 7.4 Hz) where the horizontal motion was stronger  spectral ratio in two steps following a previous study42, tilt noise due to seafloor currents and compliance noise due to pressure variations caused by ocean gravity waves63. We suggest that 0.9-Hz peak is due to microseisms , whereas the 7.4-Hz peak is probably due to the site response, as the periodicity of ocean gravity waves is too long for our analysis and particularly strong seafloor currents are not expected . The sediment thickness, H, can therefore be inferred from the dominant frequency, f, and shear velocity, Vs, such that 64. An assumed velocity between 25 m s−1 ref. 42 and 100 m s−1 ref. 65 yields a thickness of between 0.8 and 3.4 m.The OBS horizontal components may be stronger than the vertical component due to shear mode resonance in soft sediments and noise. The following three types of OBS noise are usually the strongest: microseisms driven by the local wind wavefield at frequencies near 1 HzSupplementary InformationPeer Review FileDescription of Additional Supplementary FilesDataset 6Dataset 1Dataset 2Dataset 3Dataset 4Dataset 5


Z score might effectively identify children at most risk of near‐term mortality. Wasting and stunting, driven by common factors, frequently occur in the same child, either simultaneously or at different moments through their life course. Evidence of a process of accumulation of nutritional deficits and increased risk of mortality over a child's life demonstrates the pressing need for integrated policy, financing and programmatic approaches to the prevention and treatment of child malnutrition.In 2014, the Emergency Nutrition Network published a report on the relationship between wasting and stunting. We aim to review evidence generated since that review to better understand the implications for improving child nutrition, health and survival. We conducted a systematic review following PRISMA guidelines, registered with PROSPERO. We identified search terms that describe wasting and stunting and the relationship between the two. We included studies related to children under five from low‐ and middle‐income countries that assessed both ponderal growth/wasting and linear growth/stunting and the association between the two. We included 45 studies. The review found the peak incidence of both wasting and stunting is between birth and 3 months. There is a strong association between the two conditions whereby episodes of wasting contribute to stunting and, to a lesser extent, stunting leads to wasting. Children with multiple anthropometric deficits, including concurrent stunting and wasting, have the highest risk of near‐term mortality when compared with children with any one deficit alone. Furthermore, evidence suggests that the use of mid‐upper‐arm circumference combined with weight‐for‐age  A significant proportion of wasting and stunting is present at birth and can contribute to further growth failure during subsequent infancy and childhood. Improving maternal health and nutrition in pregnancy and early life could have a critical role in the prevention of wasting and stunting.Periods of wasting leave a child more likely to experience stunting and, to a lesser extent, vice versa. Common risk factors drive an accumulation of vulnerabilities. This underlines the need for cohesive policies and the implementation of services and activities to prevent both wasting and stunting.Concurrently wasted and stunted children have an elevated risk of death and should be considered as a high‐risk group in the targeting of treatment.Z score and mid‐upper‐arm circumference may be the most effective way to identify children at highest risk of mortality, including those concurrently wasted and stunted. Further evidence is needed to understand the operational implications.A combination of weight‐for‐age 1Undernutrition remains a major public health concern in many countries and an underlying cause of almost half of global child mortality  are unclear, although some aspects of the divide have been entrenched by divergent funding and programmatic approaches in humanitarian and development contexts and the separation of wasting treatment and stunting prevention approaches  formed a technical interest group (TIG) of global experts (referred to as the WaSt TIG) to examine the relationship between wasting and stunting and published a technical briefing paper  and Sustainable Development Goals (SDG) targets as they relate to wasting and stunting.2Preferred Reporting Items for Systematic reviews and Meta‐Analyses (PRISMA) guidelines .We conducted a systematic review following the 2.1We identified search terms to describe wasting and stunting and the relationship between the two conditions, including implications for ponderal and linear growth. The search terms are listed in Figure 2.2We reviewed studies from low‐ and middle‐income countries (LMICs) where wasting and stunting are most prevalent. As wasting and stunting commonly occur in children under 5 years of age, we applied age limits from 0 to 59 months. We considered studies in the review that assessed both ponderal growth/wasting and linear growth/stunting as well as the association between the two. Included studies focused on prevalence, physiological mechanisms and outcomes related to growth and mortality. We included all types of studies that involved primary research . We also included reviews if they presented pooled analysis or new insights into the relationship between wasting and stunting. Both peer‐reviewed papers and grey literature identified through the search were considered for inclusion.We excluded studies that assessed wasting and stunting separately and that did not report on either condition in relation to the other. Also excluded were studies that focused on obesity, micronutrients, those that included children over 5 years of age and published abstracts and viewpoints.Z score of the reference), stunting as low height‐for‐age (HAZ) (<−2 Z score of the reference) and concurrent wasting and stunting as both low WHZ and HAZ at the same time (<−2 Z score WHZ and <−2 Z score HAZ). However, in literature referring to treatment programmes targeting wasting, standard WHO mid‐upper‐arm circumference (MUAC) criteria for defining wasting may also be included. We have endeavoured to specify literature for which the latter is the case.In most of the literature reviewed, wasting is defined as low weight‐for‐length/height (WLZ/WHZ)  and duplicates removed. Initial screening of titles and abstracts was conducted by ST to identify studies unrelated to the scope of the review. The remaining studies were then independently screened by ST and NS by reading the full texts. Discrepancies were resolved via discussion and, where necessary, a third reviewer (TK) was consulted. A data extraction template was developed in Excel, piloted and reviewed by members of the research team before full extraction took place.We identified three main themes before extraction: physiological understanding of the similarities in wasting and stunting, the interrelationship between the two conditions and the implications of this relationship and then extracted data along these lines. We extracted data into an Excel spreadsheet including information on authors, titles, dates of publication, sample size, data/information relevant to each theme and any research recommendations and conclusions.2.4https://www.sign.ac.uk/what-we-do/methodology/checklists/). We selected the SIGN checklists as they provide a checklist of items for case–control and cohort studies, study designs commonly used in the studies selected for this review. Adaptation was necessary due to the varied nature of the studies included. We assessed factors such as clearly defined objectives, study design, definition of participants, exposures and outcomes, statistical methods, addressing of bias and potential confounders and the presentation of results.We assessed the quality of included studies using an adapted version of the SIGN checklists . These included 21 cross sectional and 18 longitudinal studies. In total, 14 countries were covered in studies and reports conducted in single countries while 18 studies covered multiple countries—the largest analysis covering 84 countries  can be regained or maintained during nutritional stress at the expense of linear (height/length) growth  at 