To investigate how and why different aspects of small-scale heterogeneity affect the amount of capillary trapping during buoyancy-driven upward migration of CO2, we conducted modified invasion percolation simulations on heterogeneous domains. Realistic simulation domains are constructed by varying two important aspects of small-scale geologic heterogeneity: sedimentary bedform architecture and grain size contrast between the matrix and the laminae facies. Buoyancy-driven flow simulation runs cover 59 bedform architecture and 40 grain size contrast cases. Simulation results show that the domain effective CO2 saturation is strongly affected by both grain size and bedform architecture. At high grain size contrasts, bedforms with continuous ripple lamination at the cm scale tend to retain higher CO2 saturation than bedforms with discontinuous or cross lamination. In addition, the “extremely well sorted” grain sorting cases tend to have lower CO2 saturation than expected for cross-laminated domains. Finally, both a denser CO2 phase and greater interfacial tension increase CO2 saturation. Again, variation in fluid properties seems to have a greater effect on CO2 saturation for cross-laminated domains. This result suggests that differences in bedform architecture can impact how CO2 saturation values respond to other variables such as grain sorting and fluid properties.Small-scale  heterogeneity has been shown to significantly impact CO Prior research has shown that even small-scale  geologic heterogeneity can greatly affect CO2 flow and trapping21. Depositional laminations and baffles are examples of such small-scale heterogeneity, and they have been shown to form effective capillary barriers that can retain a substantial amount of above-residual CO2 saturation during both the injection (drainage) and the post-injection (imbibition) stages through the mechanism known as local capillary trapping (LCT), also called capillary heterogeneity trapping22. Hence, small-scale heterogeneity can greatly impact how much CO2 is retained in the geologic material (the storage capacity of the reservoir) and it is also crucial in controlling the CO2 plume migration speed and extent24. Therefore, it is important to conduct simulations that are capable of correctly incorporating this extra amount of CO2 residual or capillary trapping in order to accurately predict how the CO2 plume migrates through heterogeneous domains.CO2 plume migration and trapping employ coarse  grid blocks or cells greatly above the resolution of small-scale heterogeneity to save computational time and resources, but consequently run the risk of obtaining inaccurate simulation results without proper upscaling20. Furthermore, conventional full-physics simulators use continuum-scale Darcy-flow physics and have convergence issues modeling low-rate CO2 flow through highly heterogeneous domains when LCT is incorporated25. On the other hand, the modified invasion percolation (MIP) method can easily handle large, high-resolution, heterogeneous domains with LCT effects thanks to its simplified physics27. Invasion percolation (IP) simulation methods originated from simulating multiphase fluid flow in pore networks. MIP methods extend the IP algorithm to the continuum scale, allowing the usage of continuum-scale grid properties  and also include gravity forces31. MIP simulators significantly reduce the complexity of the fluid flow physics by assuming viscous forces to be negligible, so they can run several orders of magnitude faster than full-physics simulators27.Conventional reservoir simulations used to study CO2 geologic storage because capillary and gravity forces strongly dominate the vast majority of the plume over the entire post-injection time period, which can last for hundreds of years or longer33. Under such flow regimes that are most relevant to CO2 geologic storage, the impact of small-scale heterogeneity is especially pronounced due to the lack of viscous forces34. Hence, the ability to correctly quantify the resulting LCT is highly important in accurately predicting plume migration and storage capacity for CO2 geologic storage35. Therefore, in this study, we run MIP simulations on fine-grid heterogeneous domains to easily incorporate the effect of LCT and obtain the domain effective CO2 buoyant flow saturation value, which is highly heterogeneity dependent.The MIP method can be applied to simulate CO2 buoyant flow saturation is the saturation at which buoyancy-driven CO2 breaks through or percolates the fine-grid domain during primary drainage. It is therefore also the critical CO2 saturation for the equivalent upscaled coarse cell at which CO2 begins to form a continuous phase in the current cell and can start flowing to the next cell. For the coarse cell, this is the lowest nonzero CO2 saturation point  on a CO2 relative permeability curve and corresponds to the threshold capillary pressure (P2) value on the drainage capillary pressure curve as shown in Fig. 2 saturation values to coarse field-scale simulation cells is essential, as critical CO2 saturation directly affects the estimated CO2 dynamic storage capacity. For example, using a flume tank deltaic geologic model, field-scale MIP simulation results obtained on a high-resolution heterogeneous domain demonstrate that by varying the critical CO2 saturation from 3 to 48% in the cells, the total amount of CO2 retained in the system is doubled36. Although the critical CO2 saturation is located on the drainage capillary pressure curve, if imbibition occurs at this point, the final residual CO2 saturation retained is still quite close to the original critical CO2 saturation. Previous tank-scale beadpack experimental results show that 74–89% of the drainage critical CO2 saturation is residually trapped after spontaneous imbibition37. Therefore, the critical CO2 saturation provides a close upper limit on post-imbibition CO2 residual trapping resulting from buoyancy-driven flow.This domain effective CO2 buoyant flow saturation in the context of realistic sedimentary bedform architectures and matrix/laminae grain size contrasts. This work builds upon a previous study by Trevisan et al. in which MIP simulations were conducted on eight three-dimensional (3D) domains with realistic sedimentary bedforms18. Trevisan et al. discovered the strong dependence of CO2 saturation on both grain size and bedform architecture. CO2 saturation is found to increase nonlinearly with grain size contrast in a predictive manner, forming a distinct S-shaped curve. It is also found that grain sorting has an impact on how well the CO2 saturation values conform to the fitted S curve. However, with just eight bedforms, the limited size of the simulation results hinders deeper understanding. Therefore, the novelty of the current work is that it not only significantly expands the simulation dataset compared to the previous study (from 8 to 59 bedforms), but it also explains how the difference in small-scale bedform architecture (ripple vs. cross-lamination) affects CO2 saturation at different grain size contrast, grain sorting, and fluid property values.The goal of this work is to conduct MIP numerical fluid flow simulations to explore how different types and degrees of small-scale capillary heterogeneity affect CO38 have previously compiled a series of realistic bedform architecture models (BAMs). Each BAM consists of a coarse-grained matrix facies and a fine-grained laminae facies. The BAMs used in the study represent a wide variety of cm- to dm-scale heterogeneity patterns seen in sandstone formations resulting from different depositional environments. This study applies the same simulation methods used by Trevisan et al.18 to 59 of the 62 available BAMs. Note that three BAMs  are excluded from this study because they do not have lamination structures within them. Some example BAMs are shown in Fig. 38.Rubin and Carter41 as shown in Fig. 43. In order to populate the BAMs with realistic petrophysical input parameters in the simulator, we assign lognormal grain size distributions based on experimental data to both the matrix and the laminae facies. By mixing unconsolidated sands into different categories of grain size and sorting45. 2 and the water phase in N/m, taken to be 0.03 N/m to represent typical reservoir conditions46. 16.3 is the constant associated with pore geometries and unit conversion45. Finally, the mean (m.) and standard deviation (s.d.) of the lognormal distribution of After the grain size distributions for different types of sands are properly defined, we convert the lognormal grain size distributions into lognormal distributions of threshold capillary pressure, ing Eqs.  to 6),4,4\documeith Eqs.  and 8)..\documenBecause natural sediment depositional processes cause the grains to segregate and form coarse-grained matrices and fine-grained laminae, it is necessary to assign each resulting 37,The dimensionless grain size contrast parameter, mentclass2pt{minim in Eqs.  and 5)..\documen47. Permedia’s MIP simulator is based on the classical IP algorithm. The classical continuum-scale IP algorithm replicates the primary drainage process of a nonwetting phase fluid emitted from a point source by modelling a continual invasion of the neighboring grid block with the next lowest threshold capillary pressure  within the pool is determined by Eq. (Past studies have shown that under strongly capillary-dominated flow regimes (i.e. regimes with low flow rates), the nonwetting phase fluid migrates by pulsing and forms fragmented finger flow paths that have low saturationd by Eq. ,11\docum2 that are in the fingers will be assigned the fine-grid critical CO2 saturation value  phase to span the fine-grid cell. This is the CO2 saturation below which the CO2 phase is no longer continuous in the cell. 2 saturation that the cell can contain. However, this is only the case at the field scale. For smaller scales such as the case in this study, all invaded cells are assigned the CO2 saturation value 2 saturation output is dependent on the input value for 2 saturation, 2 plume distribution in the domain.Saturation-wise, any cells invaded by COmentclasspt{minimaAll input parameters entered into the Permedia MIP simulator are listed in Table 18. Results have shown that any subvolumes extracted from the BAMs that are above this size demonstrate consistent matrix-to-laminae ratios, which are equivalent to the net-to-gross sand/shale ratios53. The cell size is selected to be above the scale of the representative elementary volume so that petrophysical properties such as porosity and capillary pressure are well defined at the cell level55.As shown in Table 2 geologic storage conditions and is also kept constant across simulations18. Simulation For both matrix and laminae facies, grid properties such as porosity (2 enters the domain through a planar source at the bottom and rises through the domain through buoyancy. The MIP simulation stops when CO2 percolates the top of the domain (percolation threshold). To ensure that simulation continues until the percolation threshold, the domain is set to have closed boundaries on all sides. For each of the 40 grain size contrast cases, 50 stochastic The domain is initially assumed to be completely water filled. CO2 saturation averaged over 50 stochastic simulation runs  value of 0.927. Fitted S curves for selected BAMs are shown in Fig. Trevisan et al.n in Eq. ,11\docum2 saturation distribution for the same set of BAMs featured in Fig. 2, whereas BAMs with cross lamination  trap less CO2. Finally, BAMs with discontinuous cross lamination trap the least CO2. In Fig. Figure 2 than discontinuous cross lamination at high grain size contrast is as follows. Because ripple lamination tends to be flatter than cross lamination, this allows CO2 to spread to a much greater area laterally before building up enough column height vertically to break through the lamination. Whereas for cross lamination, CO2 can easily rise along a cross lamina and break through without spreading laterally. Therefore, fine ripple lamination that is closely spaced allows for greater CO2 sweep of the domain during its buoyant migration upward. In addition, continuous lamination forces CO2 to build up column height underneath, whereas discontinuous lamination simply lets CO2 through so