Leersia hexandra, Acroceras zizanioides, Hymenachne amplexicaulis, and Urochloa arrecta  as a control group. Green forage samples were taken in a 1 m2 frame at 30, 40 and 50 days of age and biomass production was estimated. The chemical composition was analyzed by infrared spectroscopy, and the fermentation parameters by the in vitro gas production technique. Data were analyzed using a mixed model for repeated measures. The nutritional characteristics variations were: dry matter , crude protein , neutral detergent fiber , ash (5.8–15.8%) and dry matter digestibility (DMD) between 20.8 and 60.6% from 12 to 48 h of fermentation. L. hexandra and A. zizanioides presented higher biomass, CP, ash, cellulose and Ca production. During the experimental period (30 to 50 days), the grasses did not present significant changes in nutrient availability. L. hexandra increased the concentrations of ammonia and total volatile fatty acids (VFA) and butyric acid. This last effect was also observed in the A. zizanioides grass. L. hexandra and A. zizanioides grasses constitute an alternative forage resource for ruminants during the flood seasons of the studied ecosystem.The floodplain ecosystem of the Colombian Orinoquia has a diversity of native grasses; however, there is little information on its agronomic and fermentation characteristics. The aim of this study was to determine the chemical composition and fermentation parameters of the grasses Leersia hexandra, Acroceras zizanioides and Hymenachne amplexicaulis) and a “control” grass (introduced Urochloa arrecta—Tanner grass) were sown and sampled at 30, 40 and 50 days of age. On each sampling date, biomass production in a 1 m2 frame was estimated, and the chemical composition and fermentation parameters were analyzed using near-infrared spectroscopy and the in vitro gas production technique, respectively. Data were analyzed using a mixed model for repeated measures and the least significant difference (LSD) was used for mean differentiation (p < 0.05). The grasses’ nutritional characteristics varied as follows: dry matter , crude protein , neutral detergent fiber , ash (5.8–15.8%) and dry matter digestibility (DMD) between 20.8 and 60.6% from 12 to 48 h of fermentation. Native plants such as L. hexandra and A. zizanioides presented higher biomass production, CP, ash, cellulose, and Ca levels than the control plant. During the experimental period (30 to 50 days), the grasses did not present significant nutrient availability changes. In terms of fermentation characteristics, L. hexandra increased ammonia concentrations and total volatile fatty acids (TVFA) and butyric acid. This latter effect was also observed in A. zizanioides grass. L. hexandra and A. zizanioides grasses constitute a valuable alternative forage resource during the flooding times of the studied ecosystem.Grasses from lowland ecosystems in flooded savannahs are useful to feed extensive grazing animals; however, scarce information about its agronomic and fermentation characteristics exists. This study aims to determine the chemical composition and fermentation parameters of native grasses from the floodplain lowlands ecosystem in the Colombian Orinoquia. Three native grasses ( The Orinoquia region constitutes 25.4 million hectares of the Colombian territory of which more than 5 million consist of flooded savannahs located in the Arauca and Casanare departments. The flooded savannahs are an important ecosystem to support livestock production as one of the main economic lines in the region. The breeding and raising of extensive grazing cattle are the predominant livestock activities in the zone, where native grasses and legumes make up about 90% of the food available for animals ,2. The fAcroceras zizanioides (Kunth) Dandy (1931), (blackwater straw); Hymenachne amplexicaulis (Rudge) Nees (1829) (water straw); Leersia hexandra Sw. (1788) (Lambedora grass); and Paratheria prostrata Griseb. (1866) (Carretera grass), among others, can be found [The low physiographic positions are zones that remain with a layer of water during the rainy season. The grasses and legumes within this ecosystem are adapted to the waterlogging conditions and frequently are affected during the dry period, where the layer of water disappears . The soih) Dandy 931, (blabe found . The gra) Nees 189  . AccordiForage chemical composition analysis includes the estimation of dry matter, fiber fractions , crude protein (CP), energy content, ash and some specific minerals, among others , using tThis study was conducted at the Clarinetero Territorial Division Center, municipality of Arauca, eastern Colombia. The region is characterized by flat and floodable savannah topography with the presence of a “low” physiographic position  