18–24 months if no further wasting was experienced after that time, suggesting that one episode of wasting in this age group is not enough to slow/halt linear growth. However, wasting after 6 months of age, and greater variability in WLZ in the first 17 months of life, was associated with lower LAZ at 18–24 months. Seasonal evidence also suggests that wasting is associated with further wasting whereby infants who were wasted in the first wet season of their life were more likely to be wasted in their second wet season, even after controlling for whether they were wasted during the intervening dry season , emphasising the increased vulnerability of children growing up in FCAS countries , higher rates of non‐response  but greater weight gain . MUAC gain did not differ between groups    for 378 children after SAM treatment including linear growth  and which lead to physiological and development stresses with consequences for body composition and physiological function. Although the evidence is growing and compelling, questions remain around the physiological mechanisms linking wasting and stunting and further research is warranted, particularly to better understand the critical junctures for halting the accumulation of vulnerabilities that are created as these processes interact. Some evidence suggests that, in addition to muscle and fat loss and hormonal imbalances, stunted children show deficits in the form of small organ size (Wells, Devakumar, et al., We have reviewed evidence that demonstrates that wasting can lead to stunting and, to a lesser extent, stunting can increase the risk of wasting. The former direction is supported by evidence of a mechanism whereby adequate weight and the absence and/or management of underlying morbidities is needed before linear growth can take place Garenne, . PreviouChildren identified as concurrently wasted and stunted have a dual burden of impact on body composition, which might explain the high risk of mortality associated with having both conditions. The cumulative increased risk of death from concurrent wasting and stunting undermines any rationale for different interventions addressing separate forms of undernutrition. Instead, treatment strategies need to shift focus to consider risk of death as paramount to targeting rather than specific categories of anthropometric cut‐offs to define wasting while working alongside prevention. Targeting interventions by season or by population subgroups defined by sex, socio‐economic status, maternal and child birth characteristics might help to focus preventive interventions to reduce the burden of postnatal growth failure (Mertens, Benjamin‐Chung, Colford, Coyle, et al., Most of the prevalence studies in this review reported wasting, stunting and concurrent wasting and stunting as point prevalence. We have presented evidence that demonstrates problems in the underestimation of the actual burden of wasting as children can move in and out of periods of this acute condition throughout the year. Wells, Briend, et al.  argue thOne of the secondary findings from the work identified in this review is higher prevalence of concurrent wasting and stunting in males. Although the concept of higher levels of male undernutrition is not new, the work here has renewed interest in understanding the reasons for these differences. A recent systematic review of sex differences in undernutrition showed that boys are more likely to be wasted, stunted and underweight compared with girls (Thurstans et al., The findings regarding response to SAM treatment for children who are both wasted and stunted are inconsistent. Overall, evidence suggests that outcomes are suboptimal for children with concurrent wasting and stunting. Where positive treatment outcomes are reported, this might be reflective of survivor bias. What is clear is that TFPs need to be optimised to identify most at‐risk children including those who are concurrently wasted and stunted (Bergeron & Castleman, Z score at birth were each positively associated with increased risk of mortality during the first year of life (Mwangome et al., This review highlights findings that support further operational research into the anthropometric identification and assessment of undernutrition. Evidence shows that MUAC and WAZ are the best measures to identify mortality risk (Myatt et al., The findings in this review on the peak timing of wasting and stunting suggest that policy and practice need to address undernutrition in a way that considers the life cycle of undernutrition, from preventative interventions targeted to women of reproductive age through to treatment when undernutrition occurs in the young child. A significant degree of child undernutrition is established before birth (Christian et al., The strength of the evidence has come a long way since the original review in 2014, and continued robust research on the priorities laid out above will be key to furthering our understanding of the relationship between wasting and stunting. This would serve to better prioritise prevention and treatment‐focused interventions in all contexts where undernutrition is a concern.The strength of this review lies in the systematic approach taken, but we recognise some limitations. Our search strategy might have introduced some bias in the literature that we included. The findings have demonstrated the overlap in wasting and stunting and the utility of measures of underweight in capturing this. As we did not include the term ‘underweight’ in our search, there is a chance that we missed relevant literature pertaining to underweight only. However, papers would only have been included if they also mentioned wasting and stunting and so should have been identified by the search. We also do not feel that the overall message that a child should be viewed in a more holistic way would be changed. Similarly, our findings demonstrate that, in many instances, children are born wasted and/or stunted and therefore LBW. While we did not include the terms ‘low‐birthweight’ or ‘preterm’ in our search, we have presented research that highlights the need for prevention through maternal and newborn interventions. Finally, our search may have had reduced sensitivity with the limits we applied related to relationship and association. However, we felt this was necessary to manage the large quantity of literature related to both wasting and stunting given our interest in the relationship between the two.For all of the above limitations, we feel our request to members of the WaSt TIG SWG to highlight relevant additional literature has contributed to minimising the effects given their expertise and ongoing work in the field of maternal and child health and nutrition. We recognise the limitations of cross‐sectional data throughout the text, and this is particularly relevant for assessing causal associations and incidence. In terms of associations, all cross‐sectional evidence that we have presented is supported by longitudinal data, providing robust support to the findings. Finally, there might also be a risk of survivor bias in included studies, particularly those related to long‐term outcomes of wasting and stunting.5The ongoing accumulation of evidence since the 2014 review demonstrates progress in improving the understanding and awareness of the relationship between wasting and stunting. The findings of this review are supportive of a strong relationship between these two manifestations of undernutrition and provide a better understanding of which groups should be considered at risk and therefore prioritised for treatment.Evidence on the cumulative effects of nutritional deficits, and therefore risk over the life course of a child beginning in‐utero, demonstrates the need for a more integrated approach to prevention and treatment strategies in order to interrupt this process. To achieve this, further progress is needed to overcome the divide that has typified undernutrition policy, programme, financing and research initiatives.The authors declare that we have no conflict of interest.ST led in the development of the study protocol which was reviewed by TK and CD. ST conducted the search with a second review by NS. ST led in the analysis and the write up of the manuscript with regular contributions from all authors. All authors have read and approved the final manuscript.