that it bypasses the laminae altogether. Therefore, discontinuous lamination barely retains any buoyant flow CO2.The reason that continuous ripple lamination retains more COFrom Fig. 2 buoyant flow saturation, we have conducted extra simulation runs on one particular BAM with 270 grain size contrast cases. The BAM selected is #5 as the simulation data points have a discernable spread around the fitted model. The 270 grain size contrast cases are those that satisfy the selection criteria in Section "2 buoyant flow saturation values.To investigate the effect of grain sorting on CO2. This is true regardless of the grain sorting type, but the effect is especially pronounced within the “extremely well sorted” category, which has some particularly small laminae 2 than the fitted model compared to ripple-laminated domains because the existence of cross-lamination tends to favor upward migration and hinder lateral spreading of the plume.As shown in Fig. 2 buoyant flow saturation. The base case fluid model has a density contrast of 300 kg/m3 and an IFT value of 0.03 N/m between the CO2 and the water phase. In this section, new values for the fluid model are used: (a) density difference: 100, 500, and 700 kg/m3; (b) IFT: 0.02, 0.04, and 0.05 N/m. Each fluid property is changed individually while keeping the other input parameters the same as the base case. To obtain the desired density difference, the water density is kept constant while only the CO2 density is reduced. Selected results are shown in Fig. Further simulations on eight selected BAMs have been conducted to investigate the effect of differing density contrast and IFT values on CO2 buoyant flow saturation, increasing the IFT value increases CO2 saturation. This is because a less buoyant nonwetting phase fluid can build up more column height before breaking through the capillary barrier. A higher IFT value means stronger capillary forces opposing CO2 breakthrough, which also allows for more column height buildup. Larger column height means greater rock volume invaded beneath the capillary barrier, and directly translates to higher CO2 saturation in the domain. It is also generally the case that the CO2 saturation of ripple-laminated domains is less affected than cross-laminated domains by changes in the density difference and IFT at high grain size contrast. This is because the existence of cross lamination compartmentalizes the domain, increasing the number of possible migration paths that the CO2 can take before it breaks through the domain at the top.As shown in Fig. 2 LCT for buoyancy-driven flow because of the complexity of such heterogeneities and lack of data. However, as shown by the simulation results, the impact of small-scale heterogeneity on critical CO2 saturation is significant. Depending on the types and degrees of the heterogeneity, the resulting critical CO2 saturation value can vary between 2 and 77%. As explained in the introduction, such a wide range of critical CO2 saturation values can greatly affect the field-scale CO2 trapping capacity36. Therefore, it is of interest to develop prediction models that can accurately quantify the influence of small-scale heterogeneity. The value of this study is then to provide a comprehensive simulation dataset, upon which said prediction models can be built for upscaling purposes in field-scale simulations.Previously, it has been difficult to quantify the effect of small-scale heterogeneity on CO2 saturation values are averaged over 50 stochastic runs, the associated uncertainty can be represented by the standard deviation across the stochastic runs. The standard deviation values associated with each bedform and grain size contrast case are generally small, with a mean value of 3% and a maximum value of 12%. In this study, only vertical flow simulations are conducted, and the resulting domain effective critical CO2 saturation values can be treated as isotropic. However, this will only be accurate if the grain size contrast is low. At high grain size contrast, the trapping amount tends to be different if horizontal flow simulation is also conducted. This is particularly likely to be the case for bedforms with higher degrees of anisotropy, or ripple bedforms. Therefore, future simulation studies should focus on further simulations to quantify the effect of anisotropy on critical CO2 saturation. For any critical CO2 saturation prediction model built upon this simulation dataset, another source of uncertainty would be the effect of the fluid properties. The assumption is that typical geologic CO2 storage conditions should be similar to the simulation setup in this study. And as Fig. 2 saturation.Because the reported simulated critical CO56. These are physical fluid flow experiments conducted in Hele-Shaw cell type sand tanks with analog fluids with similar fluid properties to this study and at low flow rates so that the flow regime is strongly buoyancy- and capillary-dominated as is the case for the simulation. Realistic sedimentary bedforms were packed in the sand tank with different grain size contrast cases. Not only do the experimental results demonstrate the same S-curve with increasing grain size contrast, but the critical CO2 saturation values also roughly match the simulation values for the specific BAM #537. In the future, more physical experiments should be conducted to verify the simulation results for the other BAMs.In order to verify that the simulation results are reliable and accurate, we compare the simulation results to previous experimental results2 saturation values in this study are obtained at vanishing flow rates with no viscous forces, one concern may be how the addition of viscous forces would affect the upscaling of this parameter. At low degrees of heterogeneity, we would likely have a viscous fingering pattern with early breakthrough at high flow rates, which would lead to lower critical CO2 saturation values58. However, at high degrees of heterogeneity, higher viscous forces (greater injection rates) would increase the critical CO2 saturation at domain percolation, according to another set of physical experimental results for BAM #522. Therefore, the interplay between heterogeneity and flow rates determines the domain effective critical CO2 saturation values.Because the domain effective critical CO2 buoyant flow saturation, we ran 118,000 MIP simulations, covering 59 BAMs, 40 grain size contrast cases, and 50 stochastic variations. Simulation results show that grain size contrast has a considerable impact on the effective CO2 saturation for heterogeneous domains, whereas bedform architecture only becomes important at high grain size contrast values. Different grain sorting as well as varying density differences and IFT values also affect the simulated domain effective CO2 saturation. More specifically, the following conclusions can be reached.2 buoyant flow saturation value increases nonlinearly with increasing grain size contrast values. The relationship can be described by a parametrized S-shaped curve for bedform architectures with continuous lamination layers.The domain effective CO2 saturation values for different BAMs all have similar values. However, as grain size contrast values increase, different BAMs reach different maximum CO2 saturation values. Domains with continuous ripple lamination tend to have greater maximum CO2 saturation values than domains with discontinuous cross lamination.At low grain size contrast values, effective CO2.Simulations of the “extremely well sorted” grain sorting type often tend to deviate from the S-shaped curve for domains with cross lamination. At the same grain size contrast value, domains with finer lamination grains tend to retain more CO2 saturation decreases with increasing density differences between the CO2 and the water phase, and increases with increasing IFT values between the two phases. The strength of the influence that the fluid model has on CO2 saturation is bedform dependent.Domain effective COTo investigate how grain size and bedform architecture affect COSupplementary Information.


While pair housing macaques is known to improve welfare over single housing, the process by which animals are socialized can be stressful if the animals do not get along. To assess whether or not potential partners are compatible, they typically need to be located in visual contact with one another, which often involves moving one or both animals to a new location. Such relocations can cause stress for animals, particularly if the introduction attempt is not successful. In this study, we examined whether allowing rhesus macaques to interact via video conferencing technology (Zoom) could help us determine compatibility before they were relocated. We provided a Zoom session between partners for 42 pairs of monkeys and coded their behavior. We then examined whether these behaviors predicted future pair success. The monkeys in our study spent surprisingly little time attending to the screen, and attention did not predict pair success. However, the similarity of attention shown by individuals in the pair  did. Pairs in which attention was primarily shown by one animal were more likely than others to be successful. While additional work is needed, video conferencing technology may help staff determine compatibility between potential partners.p = 0.03). Neither prosocial  nor anxiety  behavior correlated with pair success. While preliminary, our data suggest that video conferencing technology may be useful as a tool for introducing unfamiliar partners prior to a socialization attempt.Pair housing is known to promote welfare for macaques in captivity. However, finding compatible partners can be challenging, particularly when animals are not located near one another. Because macaques show interest in videos of conspecifics, we examined the use of video conference technology (Zoom) as a potential tool to assess compatibility in 84 rhesus macaques (2–22 years old) prior to pair introduction. Monkeys involved in the pairs  were unfamiliar with each other. We set up a 10 min Zoom session between potential partners (on an iPad in front of the cage). We scored attention to the screen, anxiety, and prosocial behaviors and examined whether these behaviors predicted future pair success. Monkeys spent relatively little time attending to the tablet (median = 13.3%), and attention did not predict pair success . However, pairs in which attention was primarily shown by one animal had a higher chance of success than those in which both individuals showed similar levels (B = −4.66.  