which constitute the lowest areas of the flooded savannah ecosystem , and preDuring the research period , climate data were collected with a portable weather station located approximately 600 m from the experimental site. The highest and lowest average temperatures occurred in August . The total precipitation during the grass growth cycle was 612 mm, and the average relative humidity was 91.8%Urochloa arrecta was included in the experiment as a “control” grass, as it is an introduced species commonly used in livestock systems in the region for grazing animals. The names and characteristics of the evaluated grasses  under a completely randomized experimental design.At the start of the rainy season in the area (May 2022), each species was triplicate established in the “low” physiographic position in 9 m2O5) was added to each plot at planting to ensure adequate N and P availability for the plants [The agronomic management for the establishment of each experimental plot included manual soil preparation, sowing, irrigation and weed eradication. Approximately 500 g of diammonia phosphate . The forage samples’ nutritional values were analyzed using near-infrared reflectance spectroscopy  sampled from 2014 to 2016 from different livestock regions of Colombia [In each experimental plot, height measurements were taken from 15 random plants at 30, 40 and 50 days of age; the measurement was taken from ground level to the longest leaf using a metric rule. Then, three sampling points within each plot were randomly selected during plants emergence, and the cut-off ages  were assigned to each measurement point randomized. These points were used to take grass samples, using a 1 mpy  at 30 days of age were taken to submit them to short-term in vitro fermentation tests (48 h) following the methodology proposed [Cenchrus clandestinus) (Hochst. ex Chiov.) Morrone [2 pumping to maintain the anaerobiosis. The vials were sealed with rubber stoppers and aluminum clips and placed in a forced ventilation oven at 39 °C for 48 h.Samples of the grasses (proposed . Forage proposed  and 10 m Morrone . The inoVariables such as dry matter degradation (DMD), gas volume and pH were evaluated at 12, 24 and 48 h, while ammonia, methane, total volatile fatty acids (TVFA) and the acetic, propionic and butyric acids were studied at 24 and 48 h.3 selective electrode. CH4 quantification was carried out by gas chromatography using Shimadzu GC-2014 equipment  with an FID detector in a GC-Solution data station and manual injection with 25 µL VICI® syringes. A 30 m long Stabilwax®—DA, 0.53 mm internal diameter and 0.25 µm film thickness was used. The standard used was 9.99% gaseous methane in balance with grade 5 nitrogen certified by Cryogas instrumental analysis. Nitrogen was used as the carrier gas with a flow set to 37.9 mL/min and an injector temperature of 150 °C. The methane production was estimated as a proportion of total gas volume. Finally, the VFA was also determined using gas chromatography with the same equipment and characteristics mentioned above. However, the standards used in this case were acetic, propionic and butyric acid, Fluka® analytical grade. Nitrogen was used as the carrier gas with a flow set to 42 mL/min and an injector temperature of 250 °C. The fermentation process was carried out in the ruminal biotechnology laboratory (BIORUM) of the National University of Colombia—Medellín [The DMD was determined using the gravimetric method. Gas volume was estimated using a pressure digital transducer . The pressure data were used in a quadratic regression model estimated in the laboratory, to calculate the gas volume corrected for atmospheric pressure. The ammonia in ruminal fluid was determined using the potentiometric method with an ISE NHMedellín .ijk represents the agronomic, compositional or fermentation variables observed in the “it” grass type and “jt” cutting age or fermentation time; μ represents the general average of the observed variable; Gi represents the fixed effect of “it” grass type  and H. amplexicaulis); Tj represents the fixed effect of “jt” cutting age  or fermentation time ; (GT)ij represents the interaction effect between grass type and cutting age or fermentation time; rk represents the random effect corresponding to the kth repetition of each GT interaction; and εijk represents the random error term. It was assumed that rk and εijk were independent and distributed ~N . When it was required, the evaluated models were corrected for heteroskedasticity using functions that related residual variances to the mean. Three covariance structures were analyzed to consider the relationships between repeated measures . The best model was selected using the Akaike and Bayesian information criteria. The