The ceramide transport protein (CERT) delivers ceramide from the endoplasmic reticulum (ER) to the Golgi apparatus, where ceramide is converted to sphingomyelin (SM). The function of CERT is regulated in two distinct phosphorylation-dependent events: multiple phosphorylations in a serine-repeat motif (SRM) and phosphorylation of serine 315 residue (S315). Pharmacological inhibition of SM biosynthesis results in an increase in SRM-dephosphorylated CERT, which serves as an activated form, and an enhanced phosphorylation of S315, which augments the binding of CERT to ER-resident VAMP-associated protein (VAP), inducing the full activation of CERT to operate at the ER–Golgi membrane contact sites (MCSs). However, it remains unclear whether the two phosphorylation-dependent regulatory events always occur coordinately. Here, we describe that hyperosmotic stress induces S315 phosphorylation without affecting the SRM-phosphorylation state. Under hyperosmotic conditions, the binding of CERT with VAP-A is enhanced in an S315 phosphorylation-dependent manner, and this increased binding occurs throughout the ER rather than restrictedly at the ER–Golgi MCSs. Moreover, we found that de novo synthesis of SM with very-long acyl chains preferentially increases via a CERT-independent mechanism under hyperosmotic-stressed cells, providing an insight into a CERT-independent ceramide transport pathway for de novo synthesis of SM. Intracellular trafficking of lipids is an essential event for the metabolism of lipids and membrane biogenesis in cells. In eukaryotic cells, the endoplasmic reticulum (ER) is the center of de novo synthesis of various lipid types, and lipid transport mediated by lipid transfer proteins (LTPs) at organelle membrane contact sites (MCSs) is the predominant mechanism to transport lipid from the ER to other organelles ,2. HowevIn the synthesis of sphingomyelin (SM) in the Golgi apparatus, the ceramide transport protein (CERT), a typical LTP, delivers the precursor ceramide synthesized in the ER to the Golgi apparatus, where ceramide is converted to SM . CERT coThe function of CERT is regulated in at least two distinct phosphorylation-dependent events. One of the phosphorylation sites is a serine-repeat motif (SRM), which resides downstream of the PH domain. Once the first serine (S132) in the SRM is phosphorylated by protein kinase D , the subIn the present study, we found that hyperosmotic stress induces the phosphorylation of CERT S315 residue while the phosphorylation state of SRM remains multiply phosphorylated, which indicates that the two phosphorylation events can occur independently. Under hyperosmotic conditions, the interaction of CERT with VAP-A is enhanced in a CERT S315 phosphorylation-dependent manner, and this increased binding occurs throughout the ER rather than at the ER –Golgi MCSs. In addition, we unexpectedly found that de novo synthesis of SM with very-long acyl chains increases in a CERT-independent manner in hyperosmotic stressed cells, providing a feature of a CERT-independent ceramide transport pathway for de novo synthesis of SM.In this study, to employ retroviral transfection, we used the HeLa mCAT#8 cell line, which stably expresses the mouse ecotropic retroviral receptor mCAT-1. We employed a HeLa CERT knockout (KO) cell line to prevent any interference with the endogenous wild-type (WT) CERT. In addition, when various CERT variants were expressed, CERT KO/shCERT cells that stably express a short hairpin RNA matching to human CERT were used as the parent HeLa KO cells to prevent overexpression artifacts unless otherwise noted. Using the cell system, we could control the protein expression levels of CERT variants in the CERT KO background near to the endogenous CERT level in wild-type HeLa cells A. To exaA previous study using CERT-overexpressing HeLa cells showed that when cells were treated with ISP-1/myriocin, a potent inhibitor of serine palmitoyltransferase , S315 of HA-CERT WT was shifted to be phosphorylated . In agreWe next examined whether the elevation of CERT S315 phosphorylation depended on the strength and duration of hyperosmotic stress. When cells were treated with various concentrations (100–800 mM) of sorbitol, and for different time periods, the phosphorylation states of the SRM were not altered F. In conThe osmolarity of the medium supplemented with 800 mM sorbitol is about 1100 mOsm, which should burden cells with severe hyperosmotic stress. This invoked the concern that the enhanced S315 phosphorylation was simply an irreversible event occurring in dying cells. However, the S315 phosphorylation elevation was found to be reversible A: When cWe also tested whether the phosphorylation states of CERT were responsive to hypoosmotic stress. When cells were exposed to a 1.5-fold diluted culture medium for various time periods, the states of multiply phosphorylated SRM and the S315 phosphorylation in CERT were unchanged B. A prevN-terminal mVenus-tag were ectopically expressed within the parental HeLa KO cell line  (DSP) to stabilize the CERT–VAP complex. Then, cell lysate treated with a mild detergent was subjected to co-immunoprecipitation with anti-GFP monoclonal antibody. A larger amount of VAP-A was co-precipitated with mVenus-CERT S315E compared with the CERT WT control  to assess the interaction between CERT and VAP-A. PLA detects the physical closeness between two proteins within 40 nm of each other as spot-like signals ,13. Althcis-Golgi marker GM130, or the trans-Golgi network marker TGN46, were analyzed by coimmunostaining. We observed no marked differences in the subcellular distribution of these Golgi markers between the osmotically stressed and the isosmotic control HeLa cells , and the lower bands represent SM with long N-acyl chain lengths (16 or 18 carbons). Under hyperosmotic conditions, the upper bands of SM were preferentially produced in the metabolic labeling -sphingosine in the presence of C24:1- or C16:0-acyl CoA, and the quantities of d7-sphingosine-labeled C24:1- and C16:0-ceramides were determined by LC–MS. The membrane fractions from parental HeLa cells and CERT KO cells exhibited a hyperosmotic stimulus-dependent increase in the synthesis of C24:1-ceramide, while no synthesis of C24:1-ceramide was detected in CERS2 KO cell-derived membranes (Acyl chain lengths of de novo synthesized SM are determined in the step of ceramide production. There are six isozymes in ceramide synthase CERS1-6) that exhibit different substrate specificity with distinct lengths of acyl-CoA, and CERS2 mainly catalyzes the formation of ceramide species with very-long acyl chains in HeLa cells . To eluc that exhembranes C. On theembranes D. These cis/medial Golgi regions in mammalian cells -serine  for 1 h under various osmotic conditions. The relative values of each lipid are presented as the ratios of the band intensities normalized to the amounts of proteins used in each condition. The sum of the ratios in each condition was normalized to 12 , the supernatant fraction was centrifuged . The obtained participant, referred to as the membrane fraction, was suspended in cell suspension buffer and used as the enzyme source for the in vitro ceramide synthesis assay as follows. The membrane fraction (40–50 µg protein) was incubated with 5 µM deuterium-labeled (d7)-sphingosine  and 25 µM C24:1- acyl CoA  or C16:0-acyl CoA  in 100 µL of the reaction buffer (the cell suspension buffer containing 2 mM MgCl2 and 0.1% digitonin) for 30 min at 37 °C. Following the addition of 1 nmol of C17 ceramide  to the reaction mixture as an internal standard for LC–MS/MS, lipids were extracted using Bligh and Dyer’s method [m/z = 545.6–271.4 and 655.6–271.4, respectively. Each ion pair of the molecular species in MRM was monitored for 30 ms with a resolution of unit. The contents of individual ceramide were calculated by the peak area of analyte to the peak area of the internal standard. Data acquisition and analysis were performed using Analyst Software version 1.6. (SCIEX). The sum of the signals of each condition was normalized to 6  in each experiment.HeLa cells were suspended in cell suspension buffer (50 mM HEPES-NaOH (pH 7.4), 150 mM NaCl, 10% glycerol, 1 mM DTT, 1% Phosphatase Inhibitor Cocktail 2 (Sigma-Aldrich), 1% Phosphatase Inhibitor Cocktail 3 (Sigma-Aldrich), and cOmplete™ Protease Inhibitor Cocktail (Roche)) and then lysed by sonication. After removing the cell debris from the lysate by centrifugation (300× s method , and thes method . The levt-test was used for two-group comparisons, setting p < 0.05 as a statistical significance criterion.Statistical analyses were carried out using R software. The Student’s