Social housing is an essential tool for promoting the psychological wellbeing of nonhuman primates (NHPs) in research. Studies have shown a variety of physiological and behavioral benefits associated with socialization e.g., ,2,3,4,5),3,4,52,3The process of pair introduction is often performed in a series of steps , beginnibefore moving the monkeys may decrease the need for relocations and reduce the stress associated with social introductions.The decision about whether to proceed with the social introduction is usually based on the observed interactions between the partners; introductions in which the two partners display many affiliative behaviors toward one another are likely to proceed, while those in which the partners engage in aggressive or overly fearful behavior may be aborted. In order to interact, the monkeys clearly need to be in visual contact with one another. However, in many primate facilities, potential partners may be housed in separate rooms or even different buildings from one another, necessitating the relocation of one or both partners for the introduction. Being moved to a new and unfamiliar environment can be stressful for macaques ,15, and Video conferencing technology offers the opportunity for animals to communicate virtually. Many organizations have dramatically increased the use of video conferencing in the past couple of years to improve communication, and there is evidence to suggest that macaques may also be able to communicate in this way. Studies have demonstrated the ability of rhesus macaques to recognize and process faces, particularly facial expressions, of conspecifics in still images by measuring activation in the amygdala ,17. MacaIn this study, we examined the use of the Zoom video conference app (on tablets) as a potential tool to assess pair compatibility in male and female rhesus macaques prior to relocation for pair introduction. Given that prosocial behavior exhibited early in the introduction can predict pair success , we hypoMacaca mulatta; 51 males, 33 females), 2.3–22.1 years old (mean age = 8.8 +/− SD 5.14 years), housed at the Oregon National Primate Research Center (ONPRC). Each monkey had been identified as a potential partner in a socialization attempt by the ONPRC behavioral management team based on factors such as age, weight, and temperament. Potential partners were provided with a Zoom conference session prior to the pair attempt (see below). Pairs were either isosexual  or heterosexual (n = 9), but in all cases, the partners were unfamiliar with one another and were housed in different rooms. At the time of the Zoom session, monkeys were single-housed  in standard, appropriately sized primate cages based on their weight, in accordance with the Guide for the Care and Use of Laboratory Animals )Despite these confounds, our initial data suggest that video conferencing technology may be useful as a tool for introducing unfamiliar partners ahead of their pair attempt. There are also other uses for this technology. In many facilities, partners need to be temporarily separated from one another for research or clinical-related reasons. In these situations, Zoom sessions may be set up between the familiar animals so that they may communicate, thus minimizing the impact of physical separation. At the ONPRC, group-housed rhesus macaques who are injured or sick may be removed from their pen and brought to the clinical hospital. We have used Zoom to allow these monkeys to communicate with their groupmates while they are under clinical care. While we have only used this for a handful of animals so far, we have found that both the sick/injured monkey as well as the groupmates pay a great deal of attention to the screen. More work is needed to examine whether the ability to interact with groupmates affects how well an individual responds to medical treatment and the individual’s integration back into the group after medical treatment is completed.In order to assess the compatibility of macaques prior to social introductions, the animals need to have the opportunity to interact with one another. At many facilities, that means moving animals from one room to another. These movements can be disruptive to the monkeys, and the stress can be compounded if the introduction is not successful. Having a way to assess the compatibility of potential partners prior to moving them could help to reduce unnecessary moves , therefore reducing stress. Video conference technology provides opportunities for nonhuman primates to interact with one another in real time. Studies have demonstrated that rhesus macaques show interest in video images of conspecifics e.g., ,17,18); ,18; 17,1


Bioelectrical impedance vector analysis (BIVA) is a non-invasive and low-coststrategy. The methods used to assess malnutrition in patients undergoing HDare still a challenge. The aim of the present study was to compare BIVA to7-Point Subjective Global Assessment (7-point SGA) to identify malnutrition.We also investigated the sensitivity and specificity of the previouslyproposed cutoffs point for BIVA parameters.Patients of both sexes, over 20 years of age, on HD treatment were included.Anthropometric parameters, laboratory data, and bioelectrical impedanceanalysis (BIA) were evaluated. Values of resistance (R) and reactance (Xc)obtained by mono-frequency BIA were normalized to body height (H) togenerate a graph of the bioimpedance vector with the BIVA software. Theanalysis of the area under the receiver operating curve ROC (AUC) wasperformed.Among the included 104 patients, the mean age was 51.70 (±15.10) years, and52% were male. The BIVA had a sensitivity of 35% for diagnosingmalnutrition. The specificity of BIVA for identifying the well-nourishedpatients was 85.7%. The diagnostic accuracy between the BIVA and 7-point SGAwas AUC=0.604; 95%CI 0.490-0.726, higher than the previously establishedcutoff values . The 95% confidence ellipsesdid not overlap (p<0.05).Our study showed low accuracy of BIVA for diagnosing malnutrition using a7-point SGA as a reference standard. However, it is a complementary methodfor assessing nutritional status as it provides data on cellularity andhydration, which are important aspects for the HD population. Malnutrition results from the decrease in ingestion or absorption of nutrients thatlead to changes in body composition and cellularity, with a consequent decrease infunctional capacityThe criteria or methods used in the screening, assessment, and diagnosis ofmalnutrition in this group remains a challengeOn the other hand, the hydration status of individuals on HD can complicatenutritional diagnosis due to hypervolemia, which can mask some anthropometricparameters and, therefore, affect their interpretationIn this regard, the bioelectrical impedance vector analysis (BIVA) is a non-invasiveand low-cost strategy for evaluating HD patientsThe available scientific evidence supports the use of BIA for the assessment of bodycomposition in accordance with the KDOQIA cross-sectional study with a convenience sample recruited from two HD centerswas conducted. Data collection occurred in 2015. Patients of both sexes, in HDfor at least 3 months, and over 20 years of age were included. The exclusioncriteria were recent hospitalization (<3 months), use of a pacemaker,presence of infectious diseases, hepatitis, or cancer, and physicaldisabilities, cognitive impairment, or refusal to participate. The protocol wasapproved by the Ethics Committee of our institution (54523116500005083).Clinical data, the etiology of chronic kidney disease (CKD), the presence ofcomorbidities, biochemical parameters  and KT/v were taken from the patients' medical records. Hemoglobin wasdetermined by the electronic counting method, serum albumin by the enzymaticcolorimetric method, creatinine by optical microscopy, and urea by UV kinetics.Anthropometric measurements were taken after the second HD session of theweek,,The 7-point SGA((r)), CA, USA, 50 kHz, 800 µA). The electrodes wereplaced on the midline between the protruding ends of the radius and ulna of thewrist and midline between the medial and lateral malleoli of the ankle on theside of the body without vascular access. Each patient underwent bioelectricalimpedance measurements at the beginning of and after the intermediate HD sessionfollowed by 20 min of rest, according to a previously published method,The body composition of patients was obtained using tetrapolar mono-frequency bioimpedance (QuantumII - RJL SystemsThe impedance media of the groups were compared using the T² Hotelling test (BIVA2002 software). Area under the receiver operating curve ROC (AUC) analysis wasperformed to verify the diagnostic accuracy of BIVA parameters in theidentification of patients with malnutrition, based on the 7-point SGA as thereference standard positive and negative likelihood ratios (LR+ and LR−respectively).The cutoff point performance proposed for the total sample in a previousstudyWe evaluated 104 HD patients; their mean age was 51.7 (±15.1) years, 52% were male,the main etiology of CKD was hypertensive nephrosclerosis (26%), and the mostprevalent comorbidity was Systemic Arterial Hypertension (55%). The average KT/v was1.37 (±0.25). The age and duration of HD in the group with malnutrition were higherthan those in the well-nourished group.Patients with malnutrition had lower serum albumin and creatinine concentrations. Theprevalence of malnutrition by BIVA was 25.96% while that by the SGA was 19.23%. In addiBIVA diagnosed malnutrition with a sensitivity of 30.00% using the 7-point SGA as thereference standard. The specificity for identifying the well-nourished was 85.70%.The accuracy  and agreement  betweenthe BIVA criteria and 7p-SGA in diagnosing malnutrition were low and regular,respectively. The estimated prevalence of malnutrition based on the combination ofthe established cutoff points for R/H and Z/H was 70.00% among the total sample. Thesensitivity obtained by the analysis using the pre-established cutoff was 72.20% andthe specificity was 30.50, the accuracy was low ,agreement was week , and the positive predictive value was 18.60.The confidence ellipses representative of the R/H and Xc/H values showed that therewas a difference between the vectors of the malnourished and well-nourished groupsby the 7-point SGA, since they did not overlap (p<0.05) . In pracIn the evaluation performed before and after HD, the scores of Xc and R showed asignificant increase after HD. In the intergroup analysis, only the Xc variableshowed a significant difference, being greater in the well-nourished group bothbefore and after HD .Our results show low accuracy and week agreement between BIVA and the 7-point SGAresults. The BIVA parameters R/H and Z/H demonstrated greater sensitivity thanspecificity in the identification of malnutrition, but low AUC. These results mayhave occurred because the 7-point SGA assesses aspects of up to six previous months,whereas BIVA assesses the cellularity and hydration of the present time in theanalysis. Furthermore, the vector position of each patient shows the shift of thequadrant before and after the HD session, regarding hydration.,Patients who were malnourished according to the 7-point SGA showed less cellularityby the BIVA. This result was expected since cellularity is evaluated by means of Xc(which offers a measure of opposition to the current flow caused by the capacitanceproduced by the cell membrane),-The prevalence of malnutrition based on the 7-point SGA 19.23%) in the present studywas lower than those reported in the literature for these patients, which rangedfrom 30 to 50%% in the ,Two studies in Italian patients compared th BIVA with the three-point and 7-pointSGA,As most patients on HD are anuric, fluid management, interdialytic weight gain (IWG)determination and hydration status after dialysis are information that wouldcontribute to the treatment and quality of life of the patientThe state of hypervolemia present in the patients before the HD session and confirmedby the IWG did not affect the sensitivity of the BIVA for malnutrition detection inthe present study. Confidence ellipses from before and after the HD session show amigration of all patients  to the quadrantindicating lower hydration. This difference in position was expected as excessextracellular fluid is removed during the HD session. Therefore, the decrease intotal body water (TBW) results in increased resistance. As with Xc, the increaseindicates a greater number of cells per tissue unitA limitation of the study is the use of the 7-point SGA as the only parameter toassess nutritional status and sensitivity of BIVA. Although the 7-point SGA is therecommended parameterThis study showed that there are differences in the use of the 7-point SGA and theBIVA to detect malnutrition in CKD patients on HD. Our results show that BIVAparameters R/H and Z/H provide low accuracy in diagnosing malnutrition using the7-point SGA as reference standard. However, the analysis indicated thatwell-nourished and malnourished individuals (7-point SGA) had different cellularityaccording to the BIVA. Therefore, BIVA must be used in conjunction with othermethods to diagnose malnutrition, as BIVA can provide cellularity and dehydrationdata that complement the 7-point SGA and are especially important for monitoring themedical history of the HD patient.Longitudinal studies using BIVA are needed to monitor the nutritional status of HDpatients. We believe that this monitoring will improve the quality of life ofpatients as BIVA provides cellularity and hydration data which are important fordetermining dry weight. We recommend BIVA for monitoring cellularity and hydration,two important aspects for the HD population. Therefore, the practical application ofBIVA is to monitor nutritional status at the cellular level and hydration. These aretwo important parameters for medical history, because they prevent the risk ofnutritional and cardiovascular complications by revealing changes that cannot bedetected in the short term by other methods, such as 7-point SGA.