ErbBs are receptor tyrosine kinases involved not only in development, but also in a wide variety of diseases, particularly cancer. Their extracellular, transmembrane, juxtamembrane, and kinase folded domains were described extensively over the past 20 years, structurally and functionally. However, their whole C-terminal tails (CTs) following the kinase domain were only described at atomic resolution in the last 4 years. They were shown to be intrinsically disordered. The CTs are known to be tyrosine-phosphorylated when the activated homo- or hetero-dimers of ErbBs are formed. Their phosphorylation triggers interaction with phosphotyrosine binding (PTB) or Src Homology 2 (SH2) domains and activates several signaling pathways controling cellular motility, proliferation, adhesion, and apoptosis. Beyond this passive role of phosphorylated domain and site display for partners, recent structural and function studies unveiled active roles in regulation of phosphorylation and interaction: the CT regulates activity of the kinase domain; different phosphorylation states have different compaction levels, potentially modulating the succession of phosphorylation events; and prolines have an important role in structure, dynamics, and possibly regulatory interactions. Here, we review both the canonical role of the disordered CT domains of ErbBs as phosphotyrosine display domains and the recent findings that expand the known range of their regulation functions linked to specific structural and dynamic features. ErbB1/EGFR/HER1, ErbB2/neu/HER2, ErbB3/HER3, ErbB4/HER4. ErbB receptors ensure a wide range of roles in development and adult organ function [Receptor tyrosine kinases (RTKs) are key players in cell signaling, and are essential for cell proliferation, motility, differentiation, and apoptosis . ErbBs ffunction , and knofunction . At the function ,8, but afunction , motilitfunction ,11, and function . They wefunction . Misregufunction ,15, and function . Additiofunction , as wellfunction .ErbBs, like all RTKs, are made up of an extracellular domain (ECD) that generally binds ligands, a transmembrane helix (TMH), an intracellular juxtamembrane region (JM), a tyrosine kinase domain (TKD), and a C-terminal tail (CT), sometimes called regulatory domain, that undergoes autophosphorylation of its tyrosines to interact with downstream partners . This stIn this review, we first contextualize the role of the CT of ErbBs in the different steps of signal transduction, and review their role as phosphorylation and interaction domains. We then review structural and functional information that was gathered these last few years that revisit their role beyond passive phosphotyrosine anchors, as active regulators of signal transduction. Finally, we compare CTs of ErbBs to the tails of other RTKs, showing their specificities and possible common regulation functions.EGFR is often used as the canonical ErbB, and ErbB2 is also widely studied due to its connection to breast cancer . DespiteFrom the evolutionary point of view, it was found that in invertebrates there is only one ErbB-like protein. Following three steps of gene duplication , the four distinct ErbBs appeared in vertebrates . AlthougThe numbering used in this review is given in Given their implication in both important physiological functions and cancer, a lot of effort was put into understanding the mechanism of signal transduction by ErbBs. Structural studies were key to this understanding, and each step of signal transduction down to the kinase domain was determined or confirmed with crystal and/or NMR structures. No high-resolution structure of a full-length ErbB exists, so the series of steps were inferred from fragments of the receptors and connected to functional data. A full-length model, lacking only the CT domain, was reconstituted by Jura et al. , and is Briefly, ligand binding to ErbB1, ErbB3 or ErbB4 ECD is followed by a conformational change that enables formation of active homo- or hetero-dimers of the receptors. ErbB2, on the contrary, does not bind any known ligand and is constitutively poised to dimerize. Dimerization enables the activation of the intracellular TKD of at least one monomer of the dimer and then leads to tyrosine phosphorylation of the CT. Finally, the phosphorylated tails serve as anchors for interaction with adaptor proteins that trigger signaling. A schematic view of the steps is given in Understanding the general mechanism of signal transduction by ErbB receptors is crucial to understand the role of their C-terminal tails, their different states , and their direct or indirect interactions with the other domains. Therefore, we briefly review all the steps below, before focusing on the CT. A more extensive review of these steps was written by Lemmon, Schlessinger, and Ferguson . We discErbBs ECD is composed of about 630 residues divided into four domains see B,D. CrysIn addition to this ligand-induced dimerization model, a ligand-induced activation of inactive dimers was also proposed ,47, in tYarden and Schlessinger, two key investigators of ErbBs since their discovery, determined that dimerization of the ECD itself is not sufficient for ErbB receptors signal transduction, and that other intermolecular interactions are required . In partFurthermore, mutations in the TMH are involved in overactivation of ErbBs and diseases . The oritrans-tyrosine phosphorylation of the kinase or juxtamembrane domains, which dramatically increases kinase activity by changing the conformation of the activation loop [In many RTKs, monomeric kinase activity is low. Dimerization of the receptors causes ion loop . Howeverion loop  showed tion loop  showed aion loop .A closer look at the crystal lattice of the active EGFR , and mutThe mechanisms determining the relative position of each kinase (receiver/activator) is not clear. It was proposed that it is dependent on the binding of ligand to the ECD of each monomer , but othThe C-terminal half of the juxtamembrane region (JMB) was shown to be important for kinase activity , throughtrans-autophosphorylation (phosphorylation of the tail of one ErbB monomer by the kinase of the other ErbB of the dimer) is efficient. The coexistence of trans-autophosphorylation with cis-autophosphorylation (phosphorylation by its very own kinase) was, however, never ruled out. The prevalence of trans-autophosphorylation is also consistent with the sightly higher level of phosphorylation in the tail of the activator monomer compared to that of the tail of the receiver monomer in an asymmetric dimer [From the observation that ErbB3 tail can be phosphorylated even though it is kinase dead, and other experiments with kinase-dead variants , trans-aic dimer .All the conformational changes from the extracellular domains to the kinase domain eventually lead to phosphorylation of the CT. This is the final and decisive step that triggers the interaction with different partners, each starting a different signaling pathway and determining cell fate.At the end of the chain, the role of the CT in ErbB receptors is to ensure signal transduction through interactions of phosphorylated tyrosines with signaling proteins (containing mainly SH2 and PTB domains) that are shown in Similarly, five autophosphorylation sites (one containing two adjacent tyrosines) out of 9 tyrosines were identified in ErbB2 ,82: Y102 partner .The phosphotyrosine interaction sites of ErbBs include autophosphorylation sites, but also some tyrosines that can be phosphorylated by other kinases. Partners of all these ErbB phosphotyrosines include Grb2 and Shc (MAP kinase and Akt pathways), PI3K (Akt pathway), Src and STAT5 (MAPK and Akt pathways) ,84. In pThe few experimental structures that exist of proteins bound to ErbB phosphotyrosine peptides are shown in CT-ErbBs ,89.In most studies, molecular details of the interactions of CT-ErbBs with downstream partners are studied using short peptides of ErbBs see . This leFrom the role of the CTs described above, as phosphorylation and interaction domains, they may be classified as “display sites”, as defined in the functional classification by van der Lee et al. : “flexibContrary to the extracellular and kinase domains, the C-terminal tails are poorly conserved amongst the four ErbB receptors: only 11 to 25% identity, compared to 59 to 81% identity for the kinase domain . HoweverThe difference of disorder prediction for ErbB2/ErbB3 compared to ErbB1/ErbB4 correlates with a significantly higher glycine content in the latter pair, which probably affects flexibility substantially. The other parameters we looked at are in favor of an extended conformation of the chain. All ErbBs have a very high content in proline, the most disorder-promoting amino acid. Additionally, electrostatic repulsion might arise from the charge unbalance in favor of negatively charged amino acids, with a striking depletion in lysines, resulting in negative net charge and low pI values. Finally, CT-ErbBs are rich in tyrosines, which is expected from their nature of RTKs but relatively rare in IDPs, in which most commonly phosphorylated residues are serines and threonines.Tyrosines were shown to make up less than 1% of all phosphorylated residues in a cell . This isAn interesting study of the interdependence of phosphorylation sites was performed by Dankort et al., in the laboratory of Muller, on neu (the rat ErbB2). They studied in depth the autophosphorylation sites of neu ,106,107.utations ,106,107.observed . These dThe CT is believed to participate in the regulation of the activity of the kinase domain. Deletion of some parts of the CT modulates kinase activity. However, the results of the different studies ,110,111 Crystal structures of EGFR A show thThe AP-2 helix is followed by an extended, negatively charged part (1003–1014) called the “electrostatic hook” that interacts with the C-lobe of the same monomer in the inactive conformation. There is therefore a competition, shown in e kinase . Molecule kinase , as welle kinase  support e kinase . The negFrom structural data, the first part of the tail seems to prevent formation of the active asymmetric dimer, which is consistent with the study by Pines et al. that shows that deletion of the first 70 to 80 residues of the CT leads to constitutive dimerization, increase in kinase activity and activation of signaling pathways . These dA study using EGFR-ErbB3 chimera demonstrated that the inhibition of the kinase was C-terminal sequence dependent, since ErbB3 tail grafted on ErbB1 kinase domain did not induce kinase inhibition, contrary to the nonchimeric kinase-CT variants .The C-terminal tail of ErbBs was shown to have a crucial role in the receptor internalization for recycling or degradation, since deleting it  or createlm.eu.org [These internalization mechanisms are linked to the sequence and structure of ErbB tails. A prediction of Eukaryotic Linear Motifs  complex  that is The phosphorylation of serines and threonines of CT-EGFR was linked to receptor trafficking. Tong et al. studied phosphorylation patterns in model systems reproducing the conditions after EGF stimulation and before or after internalization. They showed that many serines/threonines were highly phosphorylated after EGF stimulation and EGFR internalization, or after stress induced by anisomycin . In partPhosphotyrosine sites are also involved in receptor trafficking, for example with Grb2 binding the tail of EGFR, recruiting Cbl for ubiquitination and subsequent internalization in CCPs . FinallyAs receptor oligomerization is crucial for ErbB activation, the regulation of their local concentration is of interest. Liquid-liquid phase separation (LLPS) is emerging as a major regulation mechanism in the cell, especially for IDPs. Can the sequence properties of CtErbB2 drive local LLPS, and clustering of the receptors in the membrane? Indeed, a high concentration of tyrosines, arginines  or glyciIn our NMR study of isolated CT-ErbB2 , we did eceptors . When thA lot of structural information is available on the CT in the regions where it is in close interaction with the kinase domain, and is therefore seen in crystal structures. That is the case up to residue 1016 of EGFR. However, there is still about 200 residues with unknown conformation, except in very short segments in interaction shown in Keppel et al. were the first ones to show experimentally that the tails of EGFR and ErbB3 are indeed disordered, using a wide variety of biophysical techniques . They usOur group investigated CT-ErbB2 in more depth at the local level in the unphosphorylated state using NMR ,30. We sshown in  for EGFRUpon phosphorylation , the CD spectrum of CT-EGFR was slightly modified, and suggests local and/or small degree of loss of structure ,29,35.In the unphosphorylated state, Keppel et al. showed that isolated CT-EGFR and CT-ErbB3 were rather extended, consistent with their disordered nature . HoweverThe first long-range contact to be noted in CT-EGFR is the one detected with the kinase core. Fluorescence studies showed that this contact was partially disrupted upon autophosphorylation of the intracellular domain , which lThe contacts identified within CT-EGFR and CT-ErbB2 both bring regions that are separated by around 200 residues closer. The exact nature of these contacts is not obvious from amino-acid sequence, and the absence of salt-concentration dependence excludes electrostatic interactions in EGFR . CompariThe whole activation mechanism of ErbBs is strikingly different from the prototypical mechanism for RTKs. Firstly, the ligand-induced dimerization of ErbBs is solely receptor-mediated, meaning that the ligands do not participate in the dimerization interface necessary for activation of the receptors ,140. AmoThe beginning of the CT (including the AP-2 helix region and the electrostatic hook) of ErbBs was shown to be well-conserved amongst ErbBs, to have co-evolved with regions of the kinase domain, and to have diverged from other RTKs , which iStudies of interaction motifs in CTs of RTKs have focused on phosphotyrosines. Could it be that other motifs were missed? The unusually high content of prolines in ErbBs tail  domain could bind proline-rich segments of FGFR2 , not onlgulation . Very regulation .ErbBs are a beautiful example of how structural biology, with the successive crystallization of the different domains of the receptors, can help understand the molecular mechanism governing the function of a complex protein with multistep activation. CT-ErbBs are also a prototypical example of how IDRs were ignored in such studies for decades. CT-ErbBs have features expected from IDRs involved in signaling and regulation: they have multiple phosphorylation sites embedded in interaction motifs and a lot of partners that can bind to different motifs or compete for the same one.Biochemical and structural studies of CT-ErbBs also highlight specificities that are still overlooked in IDPs and IDRs, and are still very open questions that the studies reviewed here only started to answer: the role of prolines on conformation; the role of LLPS phenomena in receptor clustering; the regulation of (multiple) tyrosine phosphorylation and its conformational impact; the interaction between IDRs and folded domains of the same protein; and finally, the direct or indirect interaction  between different motifs of the IDR, be it for post-translational modification or interaction with partners, which can only be studied by using full-length protein variants and not short peptides. These new questions are condensed in the summary These questions apply to ErbBs, other RTKs, but also other types of receptors. More and more publications studying intrinsic disorder in receptors were published recently, for example on the N-methyl-D-aspartate (NMDA) receptor , class 1


It is generally accepted that the number of neurons in a given brain area far exceeds the number of neurons needed to carry any specific function controlled by that area. For example, motor areas of the human brain contain tens of millions of neurons that control the activation of tens or at most hundreds of muscles. This massive redundancy implies the covariation of many neurons, which constrains the population activity to a low-dimensional manifold within the space of all possible patterns of neural activity. To gain a conceptual understanding of the complexity of the neural activity within a manifold, it is useful to estimate its dimensionality, which quantifies the number of degrees of freedom required to describe the observed population activity without significant information loss. While there are many algorithms for dimensionality estimation, we do not know which are well suited for analyzing neural activity. The objective of this study was to evaluate the efficacy of several representative algorithms for estimating the dimensionality of linearly and nonlinearly embedded data. We generated synthetic neural recordings with known intrinsic dimensionality and used them to test the algorithms’ accuracy and robustness. We emulated some of the important challenges associated with experimental data by adding noise, altering the nature of the embedding of the low-dimensional manifold within the high-dimensional recordings, varying the dimensionality of the manifold, and limiting the amount of available data. We demonstrated that linear algorithms overestimate the dimensionality of nonlinear, noise-free data. In cases of high noise, most algorithms overestimated the dimensionality. We thus developed a denoising algorithm based on deep learning, the “Joint Autoencoder”, which significantly improved subsequent dimensionality estimation. Critically, we found that all algorithms failed when the intrinsic dimensionality was high (above 20) or when the amount of data used for estimation was low. Based on the challenges we observed, we formulated a pipeline for estimating the dimensionality of experimental neural data. The number of neurons that we can simultaneously record from has increased exponentially for decades; today that number is in the thousands of neurons. However, the individual firing rates are highly redundant. One approach to identifying important features from redundant data is to estimate the dimensionality of the neural recordings, which represents the number of degrees of freedom required to describe the data without significant information loss. Better understanding of dimensionality may also uncover the mechanisms of computation within a neural circuit. Circuits carrying out complex computations might be higher-dimensional than those carrying out simpler computations. Typically, studies have quantified neural dimensionality using one of several available methods reflecting a lack of consensus on which method would be most appropriate for neural data. In this work, we used several methods to investigate the accuracy of simulated neural data with properties mimicking those of actual neural recordings. Based on these results, we devised an analysis pipeline to estimate the dimensionality of neural recordings. Our