The prevalence and prognostic significance of malnutrition risk remain unclear in Chinese patients with pulmonary tuberculosis. Therefore, we aimed to investigate the malnutrition risk in Chinese patients and explore the relationship between malnutrition risk and follow-up outcomes.We conducted a hospital-based cohort study from January 2020 to December 2020. Malnutrition risks were evaluated using nutritional scales, including the Nutritional Risk Screening 2002 (NRS-2002), the controlling nutritional status score (CONUT), the geriatric nutritional risk index (GNRI), and the prognostic nutritional index (PNI). The primary outcome was all-cause mortality at a one-year follow-up. Malnutrition risk was calculated, and the relationship between malnutrition and follow-up outcomes was analyzed. We assessed the performance of malnutrition risks to predict clinical outcomes in prognostic models.P = 0.033), GNRI , or PNI  and death before 1-year follow-up remained significant. The addition of the nutritional scales to prognostic models improved death prediction, as validated by the integrated discrimination index .A total of 1,075 patients were included. According to NRS-2002, CONUT, GNRI, and PNI, 818 (76.09%), 954 (88.74%), 682 (63.44%), and 364 (33.86%) patients were at risk of malnutrition, respectively. Before 1-year follow-up, a total of 99 patients (9.2%) had died. After adjustment for risk factors, the association between severe malnutrition in CONUT (HR = 4.78, 95% CI: 1.14–20.11, Malnutrition in patients with pulmonary tuberculosis was associated with an increased risk of all-cause death in the long-term follow-up. Our findings provided evidence for the use of admission nutrition screening in patients with pulmonary tuberculosis. Tuberculosis (TB) was the leading cause of mortality worldwide from a single infectious disease, with 10 million new cases and 1.2 million deaths in 2019 . MoreoveIn 2013, the WHO published the first nutritional guideline for patients with TB. The guideline used the body mass index (BMI) as a tool for assessing nutritional status . The BMIRecently, several objective scales have been used for assessing malnutrition, including the controlling nutritional status (CONUT) score , the gerIn recent years, several predictive models of TB mortality have been developed , 21. HowThis study was based on single-center, prospective data from January 2020 to December 2020. The inclusion criteria were patients diagnosed with pulmonary tuberculosis and those aged older than 18 years. The diagnosis of pulmonary TB had to be based on the WHO guidelines  and was 2] was used to calculate BMI.Demographic characteristics, body height or weight, and medical history were collected at baseline. Clinical features and nutrition status were evaluated by an experienced nutritionist from the department of nutrition in our hospital. Laboratory parameters were collected from a hospital-based database. Weight divided by height square /2); and for men, ideal body weight = height in cm – 100 – ([height in cm−150]/4); malnutrition assessed by GNRI were defined as follows: normal (score >98), mild risk (score 92–98), moderate risk (score 82–91) and severe risk (score<82) .3) + 10×serum albumin (g/dl). Malnutrition assessed by PNI was defined as follows: normal (score>38), moderate risk (score 35–38), and severe risk (score <35) , and the score was calculated using the formula: 0.005×total lymphocyte count (mmore <35) .A face-to-face follow-up or telephone interview was performed 1 year after admission. We obtained follow-up information on all-cause death from relatives of patients or a death certificate from our hospital records.2 test, Fisher exact test, Student's t-test, or the Mann–Whitney U-test were performed for statistical analysis whenever deemed appropriate. The malnutrition risk was assessed using the nutritional scales. Cox regression was used to investigate essential factors of mortality at a 1-year follow-up. The following covariates were adjusted in the Cox regression model: age, gender, hypertension, coronary heart disease, chronic obstructive pulmonary disease, meningeal tuberculosis, and tuberculosis drug resistance . We calculated the net reclassification improvement (NRI) and integrated discrimination improvement (IDI) to quantify the correct reclassification and sensitivity improvement with the addition of nutritional scales in the predictive model. Sensitivity analyses were performed by different adjusted models. Significant improvement was recognized in the prediction model when NRI >0 or IDI >0 , patients with a medical history of retreatment tuberculosis (n = 392), patients with cancer or other end-stage diseases (n = 9), and patients with missing data on body height, weight, or other parameters used to calculate malnutrition (n = 38), a total of 1,075 patients were analyzed in the study , 954 (88.74%), 682 (63.44%), and 364 (33.86%) patients were at risk of malnutrition , whereasA more detailed stratified analysis of the severity of malnutrition was performed. Of these patients, 317 (29.48%) and 187 (17.39%) were at mild malnutrition risk, as assessed by CONUT and GNRI; 408 (37.95%), 264 (24.55%), and 96 (8.93%) patients were at moderate malnutrition risk, as assessed by CONUT, GNRI, and PNI; and 229 (21.30%), 231 (21.49%), and 268 (24.93%) patients were at severe malnutrition risk, as assessed by CONUT, GNRI, and PNI, respectively.P = 0.033), GNRI , or PNI  and death before 1-year follow-up remained significant.Before 1-year follow-up, a total of 99 patients (9.2%) had died. Univariable analyses suggested that malnutrition was significantly associated with follow-up death . Any malP < 0.05).We investigated the performance of models to predict clinical outcomes in patients with TB . The preThe present study investigated the nutritional status of Chinese patients with TB and explored the performance of nutritional scales to predict TB outcomes. The findings suggested that severe malnutrition risk ranged from 21.30 to 24.93% in patients with TB. Baseline malnutrition may be a predictor of TB mortality. The addition of nutritional scales to mortality models improved C statistics, NRI, and IDI. The results stress the importance of assessing the nutritional status of patients with TB.Our data showed that malnutrition risk ranged from 33.86 to 88.74% in patients with TB, the moderate risk was 8.93 to 37.95%, and the severe risk was 21.30 to 24.93%. The 2013 WHO guideline considers BMI as a tool for assessing malnutrition in patients with TB . This coTherefore, we compared the performance of other objective nutritional scales in our study. The malnutrition risk varied between different objective scales. The malnutrition risk calculated by CONUT was 88.74%, whereas 63.44% was calculated by GNRI and 33.86% by PNI. The GNRI  involvesSeveral studies examined the association between nutritional status and TB mortality , 4. A prSeveral prognostic models have been developed to predict TB mortality. However, nutritional status was not involved in these studies , 21. We Our study has its limitations. First, due to the unavailability of necessary variables, we failed to investigate malnutrition according to the European diagnostic criteria  and coulIn conclusion, severe malnutrition risk ranged from 21.30 to 24.93% in Chinese patients with TB. Malnutrition risk was related to an increased risk of death in the long-term follow-up. Nutritional scales may be significant indicators for predicting clinical outcomes in patients with TB.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by the Ethics Committee at Beijing Chest Hospital. The patients/participants provided their written informed consent to participate in this study.Conceptualization: J-JM and W-ML. Data collection: J-JM, ZL, YC, and HH. Formal analysis: Y-JG. Funding acquisition: W-ML. Methodology: Y-JG and W-ML. Data supervision: J-JM and W-ML. Writing—original draft: J-JM and Y-JG. Writing—review and editing: J-JM, Y-JG, ZL, YC, HH, and W-ML. All authors contributed to the article and approved the submitted version.


Obtaining valid parental informed consent for pediatric procedures in the emergency department (ED) is challenging. We compared a video-assisted informed consent intervention with conventional discussion to inform parents about pediatric procedural sedation in the ED. We conducted a prospective randomized controlled trial using a convenience sample including the parents of children in the ED in whom procedural sedation for facial laceration was recommended. The video group watched an informational video. Conventional group participants received information from physicians during conventional discussion. The primary outcome was knowledge improvement of the video intervention compared with conventional discussion. The secondary outcome was parental satisfaction. Video and conventional groups comprised 32 and 30 participants, respectively. Mean knowledge scores of parents after intervention [±standard deviation] were higher in the video group (91.67 ± 12.70) than in the conventional group (73.33 ± 19.86). Knowledge score differences were significantly bigger in the video group . Video group participants reported greater satisfaction than conventional group participants. Parents’ comprehension of and satisfaction with the informed consent process for pediatric procedural sedation may be improved with the use of an educational video. Standardized approaches should be developed by healthcare institutions to better educate parents, facilitate treatment decisions, and boost satisfaction in the ED. For ethical and legal reasons, it is crucial that physicians provide patients with information about procedures, risks, benefits, and treatment alternatives during the informed consent process ,2,3. ObtObtaining legitimate informed consent in the emergency department (ED) is a difficult process in that it usually takes longer to communicate and discuss relevant information with patients or their families . ProvidiProcedural sedation involves the use of sedative, analgesic, and dissociative drugs to produce analgesia, sedation, and motor control during uncomfortable or painful diagnostic and therapeutic procedures . It is aDuring the traditional informed consent process in the ED, patients and their family members may struggle to understand how the treatment procedure or surgery will progress as well as how to make treatment decisions; this is because a large amount of information is provided to them during this process ,17. TherThe aim of the present study was to develop an educational video intervention tool to assist the informed consent process and to determine whether using an educational video could improve routine discussion with parents about the risks, benefits, and alternatives of procedural sedation for their children in the ED.The conceptual framework and study design, developed by Lin et al.  to evaluA portable computer preloaded with the information program was used, and the volume was appropriately adjusted so that participants could hear the content. A research associate was present during the participant’s navigation through the program to ensure completion and help as needed. Watching the entire video took approximately 6 min, after which participants could ask a physician questions about the procedure.A questionnaire assessing knowledge regarding the purpose, benefits, and risks of procedural sedation was compiled based on previous questionnaires ,25,26,27During the second stage of the study, we conducted a prospective randomized controlled trial using a superiority study design in a tertiary university hospital ED. Study participants included the parents of children recommended for procedural sedation for a facial laceration. Ketamine  was provided for pediatric procedural sedation. Participants randomized to the video group received the video-assisted informed consent intervention, which explained the procedure and its risks, benefits, and alternatives. The conventional group participated in routine discussion with a physician, who provided information about the procedure. All participants completed the knowledge questionnaire  before aAll parents of pediatric patients older than 1 year and younger than 7 years of age in whom procedural sedation for facial laceration had been recommended were eligible for enrollment if a trained research associate was available. Exclusion criteria included parental refusal to participate, parental inability to understand the study process, patient clinical instability, or parents’ inability to speak and read Mandarin. If an eligible participant was missed, the reason, such as the unavailability of the research associate, was recorded in a study logbook. p < 0.05); (e) t-test is two-tailed; and (f) 20% dropout rate is used to analyze the data. Given these assumptions, we determined that a sample size of 29 in each group was needed to achieve an effect size of more than 0.8 with 90% power and a significance of 0.05. The following power assumptions were made: (a) higher education levels increase the mean questionnaire score ; (b) scores are normally distributed; (c) standard deviation is 16 for both groups; (d) level of significance is 0.05 . Additional information was collected from the hospital’s electronic medical records system and patient documents, such as arrival time to the ED and emergency physician. All participants completed the multiple-choice knowledge assessment before the educational sessions. Conventional group participants received information about the procedure from their physician in a conventional discussion. Following the discussion, conventional participants completed the knowledge assessment again and responded to questions assessing their satisfaction with the informed consent process. Participants in the video group watched the informational video on a laptop computer at the patient’s bedside. Participants could pause and/or replay the video. All video participants received the question-and-answer session after watching the video. If participants had additional questions about the procedure, they could speak with a physician after the educational session. The aim of this question-and-answer session was to provide a similar opportunity for questions as that provided to the conventional group during the informed consent process. The same knowledge assessment and satisfaction survey were completed by participants in both groups after the question-and-answer session.In our ED, informed consent for the procedure was obtained by emergency attending physicians. Physicians who obtained informed consent were not aware of the questions in the knowledge assessment. The research associate was blinded to the allocations when assessing the outcomes.The primary outcome was the participants’ understanding of pediatric procedural sedation as evaluated using the knowledge assessment. Questions were multiple-choice and equally weighted . The maxt-test was used to compare mean pre- and post-education scores on the knowledge questionnaire between groups. The paired t-test was used for within-group comparisons.Data were recorded using participant numbers, with no individual identifying information to maintain participant anonymity. Collected data included participant demographics, ED arrival time, and the treating physician. Demographics of the conventional and video groups were analyzed using descriptive statistics. Continuous variables were analyzed using mean and standard deviation, and categorical variables were analyzed using proportions. Binary, ordinal, and categorical variables were calculated using Fisher’s exact test. The Student p < 0.05 was used to indicate statistical significance, and 95% confidence intervals were computed. Stata version 14.0  was used to analyze relevant data.We calculated the knowledge score difference by subtracting pre-education scores from post-education scores. If independent factors were associated with knowledge score difference and satisfaction in univariate analysis or if these were clinically significant, they were included in the multivariable regression models. We used two regression models with predefined covariates for knowledge score differences, including a multiple linear regression model and a multiple ordinal logistic regression model. The knowledge score differences were categorized and applied to the analysis of the multiple ordinal logistic regression model. A multivariable ordinal logistic regression model with preset factors for parental satisfaction was used. Likelihood ratio tests were used for the multivariable models. A threshold of During the 1-year study period, 118 children received procedural sedation . Forty-fIn the subgroup analysis of baseline knowledge scores, there was no statistically significant difference between the conventional and video groups . Post-edWe found that participants who received a video-assisted informed consent intervention had greater knowledge of pediatric procedural sedation. Referring to the multimedia principle, a theory of multimedia learning, Mayer proposed that people learn at a deeper level through pictures and words than through words only . CompareThe two core principles of patient autonomy and patient well-being are promoted in the informed consent process ,17, and It is difficult and time-consuming to obtain legitimate informed consent in the ED. Owing to time limits, strong emotions, and stress brought on by pain or acute symptoms, it is frequently hard for patients and their family members to comprehend and take in sufficient information to be able to give their informed consent for emergency procedures ,16,17,19Research on the decision-making framework for informed consent has identified many factors that affect patients’ understanding of the process. Patient factors , physician factors , disease context , and environmental factors  may affect information exchange, patient deliberation, and voluntarism regarding treatment decisions and consent ,42. Our A previous study reported that parental educational level and sex affect risk counseling recall; children’s mothers showed better risk recall than fathers . HoweverThe entire consent process is of great importance. A good consent process may improve patient satisfaction and foster a good patient–physician relationship. Several strategies have been proposed to improve the consent process in the emergency setting ,45. One We found that parental knowledge and comprehension were significantly predicted by baseline knowledge score and usage of an educational video. This echoed the previous study . Some prRecent advancements in tablet and portable computer technologies have given rise to the excellent potential for improving preoperative parental education regarding pediatric emergency surgery . The newWe did not measure the time spent in the informed consent process for pediatric procedural sedation in the two groups. Time spent in the informed consent process may affect participant comprehension and satisfaction ,47,59. HFurthermore, the importance of adequate education and training for healthcare providers to deliver structured and comprehensive patient information should not be underestimated ,66. No sOur study has several limitations. First, this study focused on one intervention for a particular population at one institution; thus, the findings may not be generalizable to other situations or populations. Second, although the video-assisted informed consent intervention improved participant understanding, it remains unclear how the intervention influenced the decision to provide consent for the procedure. No participants refused consent after being informed through either the conventional process or the video-based process. However, the choice of study design and population may make it more likely that participants were already aware of the need for procedural sedation and had made an initial decision to accept it prior to study recruitment. Further studies are needed to examine the effect of video-assisted informed consent intervention on decision-making. Moreover, there are no reliable and valid measures of participants’ understanding of the risks, benefits, and alternatives of pediatric procedural sedation. In this study, the video and knowledge assessment developed by a panel of experts demonstrated face validity and included information that we believed participants should know before providing consent for this particular procedure. Knowledge assessment focused on the purpose, risks, and benefits of pediatric procedural sedation, and participants showed substantial improvement in their understanding of these aspects. However, knowledge about alternative treatments was not measured. It would have been useful to assess this and to determine whether the intervention improved participants’ understanding of the alternatives. Moreover, the test question type and literacy level might have influenced the results of the knowledge assessment. However, it remains unclear as to whether the use of percentages for possible complications is easily understood and appreciated by parents in the knowledge assessment, although we believe one of the strengths of the questionnaire would be the secondary outcome of satisfaction we measured, which was more generalizable. Furthermore, seven eligible parents refused to participate. They expressed that they were too anxious to participate in the study. We did not evaluate the effect of video education on parents’ anxiety. A previous study reported that a multimedia intervention significantly reduced anxiety and increased knowledge among parents . FurtherThe use of an educational video can improve the process of informed consent for pediatric procedural sedation in the ED. Intervention using an education video may improve participant comprehension of the procedure and satisfaction with the informed consent process. To confirm these initial findings, additional studies are needed among patients receiving different types of procedures or surgeries. To better inform parents in the ED, healthcare institutions should develop organized and standardized methods for providing informed consent. Educational videos may be beneficial to improve communication among healthcare providers, patients, and families and facilitate treatment decisions.


To test whether this mild phenotype is recurrent in other insect species, we analysed resilin in the spotted‐wing fruit fly Drosophila suzukii. As quantified by DT autofluorescence by microscopy, DT intensities in the trochanter and the wing hinge are higher in D. suzukii than in D. melanogaster, while in the proboscis the DT signal is stronger in D. melanogaster compared to D. suzukii. To study the function of Pro‐Resilin in D. suzukii, we generated a mutation in the proresilin gene applying the Crispr/Cas9 technique. D. suzukii pro‐resilin mutant flies are flight‐less and show a hdw phenotype resembling respective D. melanogaster mutants. DT signal intensity at the wing hinge is reduced but not eliminated in D. suzukii hdw flies. Either residual Pro‐Resilin accounts for the remaining DT signal or, as proposed for the hdw phenotype in D. melanogaster, other DT forming proteins might be present in Resilin matrices. Interestingly, DT signal intensity reduction rates in D. suzukii and D. melanogaster are somehow different. Taken together, in general, the function of Pro‐Resilin seems to be conserved in the Drosophila genus; small differences in DT quantity, however, allow us to hypothesise that Resilin matrices might be modulated during evolution probably to accommodate the species‐specific lifestyle.Resilin is a protein matrix in movable regions of the cuticle conferring resistance to fatigue. The main component of Resilin is Pro‐Rresilin that polymerises via covalent di‐ and tri‐tyrosine bounds (DT). Loss of Pro‐Resilin is nonlethal and causes a held‐down wing phenotype ( Drosophila suzukii. In proresilin mutant D. suzukii flies the di‐tyrosine signal is reduced indicating reduction or elimination of proresilin. Reduction or elimination of dityrosine and proresilin in D. suzukii causes wing posture defects preventing flight.Di‐tyrosine represents proresilin in several patches in the cuticle of the wing hinge region in  Drosophila suzukii and Drosophila melanogaster adults is similar.Resilin/di‐tyrosine distribution in D. suzukii and D. melanogaster.Reduction or elimination of Resilin/di‐tyrosine causes an identical phenotype in Besides proresilin, resilin matrices in Drosophila species probably contain also other proteins.A general positive correlation between body size and Resilin/di‐tyrosine amounts is not apparent in Drosophila species. After mass crossing of the flies generated by injected eggs, we continuously checked for the occurrence of the held‐down wing phenotype in our CCproresilin  stock. Flies with this phenotype were isolated and kept together to generate a stock for further analyses. For determination of the mutation, genomic DNA was prepared from these and control flies by standard methods to amplify and sequence the respective region using the primers ATTCCGATCAGCAGCAGTCC and GTCCGCCATTACCATTACCG. Sequences, determined by Microgen Europe B.V., were analysed by the BLAST software at the NCBI and the SignalP 6.0 software on the DTU Health Tech site.To mutagenize the 2.3For body size measurements, flies were submerged in halocarbon oil (Sigma‐Aldrich) on a glass slide and photographed on a Nikon AZ100 microscope at a magnification of 10x using the in‐built NIS software that allows length determination.For morphological analyses, flies were observed on an inverse Axio Observer Z1 (Zeiss) equipped with an Axiocam Mono camera at the systems biology group of C. Dahmann at the TU Dresden. The filters were: AF405 (DT specific filter): Ex: 357/44 nm Brightline HC, splitter HC BS 389 nm, Em: 420/40 nm ET Bandpass; DAPI: Ex: 359/48 nm, splitter: 395 nm, Em: 445/50 nm; and GFP: Ex: 470/40 nm, splitter: 495 nm, Em: 525/50 nm. These objectives were used: EC Plan‐Neofluar 5x/0.67 M27 DRY, EC Plan‐Neofluar 10x/0.3 M27 DRY, and EC Plan‐Neofluar 20x/0.5 M27 DRY. The light source was a halogen lamp. Flies were immobilised by freezing at −20°C for 4 min, then dissected and mounted in ROTI®mount Aqua. Images were taken with the Zeiss inbuilt software. They were prepared using ImageJ and Adobe Illustrator CS6.D. melanogaster wild‐type wing hinge signal extracted by the profile‐program comparing the intensities obtained by the DT and DAPI filters (DT > DAPI). The threshold of 7500 counts was used to produce the numbers of DT dots in the wing hinges of all used lines. These area counts where than compared and calculated with the total individual numbers (n) per line.In this study, we aimed at identifying Resilin matrices indirectly by DT autofluorescence. For this purpose, we used a specific filter system adjusted to the published emission range . All statistical analyses were performed using XLstat 2020 (AddinSoft®). All computed p‐values are listed in Supporting Information: Data Data were analysed using Student's δs:dt = (mean body size species 1/mean intensity species 1) – (mean body size species 2/mean intensity species 2). If there is a body size‐proportional correlation , the coefficient approximates 0. If the coefficient is different from 0, we suppose a nonbody size dependent difference of intensity. Indeed, for most cases tested in this study, we obtain a coefficient of approximately 0  that is needed for the secretion of Pro‐Resilin to the extracellular space. According to the SignalP 6.0 software, the deletion of M11A12 would shift the signal peptide cleavage site to the position between residues 