work will allow scientists to extract informative features from a large number of highly redundant neurons, as well as quantify the complexity of information encoded by these neurons. Studies that simultaneously record the activity of many neurons have shown that cortical neural activity is highly redundant . In primUnfortunately, it is surprisingly difficult to estimate the dimensionality of neural manifolds, particularly in the realistic condition of a noisy, nonlinear embedding. There is evidence of a nonlinear mapping between the recorded neural activity and the associated low-dimensional latent signals ,25–27. NThe methods that have been proposed for estimating the dimensionality of neural manifolds can be broadly categorized into linear or nonlinear algorithms, based on assumptions about the nature of the mapping between the low-dimensional representation of the latent signals and the high-dimensional space of neural activity. The most commonly used linear method for dimensionality reduction is Principal Component Analysis (PCA), based on identifying mutually orthogonal directions in the empirical neural space of recorded activity; these directions are monotonically associated with the largest data variance. PCA provides a hierarchical description in which the data projected onto the manifold subtended by the leading principal components become closer and closer to the recorded data as the dimensionality of the linear manifold is increased towards the dimensionality of the empirical neural space. Although PCA provides a useful and systematic tool for variance-based dimensionality reduction, it does not specify how to uniquely identify the dimensionality of the manifold: the typical implementation requires the choice of an arbitrary variance threshold. Other PCA-based algorithms such as Participation Ratio (PR) ,18 and PLinear dimensionality estimation algorithms may work well for linear datasets, but are likely to overestimate the dimensionality of a manifold arising from a nonlinear mapping between the low-and high-dimensional spaces ,21,33,34Most dimensionality estimation methods have been tested in the absence of noise even though it is known that linear and nonlinear methods overestimate dimensionality when the data is noisy . The robThe objective of this study was to characterize the accuracy of several dimensionality estimation algorithms when applied to high-dimensional recordings of neural activity. We evaluated previously proposed algorithms on synthetic datasets of known dimensionality to identify conditions under which each method succeeded and/or failed. Specifically, we evaluated how the algorithms handled the nature of the embedding (linear or nonlinear), the amount of noise added to the simulated neural data, and the amount of data available. We found increasing levels of noise to be a challenge for all tested algorithms. We therefore also evaluated different approaches for reducing noise prior to performing dimensionality estimation, including the “Joint Autoencoder”, a method we developed based on deep learning techniques. Together, our results allowed us to propose a methodological pipeline for estimating the intrinsic dimensionality of high-dimensional datasets of recorded neural activity.All surgical and experimental procedures that yielded the multi-electrode array recordings from non-human primates , which fd signals by randomly selecting (d x M) samples from an empirical distribution of firing rates that we obtained from multi-electrode array recordings of neural activity in the macaque primary motor cortex (M1) made while the monkey was performing a center-out task  interval, thus imposing a large degree of variance similarity across channels. In contrast, variances of real neural recordings can vary as much as 10-fold from channel to channel. To evaluate the performance of the dimensionality estimation algorithms considered here in the presence of non-uniform variances across channels, we scaled each channel of simulated neural data by a randomly chosen real number between 1 and 10. We found that most algorithms yielded lower estimates of dimensionality when applied to the rescaled data in comparison to the estimates obtained when the algorithms were applied to the data before rescaling Because of the superior accuracy of Levina-Bickel Maximum Likelihood Estimation and Two Nearest Neighbors, we focused on these two methods for the remainder of the nonlinear analyses. We also retained Parallel Analysis as a benchmark for some of the analyses, as it was the most accurate linear method for estimating the dimensionality of nonlinearly embedded data.d < 20, but inaccurate on nonlinear datasets of all dimensions, as expected . Below about d = 6, Levina-Bickel Maximum Likelihood Estimation and Two-Nearest Neighbors were accurate on both linear and nonlinear datasets. However, Levina-Bickel Maximum Likelihood Estimation began to underestimate the dimensionality of both linearly embedded  and nonlinearly embedded  datasets for d > 6. This underestimation increased with increasing d. For nonlinear datasets, the estimate saturated at d = 13, where underestimation began to get much worse. These results revealed that the intrinsic dimensionality of nonlinearly embedded datasets is hard to estimate reliably when it is large.We next evaluated how the true intrinsic dimensionality of the noise-free data influenced algorithm accuracy. Can any intrinsic dimensionality be reliably estimated? We found that the answer is no: the accuracy of all algorithms suffered when the intrinsic dimensionality of the synthetic data was too high. Parallel Analysis was accurate on linear datasets with α in Fig 6). Surprisingly, even Parallel Analysis was accurate up to levels of nonlinearity corresponding to α≈8, where it started to overestimate the intrinsic dimensionality. These results revealed that Levina-Bickel Maximum Likelihood Estimation and Two Nearest Neighbors provide accurate dimensionality estimates for wide levels of nonlinearity, whereas Parallel Analysis is accurate only for low levels of nonlinearity.We next evaluated how the degree of nonlinearity influenced the accuracy of the dimensionality estimation algorithms. We controlled the degree of nonlinearity by varying the parameter d = 6, by varying both the number of samples M and the number of recording channels N.Ideally, algorithms would require only small amounts of data, so that the intrinsic dimensionality could be estimated even during transient behaviors and for a small number of recording channels. We thus evaluated the amount of data required to estimate the dimensionality of datasets with Fig 7A). In contrast, the accuracy of both Levina-Bickel Maximum Likelihood Estimation and Two Nearest Neighbors also depended on the number of samples . Around M = 600, requiring about 30 seconds of data binned at 50 ms, was sufficient for accurate estimates of intrinsic dimensionality using either of these two nonlinear methods.On linear datasets, the accuracy of Parallel Analysis depended only on the number of channels: the algorithm was accurate if 20 or more channels were available , Parallel Analysis was not accurate  regardless of the amount of data. Both Levina-Bickel Maximum Likelihood Estimation and Two Nearest Neighbors were accurate provided that data from more than 50 channels were available . Furthermore, while Levina-Bickel Maximum Likelihood estimation required around 600 samples of data for accurate dimensionality estimates, Two Nearest Neighbors required more than twice as many samples. These results would also depend on the actual dimensionality d; here we focused on d = 6.As expected for highly nonlinear datasets . For any given noise level, estimation errors for the linear datasets  were a bit smaller than those for the nonlinear datasets . Adding noise with a power of only 1% of that of the signal (SNR = 20 dB) caused Levina-Bickel Maximum Likelihood Estimation and Two Nearest Neighbors to overestimate the dimensionality of the nonlinear data by ~200% . PA yielded consistent overestimation errors across all nonzero levels of noise for both linear and nonlinear data.Any experiment will include some amount of noise in the recorded signals. As expected, all tested algorithms overestimated intrinsic dimensionality in the presence of noise  caused the dimensionality to be overestimated by about 100%. These results were consistent for different degrees of nonlinearity. The more nonlinear the data, the more appropriate it was to use the Joint Autoencoder for denoising.We evaluated two algorithms for mitigating the effects of noise prior to estimating dimensionality: a PCA-based linear method and a Joint Autoencoder nonlinear neural network see . Both meThis study evaluated techniques for estimating the intrinsic dimensionality of high-dimensional neural recordings. We considered representative linear and nonlinear algorithms, testing their performance on synthetic datasets that captured properties of neural recordings likely to affect dimensionality estimation. The tested datasets had known intrinsic dimensionality, known levels of noise, and embeddings that were either linear or nonlinear. Our results demonstrated that none of the tested