17 and 18 concomitantly reducing the probability of signal peptide cleavage (0.810029). The new cleavage site, if functional, would cause the deletion of four N‐terminal amino acids of the secreted protein. We assume that disruption of the signal peptide prevents the efficient secretion of Pro‐Resilin into the differentiating cuticle, thereby causing the observed phenotype. In addition, N‐terminal truncation of the protein might destabilise it and/or reduce its activity. Together, despite the deletion, low amounts of aberrant Pro‐Resilin may be secreted correctly and display residual function, thereby explaining the relatively mild nonlethal phenotype. Alternatively, redundant DT‐forming proteins may act in parallel to Pro‐Resilin thereby alleviating the defect. Indeed, the genomes of many insect species harbour more than one gene coding for Pro‐Resilin‐like proteins , 13% (trochanter), 16% (wing hinge), and 25% (cibarium)   and conserved in other Drosophila species (orthology ID 2370). As Cpr56F is, probably like in D. melanogaster, not expressed in the wing hinge, at least a third DT‐forming protein may be postulated to be incorporated into Resilin matrices in at least some body parts in Drosophilae. Overall, based on our data, we conclude that the Resilin matrix composition and probably function are largely conserved between D. melanogaster and D. suzukii. Despite this, we detected minor differences in DT signal intensity and distribution  suggesting that resilin matrices may be optimized in related species during evolution to accommodate slightly different lifestyles. The biomechanics consequences of these differences, once investigated, may serve to advance in biomimetics approaches.To evaluate the effects of the r Figure  and S3. ) Figure . The DT ) Figure . In otheSteven Lerch performed most histological experiments, analysed the data and contributed to the writing of the first draft of the manuscript. Yang Yang assisted Steven Lerch in microscopy in some experiments. Justin Flaven‐Pouchon performed the statistical analyses of the data. Nicole Gehring generated the CRISPR/Cas9 flies. Bernard Moussian conceived and planned all experiments. He analysed the data and wrote the manuscript.The authors declare no conflicts of interest.Supporting information.Click here for additional data file.Supporting information.Click here for additional data file.Supporting information.Click here for additional data file.Supporting information.Click here for additional data file.Supporting information.Click here for additional data file.


BackgroundSelf-efficacy is an important determinant of treatment adherence, and peer modelling of success can provide vicarious self-efficacy. A series of patient stories  were developed with people with cystic fibrosis (CF) as part of the CFHealthHub multi-component adherence intervention, aiming to demonstrate success with daily therapy in ‘people like me’.MethodologyOne-to-one semi-structured interviews exploring patients’ experiences, barriers and facilitators of nebuliser adherence were audio and video-recorded between October 2015 and August 2016. Interview transcripts were reviewed to identify descriptions of problem-solving and sustained treatment success. Positive stories potentially providing vicarious descriptions of success were selected as video clips.ResultsIn total, 14 adults with CF were recruited from five UK CF centres. Each participant contributed a median of five (interquartile range: 3-6) video clips, and a total of 57 unique clips were uploaded onto the CFHealthHub digital platform. Nine of those clips spanned two categories, hence, there were 66 clips across 16 categories.ConclusionsThe videos were well received though some adults were concerned that comparisons with peers might create anxiety by highlighting the possibility of future decline or current relative underperformance. It is important to sensitively support choice when providing resources aiming to increase vicarious self-efficacy. Our experience may guide the development of similar videos for people with other long-term conditions. Cystic fibrosis (CF) is a genetic condition where thick mucus in the lungs and digestive system leads to progressive lung damage and malabsorption of nutrients. It is an archetypal long-term condition; there is as yet no cure for CF but highly efficacious medications exist. However, medication adherence is low in the range of 35-50% among adults with CF ,2. Develhttps://www.cfhealthhub.com/, ISRCTN14464661) is an improvement collaborative among 17 UK adult CF centres in which technical, behaviour change and implementation science approaches are explicitly used to learn and improve. Within a learning health system, patients can contribute new knowledge because data on all patients are routinely captured as an integral by-product of care delivery, and the capture of patient experience and best practices are seamlessly embedded in the care delivery process. At the heart of the Learning Health System is a tailored multi-component intervention to help people with CF master self-care with inhaled therapies using a digital platform supported by healthcare professionals. This multi-component intervention [The CFHealthHub digital Learning Health System  showed that the CFHealthHub intervention achieved higher objectively measured adherence and lower perceived treatment burden sustained up to 80 weeks . The CFHEach of the COM-B domains may be subdivided, for example, motivation may be automatic (e.g. habit of using nebuliser) or reflective (e.g. beliefs about treatment). Self-efficacy, or a person’s perception of his/her capabilities to perform a behaviour , is a crSelf-efficacy can be increased through the sharing of vicarious experiences of success provided by social/peer role models because seeing people similar to ourselves succeed raises our beliefs that we can also succeed . For exaThe CFHealthHub group, therefore, worked with the University of Oxford Health Experiences Research Group (HERG) to develop a set of ‘talking heads’ videos as part of the CFHealthHub multi-component intervention. HERG collects patient experiences of care pathways to familiarise others with the treatment journey. In the collaboration between the CFHealthHub group and HERG, the focus was on the challenge of the success of preventative self-care. The ‘talking heads’ videos aim to increase vicarious self-efficacy in success with daily inhaled treatments by showing a range of different types of people with CF managing or increasing their adherence. Thus, the videos provide a library of strategies used for successful self-care. This paper describes the method of selection and production of the ‘talking heads’ videos.The methodology used to develop the ‘talking heads’ videos is summarised in Figure Purposive sampling was conducted using the CF registry and clinical records to identify people with CF aged ≥16 years who fulfilled the inclusion criteria  in participants’ homes between November 2015 and August 2016. The topic guide (Appendix A) was designed to explore participants’ experiences of living with CF, to reflect on times when nebuliser adherence had been lower, and how and why the use of nebulised medication subsequently improved. Data were collected and analysed concurrently. Following each interview, audio recordings were transcribed verbatim by a HERG transcriber. The interview transcripts were sent to participants who had the option of removing any sections that they wished prior to analysis.Final transcripts were reviewed by the CFHealthHub research team  to identify sections that demonstrated talk about making positive changes to adherence, reflections on difficult times and overcoming challenges or examples of problem-solving that enabled sustained adherence. The research team focused on positive stories with the potential to provide vicarious descriptions of success likely to increase self-efficacy. The sections chosen were relatively short so that the resultant videos were not too time-consuming to watch. Highlighted transcripts were then sent back to SK for HERG to extract relevant video clips from video recordings.The research team reviewed the extracted clips. Transcripts of clips considered suitable for inclusion were sent to participants seeking permission to publish the video clips on the CFHealthHub digital platform. Participants had the option of being identified by their first names in the video library or using a pseudonym. Following confirmation of the agreement, a website delivery manager from HERG did the final editing of selected video clips and uploaded them onto the CFHealthHub platform.The main content of the selected video clips, for example, ‘advice to younger self’ was identified and used to tag the episode in CFHealthHub. The research team (which included a person with CF) identified gaps in the ‘library’, and purposive sampling was used to try to include as yet missing experiences identified as important, such as managing CF whilst at university or as a parent caring for a young child.https://www.cfhealthhub.com/), who can access the video clips by logging onto the CFHealthHub website or app.The research team sorted the final version of the video clips into different categories to organise the CFHealthHub digital platform video library. For ease of use, the categories were searchable within the digital platform, and each clip also had searchable hashtags, for example, #uni alongside the video thumbnails. The video library was reviewed by the Patient & Public Involvement group of CFHealthHub, who suggested short descriptions for each video clip to accompany the thumbnails. The video clips are only available for participants of the CFHealthHub digital Learning Health System  were females, and the median age was 27 years (interquartile range (IQR): 25-33 years) (Table .9% were Adapting a proven methodology developed by HERG to create videos for other long-term conditions such as asthma, diabetes and epilepsy, we have produced a series of ‘talking heads’ videos that are hosted on the CFHealthHub digital platform to support habit formation with inhaled therapies. These videos extend the multi-component behaviour change toolkit available within CFHealthHub by providing videos of positive role models and allowing CFHealthHub users insight into the experiences of others overcoming the daily challenges of living with CF. In a 607-participant multi-centre RCT, the multi-component CFHealthHub intervention was shown to achieve higher objectively measured adherence to inhaled medications sustained over 12 months, higher body mass index and lower perceived CF treatment burden, without increasing anxiety [There is emerging evidence for the role of peer modelling to increase self-efficacy and promote behaviour change in other long-term conditions. An RCT evaluating videos of patient narratives about their individual struggles and successful hypertension management showed improved blood pressure among people with uncontrolled hypertension . It is t‘People like me’ who have successfully managed behaviour change and achieved good health outcomes can also be thought of as ‘positive deviance’. Positive deviance refers to at-risk individuals who have figured things out for themselves and are able to follow beneficial practices that are uncommon within the community, thus achieving better outcomes in a population that shares similar risks . The posWe found that a key motivation for people with CF who participated in interviews to develop these videos was a desire to help others who may be struggling with similar problems by sharing their experiences. Talking openly about their previous barriers, as well as how they had overcome them, was something they felt might benefit others. In addition to the ‘talking heads’ videos, the content of the interview transcripts also generated valuable insights into difficulties that people with CF often face in motivating themselves and overcoming barriers to adhere to nebulised treatment. Useful strategies were shared such as setting achievable goals coupled with rewards for achievement also the importance of finding a treatment routine that fits around the life of the person indicating the necessity for individually tailored approaches to adherence support.In our study, there were interesting differences in emphasis when transcripts alone were compared with transcripts viewed alongside video recordings. This discrepancy highlights the importance of developers watching the video in full to ensure all relevant clips are utilised. It may be beneficial for others carrying out similar work to watch the video in full to ensure all relevant clips are utilised. The sample size of this study was limited by budget and time constraints, so we may not have reached saturation in barriers and solutions sampled. While participants using CFHealthHub generally welcomed the video library, some preferred not to view videos to avoid comparison with people like them who have more severe CF or people like them who seemed healthier . AlthougWe have developed ‘talking heads’ videos to support behaviour change as part of a complex adherence intervention. The ‘talking heads’ videos aim to increase vicarious self-efficacy with using daily inhaled treatments by showing a range of people with CF managing or increasing their adherence. These videos broaden the behaviour change intervention of CFHealthHub by providing positive role models and allowing CFHealthHub users insight into the experiences of others with CF. Some but not all those who have used the CFHealthHub digital platform have found the videos to be helpful. It is important to sensitively support choice when providing resources aiming to increase vicarious self-efficacy. Our experience may guide the development of similar videos for people with CF and other long-term conditions.