algorithms work for all possible scenarios, but they yielded important insights for when estimates of intrinsic dimensionality are likely to be valid and when they are not. As expected, we found that linear estimation methods are generally not as accurate as nonlinear methods when the mapping between the low-dimensional latent space and the high-dimensional space of neural recordings is nonlinear. Surprisingly, the linear method Parallel Analysis estimated the dimensionality of mildly nonlinear datasets well though it failed for more highly nonlinear embeddings. In contrast, the nonlinear methods worked well on both linear and highly nonlinear datasets but failed once the intrinsic dimensionality of the data became too high.Noise was a challenge for all methods, causing dimensionality to be overestimated even for signal-to-noise ratios as low as 20 dB (1% noise variance). We presented two approaches for denoising the data so as to improve the accuracy of the dimensionality estimation. These were a linear PCA-based approach and a novel nonlinear, deep learning approach that we call the Joint Autoencoder. Both denoising approaches attempted to remove signal components that were not shared across the data channels. To achieve this, the PCA-based approach simply removed Principal Components with low variance, whereas the Joint Autoencoder identified an underlying manifold that was common to two randomly sampled sets of channels. Both approaches relied on a linear, upper-bound estimate of the intrinsic dimensionality. Denoising by either method substantially improved subsequent dimensionality estimation, but the Joint Autoencoder was substantially more effective in denoising nonlinear datasets. For linear datasets, dimensionality estimates using Parallel Analysis, Levina-Bickel Maximum Likelihood Estimation, and Two Nearest Neighbors were accurate after PCA-denoising. In the nonlinear case, dimensionality estimates using the two nonlinear methods (LBMLE and TNN) were similarly accurate after JAE-denoising.Due to its computational efficiency and ease of interpretation, most studies have used PCA with an arbitrary variance cutoff to estimate the dimensionality of M1 neural recordings ,41,44,45Some of the linear methods used in neuroscience studies rely on a structure of repeated trials in the data ,46. ThesDespite the simplicity of linear algorithms, estimating the dimensionality of nonlinear manifolds requires nonlinear algorithms. There is some evidence that neural manifolds may be nonlinear. Recent studies have shown that nonlinear methods for inferring behavioral parameters from M1 neural manifolds are superior to linear methods –63. ThisNonlinear algorithms were more accurate than linear algorithms for nonlinear datasets of dimensionality below 10. However, nonlinear methods underestimated dimensionalities above 10. This is a critical concern for experimental recordings, since a low dimensionality estimate from a nonlinear method might be inaccurate if the true dimensionality were large. Multiple studies using linear methods have reported an estimated dimensionality of M1 of around 10 for simple, well-practiced behaviors ,45,59. OOne method for addressing this concern would be to use nonlinear methods to reduce the dimensionality of a dataset to that of its nonlinear dimensionality estimate, and then to assess the amount of variance that the nonlinear low-dimensional representation captures. If the VAF is high, the data may be truly nonlinearly low dimensional. If, on the other hand, the VAF is low, the true intrinsic dimensionality could be higher than estimated. For the latter case, a practical approach would be to report only the linear dimensionality estimate and emphasize that it only provides an upper bound to the true dimensionality.M be on the order of 10d/2  range. The arbitrary scaling of firing rates provided a simple means for the nonlinear datasets to have the same range as their linear counterparts, as the nonlinear activation function that we used mapped the  range onto itself. However, this modeling restriction does not reflect experimental neural firing data, since the range of neural firing can differ significantly even across neurons of the same type. We have illustrated how heterogeneity in the range of firing rates affects the reliability of dimension estimation algorithms. Soft-normalization approaches that are commonly used in neuroscience , would The latent signals used to generate simulated firing rates have the same first-order statistics as the actual data from which they were sampled, but the rescaling of simulated channel activity to a  range introduced a departure from realism. This unrealistic scenario is addressed through the random rescaling of individual simulated channels, to reflect the heterogeneity in the range of firing rates observed in actual neural recordings. The latent signals corresponding to the rescaled data no longer share common statistics. This scenario allows us to address an important problem: that low-variance latent signals can be informative . As demo. First, obtain an upper-bound estimate D of the intrinsic dimensionality of the data. We found that Parallel Analysis works well for this purpose, being both computationally efficient and the most accurate linear method in our tests. Next, the signals should be denoised. Our denoising approach worked by projecting the neural signals into a subspace of dimensionality D equal to the upper-bound dimensionality estimate, and then reconstructing them based on these projections. A PCA based reconstruction is easy to implement and interpret and may be preferable if computational efficiency is important. A nonlinear denoising algorithm, such as the Joint Autoencoder we proposed, should also be used to assess the degree of nonlinearity of the manifold. The usefulness of the denoising step was quantified through the VAF between the reconstructed signals, assumed to be denoised, and the noise-free synthetic signals before noise was added to them. Our results showed that for nonlinear datasets this VAF was higher for the Joint Autoencoder than it was for PCA. However, this VAF cannot be computed for experimental data, for which we do not have access to the noise-free signals. In this scenario, the reconstruction VAF between noisy inputs and the denoised reconstructed outputs may be useful for detecting nonlinear manifolds: a higher reconstruction VAF for Joint Autoencoder denoising than for PCA denoising would signal a nonlinear manifold. A reconstruction VAF that prefers the Joint Autoencoder indicates that this denoising method yields better denoised signals. Once the signals are denoised, and the linearity or nonlinearity of the manifold is established, either a linear or nonlinear dimensionality estimation method should be used depending on the comparative performance of the corresponding denoising algorithms. The most accurate linear method we tested was Parallel Analysis. Of the nonlinear methods, Levina-Bickel Maximum Likelihood Estimation and Two Nearest Neighbors were the most accurate; Levina-Bickel Maximum Likelihood Estimation required fewer data samples.Based on our results, we recommend the following approach for estimating the dimensionality of neural recordings Estimating the dimensionality of neural data is challenging. In this study, we tested several available algorithms and determined the conditions under which estimating dimensionality may be particularly difficult or even impractical. Noise is a confounding factor and must be removed prior to dimensionality estimation. Most existing studies have estimated intrinsic dimensionality using linear methods that are computationally efficient and easy to interpret. We showed that linear methods provide an upper-bound to the intrinsic dimensionality, and in cases of high noise, may even provide better estimates than nonlinear methods, although neither linear nor nonlinear methods will yield accurate estimates in this scenario. Nonlinear algorithms were more accurate for nonlinear datasets when noise was adequately removed. Finally, algorithms failed when the intrinsic dimensionality was high. It may be impractical or impossible to estimate the dimensionality of neural data when it is above ~20. However, estimation of the dimensionality of neural activity in the primary motor cortex may be possible, as many studies have reported its linear dimensionality to be within the practical limits for accurate estimation by the methods we tested.S1 Tabled = 6 for the intrinsic dimensionality of most of our simulated datasets.The parallel analysis (PA) estimates of the dimensionality are shown for each of the datasets J1, J2, and J3. These values determined the dimensionality to be used for denoising each dataset. The PCA-based denoising yielded reconstructions with 53%, 51%, and 56% VAF. The JAE-based denoising was slightly better for all datasets, with 61%, 59%, and 62% VAF. The better performance of the JAE-based denoising is indicative of modest nonlinearity in all three datasets. Once each dataset had been denoised using JAE, the corresponding dimensionalities were estimated using MLE and TNN. These results motivated our choice of (DOCX)Click here for additional data file.