Occupational therapy (OT) helps older adults improve their ability to perform day-to-day tasks. Veterans Health Administration (VHA) is the single largest employer of occupational therapy (OT) practitioners in the United States and a forerunner in telehealth. As a result of COVID, OT video visits increased by nearly 2000% from 2019 to 2020. To ascertain barriers and facilitators to this shift in care delivery, we conducted interviews between January and April 2021 with OT practitioners (N=27) who were high users of VA Video Connect (VVC), VHA’s videoconferencing software. OT participants were from rural and urban settings, and had completed an average of 536 VVC appointments each in 2020. Participants used VVC to deliver a variety of OT services, including mental health groups and home safety interventions. Facilitators to VVC included, a) Patient characteristics, such as positive perceptions of VVC and technological skill, b) OT clinician characteristics, like flexibility, level of experience, and desire to increase patient access to care, and, c) VHA’s telehealth infrastructure. Barriers included, a) Patients’ lack of familiarity or skills with technology, particularly older patients, b) challenges translating traditionally hands-on care to video, and c) unreliable internet connectivity, particularly for rural patients. This study broadens our understanding of video telehealth service delivery for care which has historically been delivered in brick-and-mortar settings. Understanding challenges and enablers to video telehealth highlights opportunities to increase access to those who face barriers, such as older, rural patients.


HCN4 mutation, HCN4-R666Q, in two sporadic arrhythmia patients with sinus bradycardia, QT prolongation, and short bursts of ventricular tachycardia. To determine the functional effect of the mutation, we conducted clinical, genetic, and functional analyses using whole-cell voltage-clamp, qPCR, Western blot, confocal microscopy, and co-immunoprecipitation. The mean current density of HEK293T cells transfected with HCN4-R666Q was lower in 24 to 36 h after transfection and was much lower in 36 to 48 h after transfection relative to cells transfected with wildtype HCN4. Additionally, we determined that the HCN4-R666Q mutant was more susceptible to ubiquitin-proteasome system–mediated protein degradation than wildtype HCN4. This decreased current density for HCN4-R666Q could be partly rescued by treatment with a proteasome inhibitor. Therefore, we conclude that HCN4-R666Q had an effect on HCN4 function in two aspects, including decreasing the current density of the channel as a biophysical effect and weakening its protein stability. Our findings provide new insights into the pathogenesis of the HCN4-R666Q mutation.Mutations in the hyperpolarization-activated nucleotide-gated channel 4 (HCN4) are known to be associated with arrhythmias in which QT prolongation (delayed ventricular repolarization) is rare. Here, we identified a  Sinus bradycardia is defined as a heart rate less than 60 beats per minute (bpm) in adults . AsymptoIf) carried by the hyperpolarization-activated cyclic nucleotide-gated (HCN) cation channel plays a specific role in pacing and heart rate control   (HCN4-G1097W)   , left ve, G811E) , 21, atr, K530N) . Newborn, K530N) . Almost  channel . Therefo channel . AlthougC-linker , 30, porC-linker , 17, 18,HCN4-R666Q, which was located in the non-cAMP-binding region of CNBD, in two sporadic patients with sinus bradycardia, QT prolongation, and short bursts of ventricular tachycardia. We hypothesized that HCN4-R666Q mutation was associated with the clinical phenotype of two sporadic patients and explored the mechanism of channel dysfunction caused by the mutation.Here, we identified a novel heterozygous mutation A) suffering from palpitation and syncope were admitted to Fuwai Hospital as outpatients. On arrival, the heart rate of two patients was slowed down , but their blood pressure was normal . According to the two patients’ descriptions, they were the only child in their families. Patient A had a son and an aborted fetus, while patient B had no children. Panel sequencing showed that the two patients with clinical manifestations of sinus bradycardia, QT prolongation, and short bursts of ventricular tachycardia carried HCN4-R666Q  mutation in which glutamine was substituted for arginine. In addition, no other significant mutations in arrhythmia-related genes were observed in the two patients (https://genebass.org/) suggested that the HCN4 p. Arg666Trp mutation was associated with susceptibility to atrial fibrillation (p = 9.7e-4), indicating the potential significance of this residue in HCN4 . Holter monitoring showed the two patients have lower average  and minimum  heart rates. 24-h Holter recordings were analyzed by a computer system and confirmed by an electrophysiologist. The patients were diagnosed as having sinus bradycardia with average heart rate <60 bpm concurrent with QT prolongation with QT-intervals >0.44 s  corrected QT-intervals = 0.51 s; Patient B: QT-intervals = 0.48 s; QTc = 0.51 s) accompanied by short bursts of ventricular tachycardia as assessed by 24-h Holter monitoring and 12-lead ECG (C–F). Patient A had premature ventricular complexes  and two patients had no history of coronary heart disease or drug-induced QT prolongation. No abnormal changes were observed in plasma ion concentrations of two patients, including sodium, potassium, magnesium, and calcium. Echocardiography examination of two patients showed no structural heart disease  showed that arginine 666 was replaced with a glutamine (R666Q) in the highly conserved CNBD of the HCN4 subunit (H). This mutation was not found among the 1000 genomes and EPS6500 database, and no minor allele frequency was observed in the general population in the Exome Aggregation Consortium database , PROVEAN (deleterious), FATHMM (damaging), and PolyPhen-2 (possibly damaging). A multiple alignment  showed that arginine 666 was conserved across a wide range of mammals, including Homo sapiens, dog, rabbit, zebrafish, chicken, and mouse (I).Sanger sequencing G showed  subunit H. This mdatabase . Furthernd mouse I.HCN4-R666Q mutation, we performed whole-cell voltage-clamp recordings of HEK293T cells transfected with only wildtype HCN4 or HCN4-R666Q (homomeric) or cells that were cotransfected with wildtype and HCN4-R666Q (heteromeric). A series of hyperpolarizing steps were applied to activate functional HCN4-mediated currents (A). The results showed that the amplitudes and density of wildtype HCN4 currents were significantly larger than the current amplitudes recorded from cells expressing mutant homomeric or heteromeric HCN4-R666Q channels in both 24 to 36 h and 36 to 48 h after transfection (A and B). The mean current density of homomeric and heteromeric mutant channels and wildtype channels in 24 to 36 h and 36 to 48 h after transfection were also calculated. Interestingly, the mean current density of mutant channels was lower in 24 to 36 h after transfection and much lower in 36 to 48 h (C) compared to wildtype HCN4, respectively. The voltage for half maximal activation (V1/2), which was calculated by fitting the activation curve of HCN4 currents using the Boltzmann equation, was similar for both wildtype and homomeric HCN4- R666Q channels, suggesting that the HCN4-R666Q mutation did not influence channel activation kinetics  was included into patch pipettes. After breaking into the whole-cell mode, the cells 24 h after transfection were held for 10 min at a resting membrane potential of -30 mV to stabilize the intracellular 8-Br-cAMP concentration. The values of V1/2 of activation curves for wildtype and mutant HCN4-R666Q channels were shifted about 10 mV toward the depolarization direction (F and G), whereas k values of the activation curves showed no significant difference between wildtype and homomeric HCN4-R666Q channels in the presence of 8-Br-cAMP  after transfection (A). However, total protein and membrane protein levels at 48 h and 72 h after transfection were significantly decreased in HEK293T cells expressing homomeric HCN4-R666Q compared to those expressing wildtypeHCN4 (B and C). The localization of the wildtype and homomeric HCN4-R666Q mutant protein by confocal microscopy also suggested that protein levels of HCN4-R666Q were decreased compared to wildtype (D).Electrophysiological analysis showed that HCN4-R666Q channels reduced the current density more significantly in 36 to 48 h after transfection, so we determined whether this change was resulted from the alteration of mRNA or protein levels. There were no significant differences in the sfection A. HowevetypeHCN4 , B and Cwildtype D.Figure versus 10.7 ± 1.98 h, p = 0.011; Fig. 4A), indicating the HCN4-R666Q protein was less stable and more rapidly degraded than wildtype.To understand the potential mechanism of the altered protein levels of HCN4-R666Q, we examined the half-life of wildtype and HCN4-R666Q protein. In HEK293T cells treated with the protein synthesis inhibitor cycloheximide (CHX) , the halB). This result suggested that the autophagy pathway was successfully inhibited, and HCN4 mutant protein levels were not regulated by autophagy.Next, we investigated the pathway mediating HCN4-R666Q protein degradation. In transfected HEK293T cells treated with autophagy inhibitors, HCN4-R666Q protein levels were not rescued, although p62 and the ratio of LC3-I to LC3-II were accumulated B. This rC). Furthermore, an immune-coprecipitation assay indicated that HCN4 was ubiquitinated, and ubiquitination levels of HCN4-R666Q mutant protein were higher than that of wildtype (D). Finally, we examined whether the SUMOylation, which was the reversible covalent attachment of a small ubiquitin-related modifier (SUMO) protein to a target protein, affected HCN4 protein levels. Immunoblot analysis showed that the expression levels of four SUMO isoforms (SUMO 1–4) were not significantly different between HCN4-R666Q mutant and wildtype (E). Taken together, these results indicated that the UPS pathway was considered as the potential mechanism of reduced HCN4-R666Q protein levels.We next examined whether HCN4 protein were affected by the ubiquitin-proteasome system (UPS). MG132 affected HCN4 protein levels dose-dependently, and the optimal concentration was 5 μM C. Furthewildtype D. Finallwildtype E. Taken HCN4-R666Q mutation, we determined the effect of proteasome inhibitor MG132 on rescuing the decreased protein levels and current density of HCN4-R666Q. HEK293T cells were transfected with wildtype or the homomeric HCN4-R666Q mutant and then treated with MG132 for 12 h. The half-life of the wildtype and HCN4-R666Q protein both were prolonged in the presence of MG132  (A). Interestingly, in the presence of MG132 treatment, HCN4-R666Q protein levels were similar to that seen for wildtype in the absence of MG132 treatment (B). Consistently, decreased mutant channel current density was obviously rescued by treatment of transfected cells with MG132 for 12h (C–F). However, there was no significant difference in V1/2 values after MG132 treatment, whereas the k was different apparently after MG132 treatment  A. Interereatment B. Consis for 12h , C–F. Horeatment .Figure 5HCN4-R666Q mutation in two sporadic patients with sinus bradycardia, QT prolongation and short bursts of ventricular tachycardia. No other pathogenic mutations in the known arrhythmia-related genes were found in the two patients, suggesting that the loss of function of the HCN4 mutation might be associated with sinus bradycardia, QT prolongation, and short bursts of ventricular tachycardia. We found the HCN4-R666Q mutation decreased the channel electrophysiological function and increased its protein degradation, both resulted in reduced current density of HCN4 channels. We also showed that proteasome inhibitor might has therapeutic benefits to mitigate the effects of this HCN4 mutation  carried HCN4-R666Q mutation, which has not been studied. The allele frequency of the mutation in the Non-Finnish European population was 3.72e-6. Interestingly, another mutation at residue 666, HCN4 p. Arg666Trp (R666W), appears to be associated with the susceptibility to atrial fibrillation (p = 9.7e-4). The allele frequency of HCN4-R666W in the Non-Finnish European population was 2.42e-5, indicating the significance of position 666 in HCN4. Several lines of evidences further suggested that the HCN4-R666Q mutation had functional consequences and was not simply a silent polymorphism. First, within the general population, the HCN4-R666Q variant was not found in the 1000 genomes, EPS6500, or the Exome Aggregation Consortium databases. Second, the mutation occurred in a segment that was highly conserved throughout evolution. Third, this substitution decreased electrophysiological function that underlies the clinical phenotype.Two individuals in UK biobank  suffering from palpitations and syncope were admitted to Fuwai Hospital as outpatients.According to the two patients’ descriptions, both patients were normally healthy and had no medical history and familial cardiac events, sudden cardiac death, or other diseases and did not take drugs that could prolong QT.Patients were evaluated by clinical examination, 12-lead echocardiography, 24-h Holter monitoring, and Doppler echocardiography. The two patients’ parents and patient A’s son were evaluated by 12-lead ECG and Doppler echocardiography.The patients were not followed-up in accordance with the patients’ wishes. The study was approved by the ethics committee of Fuwai Hospital, Peking Union Medical College , Beijing, and conformed to the principles outlined in the Declaration of Helsinki. Written informed consent was obtained from each patient.HCN4-R666Q mutation is pathogenic, we tested the pathogenicity potential based on in silico analyses, including Mutation Taster (http://www.mutationtaster.org/), PROVEAN (http://provean.jcvi.org/index.php), FATHMM (http://fathmm.biocompute.org.uk/), and PolyPhen-2 (http://genetics.bwh.harvard.edu/pph2/) (To determine whether the u/pph2/) , 58, 59.HCN4 cDNA (GenBank: NM_005477) was subcloned into GV230 vector containing a C terminally fused enhanced green fluorescent protein. Subsequently, the HCN4-R666Q mutation was introduced using the QuikChange II Site-Direct Mutagenesis Kit (Stratagene) into GV230 vector with appropriate mutagenic primers. All constructs were verified by Sanger sequencing.To analyze the effect of the mutation in cells, we constructed plasmids with wildtype or mutated HCN4. Human 2 at 37 °C.HEK293T cells were cultured in Dulbecco’s Modified Eagle’s Medium supplemented with 2 mM glutamine, 10% fetal bovine serum (Gibco), 1% P/S  (Gibco) in 5% COHEK293T cells, which were plated onto 35-mm gelatin-coated dishes, were transfected with 0.6 μg of wildtype or mutant HCN4-R666Q plasmid DNA using Lipofectamine 3000 (Invitrogen). In co-expression experiments, equal amounts (0.3 μg) of both plasmid DNAs (wildtype and HCN4-R666Q) were transfected. GFP-positive cells were used for whole-cell patch clamp experiments.4, 1 NaCl, 0.1 CaCl2, 1 EGTA, 3 MgCl2 and 10 Hepes (pH 7.2 with KOH). The bath solution contained (in mmol/L): 60 KCl, 75 NaCl, 10 glucose, 2 CaCl2, 1 MgCl2, 1.2 NaH2PO4, and 10 Hepes (pH 7.3 with NaOH). Intracellular and extracellular solution osmolarity was adjusted with glucose to 290 and 300 mOsmol/L, respectively. Series resistances were electronically compensated by 60%. To evaluate functional properties, HCN4 currents were evoked by hyperpolarizing steps ranging from -40 to -130 mV . For drug application experiments, the transfected HEK293T cells were treated with 5 μM MG132 (a proteasome inhibitor) for 12 h, and current density was determined by dividing the fully activated current at -120 mV at the end of the 5s pulse by cell capacitance. The voltage dependence of channel activation was analyzed by fitting the Boltzmann function (I - I min)/(I max - I min) = 1/(1 - exp [(V - V1/2)/k]), where V1/2 is the half-activation voltages, and k is the inverse slope factor.HCN4 channel-mediated currents were recorded in HEK293T cells 24 to 48 h after transfection using conventional whole-cell patch clamp techniques at room temperature (21–23 °C) as described , 60. SigHCN4-R666Q, HEK293T cells were washed three times with ice-cold PBS, and total RNA was isolated using Trizol (Invitrogen). Then, 1 μg of total RNA was reverse transcribed using Hifair II 1st Strand cDNA Synthesis Super Mix. Real-time PCR assays were run in triplicate and quantified using the Prism 7500 sequence-detection system (ABI) with Hieff qPCR SYBR Green Master Mix. Relative mRNA expression was calculated by the ΔΔCt method, and the values were normalized to the expression of actin beta (ACTB). Primers used: HCN4 forward 5′-TGGACACCGCTATCAAAGTGG-3′ and HCN4 reverse 5′-CTGCCGAACATCCTTAGGGA-3′.At 12, 24, 36, and 48 h after transfection with wildtype or For immunoblotting, HEK293T cells at 36, 48, and 72 h after transfection were washed three times with ice-cold PBS, and total proteins were lysed with RIPA buffer and membrane proteins were extracted using a Minute Plasma Membrane Protein Isolation and Cell Fractionation Kit (Invent Biotechnologies). Protein samples were denatured by heating to 37 °C for 30 min and then used for immunoblot analyses. ACTB was served as a total protein control and Na, K-ATPase  was used as a membrane protein control. All data shown represented at least three independent experiments.Immunofluorescence assay was performed as described previously , 21. At Protein half-life assay was performed as described previously . At 36 hAutophagy pathway assay was performed as described previously , 63, 64.Ubiquitination and SUMO assays were performed as described previously , 65. At g at 4 °C for 20 min. The lysates were precleared by incubation with protein A/G-agarose (Proteintech) for 1 h at 4 °C. Specific antibodies were incubated overnight at 4 °C followed by protein-A/G agarose beads incubation at 4 °C for 4 h. After the beads were washed three times with washing buffer, the immunoprecipitation was heated in 2× sample buffer at 37 °C for 30 min and then separated by SDS-PAGE. The separated proteins were analyzed via immunoblot.Co-immunoprecipitations were performed as previously described , 67, 68.t test was performed for comparison of two groups only. p < 0.05 was considered statistically significant; 2-tailed tests were used for all analyses. Analysis of the Western blot bands was performed with ImageJ image analysis software. All measurements were obtained from distinct samples. Electrophysiological data were analyzed with patchmaster software. All statistical analyses were performed using GraphPad Prism 8.0.Data are reported as the mean ± SD. Statistical analysis of the results of >2 groups was carried out by one-way ANOVA followed by the least significant difference test or Tukey test. Student’s Raw data will be available upon request.This article contains The authors declare no conflicts of interest with the contents of the article.