Recently, there has been an increase in robotic-assisted spine fusion for degenerative spondylosis of the lumbar spine. We present the case of a 60-year-old female with grade 1 spondylolisthesis at L4/5 and L5/S1 who underwent L4-S1 anterior lumbar interbody fusion (ALIF) with percutaneous robotic-assisted pedicle screw fixation. We provide a detailed analysis of the procedure including the speed of robotic screw placement and pitfalls of this surgical approach. Lumbar spondylolisthesis is an anatomical spinal disorder involving the translation of one vertebral body on another which can cause symptoms of nerve root compression. The management of lumbar spondylolisthesis depends on the etiology, anatomical imaging characteristics, and patient presentation but typically involves lumbar spinal fusion -7. RecenThe patient is a 60-year-old female with a past medical history of sarcoidosis and breast cancer treated with surgical resection and chemoradiation that presented with a six-month history of low back pain and dysesthesia radiating down her right posterior thigh and intermittently in the anterior and medial thigh after falling. She denied any left-sided symptoms. She went from ambulating independently to using a walker. She experienced nighttime urinary incontinence due to pain. She failed conservative treatment with lumbar epidural steroid injections, physical therapy, and narcotic pain medications. On examination, she was grade 4+/5 Medical Research Council (MRC) strength in her right hip flexion and knee extension, and 4-/5 right dorsiflexion, plantar flexion, and extensor hallucis longus. She was full strength on the left with normal sensation.CT of the lumbar spine demonstrated no fractures. MRI of the lumbar spine demonstrated grade 1 spondylolisthesis of L4/5 and L5/S1 with nerve root compression in the neuro-foramen, right greater than left. Figures , 1B. TheGiven our patient’s unilateral symptomatology, grade 1 spondylolisthesis, and no significant central stenosis, she was offered an L4-5 and L5-S1 ALIF with percutaneous robotic-assisted posterior percutaneous lumbar spinal fusion. A vascular surgeon was used for abdominal access.Operative procedureBoth the anterior approach and posterior fusion were performed under general anesthesia. The patient was supine for the anterior approach. We utilized a vascular surgeon to gain retroperitoneal access to the anterior spine. A bent spinal needle was placed into the L4/5 and L5/S1 intervertebral disc space and fluoroscopy was utilized to ensure the placement was midline and at the correct lumbar level. The spinal needle was removed and the midline was marked at L4/5 and L5/S1.We began the ALIF with an annulotomy at L5/S1 along the implant sizer. A discectomy was performed to the level of the posterior longitudinal ligament (PLL). Fluoroscopy was utilized to gauge the depth of the discectomy. Prior to placement of the interbody, the retractor over the iliac vein dislodged, requiring repositioning. A minor injury to the common iliac vein occurred and was repaired under direct visualization by the vascular surgeon. Under fluoroscopic guidance, a 32x23x12mm 12º cage packed with allograft was placed into the L5/S1 disc space. This was secured with a 25-mm screw into the inferior endplate of L5. Fluoroscopy was utilized to ensure the appropriate placement of both the graft and the screw. At L4/5 we placed a 32x23x14mm 8º cage with allograft. This was anchored into the L4 inferior endplate with a 25-mm screw. Lateral and anterior-posterior (AP) fluoroscopic images were obtained and demonstrated good alignment of the grafts. The vascular surgeon then performed closure of the abdominal incision. It took one hour and 15 minutes from disc incision to graft placement for the L5/S1 level and 28 minutes from disc incision to graft placement at the L4/5 level.Robotic-assisted percutaneous posterior lumbar spinal fusionThe patient was then positioned prone on a Jackson table with her arms extended towards her head. Using fluoroscopy, we identified the posterior superior iliac spine (PSIS) to mark the location for Schanz pin placement for robot mounting. The patient was then prepped and draped in the usual sterile fashion. The Mazor robot (®Medtronic) was mounted and draped sterilely. A stab incision was made at the marked location along the PSIS and the Schanz pin was placed via battery-powered drill into the right PSIS. The robotic navigation was performed in sequence with three-define spatial orientation and O-arm intraoperative CT imaging for stealth navigation. It took 27 minutes to mount the robot and drape the patient. After completion of stealth CT, software was utilized to pre-plan percutaneous screw trajectories at L4, L5, and S1 bilaterally then uploaded to the robot for guidance. Screw trajectory planning took 17 minutes  can be completed via multiple different approaches including posterior (PLIF), transforaminal (TLIF), oblique (OLIF), lateral (LLIF), and anterior (ALIF). While there are many different approaches, there is currently no consensus on which approach is superior when evaluating fusion rates and clinical outcomes . Choice While ALIF was determined to be the best approach for this patient, ALIF does have its shortcomings. One of the risks of ALIF is the potential for vascular injury due to the required retraction of the iliac vessels in order to access the disc space . This paRole of indirect decompression for lumbar radiculopathyLumbar radiculopathy can result from a multitude of etiologies causing compression of the lumbar foramen. In this particular case, grade 1 spondylolisthesis at L4/5 and L5/S1 led to nerve root compression in the neuro-foramen, greater on the right than on the left. While direct decompression utilizes the complete removal of compressive tissues, indirect decompression involves an expansion of neural corridors without directly manipulating the compressing tissues ,7. AdditPercutaneous robotic assistance vs fluoroscopic free hand screw placement vs Stealth navigationFor the posterior aspect of the lumbar spinal fusion, we chose to utilize a percutaneous robot-assisted approach rather than using traditional fluoroscopic free-hand screw placement. When choosing between fluoroscopic free-hand screw placement vs robotic assistance, there are multiple reported benefits of robotic assistance. A primary advantage of robotic assistance is a reduction in intraoperative complications. Multiple groups have reported a reduction in complications with robotic-assisted screw placement versus fluoro-guided minimally invasive screw placement, some of which report up to a five-fold reduction in complications ,9. A priIn addition to increased accuracy, percutaneous robot-assisted techniques are associated with decreased surgical time and more rapid screw placement. Multiple studies have reported decreased operative time with the use of percutaneous robotic-assisted technique versus fluoroscopic-guided free hand screw placement ,10,13,14Lastly, robotic-assisted techniques allow for minimally invasive approaches. The use of percutaneous screw placement is associated with shorter hospital stays and less post-operative pain than conventional freehand screw placement due to reduced muscle dissection and retraction ,9. At foWe demonstrate a unique approach utilizing a 360°, minimally invasive, robotic-assisted spinal fusion in combination with an ALIF for degenerative lumbar spondylolisthesis leading to improved symptoms of lumbar radiculopathy. Robotic-assisted spine surgery can be effective however pitfalls including screw misplacement can still occur.