analysis was performed using the “mixed and general linear models” option of the statistical InfoStat software [p < 0.05).As the evaluated agronomic, compositional and fermentation variables presented a longitudinal structure, data were analyzed using a mixed model for repeated measures  through software . The leaTo evaluate the relationship between the grasses’ chemical composition variables and their fermentation parameters, multivariate profile diagrams were made through the InfoStat software .p < 0.05), except for the green forage yield (GFY), dry matter (DMY) yield, forage height, cellulose content and Ca levels. Mean values of grass type x cutting age interaction are shown in Most of the evaluated productive and chemical composition variables showed statistical differences throughout the studied experimental period (p < 0.05). GFY was the highest in L. hexandra and A. zizanioides , followed by H. amplexicaulis and U. arrecta (control) . A similar behavior was observed in terms of DMY, where the highest productivity was found in L. hexandra (1.9 ton/DMY/ha) followed by A. zizanioides (1.3 ton/DMY/ha). The species U. arrecta (control) and H. amplexicaule behaved similarly (1 ton/DMY/ha approximately in both cases). During the evaluated period, GFY and DMY increased obtaining the highest yields at 50 days (p < 0.05), with average values ranging between 4 and 5.4 ton/GFY/ha and 1.1 and 1.5 ton/DMY/ha.GFY and DMY presented differences between species and cutting ages (p < 0.05). H. amplexicaulis was the tallest plant (81.2 cm) followed by U. arrecta (control—60.0 cm), L. hexandra (56.1 cm) and A. zizanioides (40.4 cm). As expected, the lowest height was found at 30 days (47.7 cm) and the highest at 50 days (67.3 cm).Plant height varied between species and cutting ages (L. hexandra species (p < 0.05) with values between 33.5 and 39.1%. A. zizanioides was superior to the control plant at 30 days ; however, this difference was not observed in the other evaluated ages. At 40 and 50 days, A. zizanioides and H. amplexicaulis were similar to the control (U. arrecta) with values between 22.5 and 26.2%.During the entire experimental period, DM percentage was higher in the L. hexandra, A. zizanioides and H. amplexicaulis, while in the control (U. arrecta) a decreasing behavior was observed. At 30 days, L. hexandra and A. zizanioides plants presented the highest CP levels  followed by U. arrecta (10.1%) and finally H. amplexicaulis (7.5%). At 40 days, A. zizanioides continued with the best performance (11.3%), while the rest of the plants were between 7.2 and 8.2%. On day 50, A. zizanioides and L. hexandra were superior to the rest of the plants with values of 12.2 and 10.6%, respectively.Crude protein levels remained relatively stable in the native species L. hexandra showed the highest ash content during the entire experimental period (p < 0.05) with values between 13.8 and 15.8%. A. zizanioides and H. amplexicaulis were comparable to the control (U. arrecta) at 30 and 40 days (6.7–9.8%). By day 50, only A. zizanioi-des was superior to the control .A. zizanioides and H. amplexicaulis, while in L. hexandra and the control (U. arrecta) a decreasing response was evidenced. In general, A. zizanioides presented the highest Ca percentages during the study . The other species were similar with values between 0.23 and 0.46%. Regarding P levels, H. amplexicaulis and A. zizanioides showed the highest values, with a statistical difference (p < 0.05) only at 40 days . The control plant (U. arrecta) and L. hexandra presented the lowest values ranging between 0.14 and 0.23%.Ca concentrations remained constant during the experimental period in H. amplexicaulis and the control (U. arrecta), while L. hexandra and A. zizanioides remained relatively constant. At 30 days, the NDF in the A. zizanioides and L. hexandra species was similar to the control (59.9 to 60.6%); however, it was lower at 40 and 50 days of growth . The H. amplexicaule plant presented the highest NDF values during the experimental period (66.3 to 69.6%). The control plant (U. arrecta) presented the lowest ADF content at 30 days (29.8 vs. 37.8–40.6%). No differences were observed between the native species and the control plant at 40 days of age, with values between 35.4 and 37.1%. Finally, at 50 days, both the control (U. arrecta) and L. hexandra attained the lowest ADF values .The NDF varied among the species, with slight increases in p < 0.05); although, at 40 and 50 days no differences were observed among the studied species with values ranging between 8.1 and 9.6%.At 30 days of age, the lignin content was the lowest in the control plant . The cellulose concentration only varied between species (p < 0.05) and was highest in native species (28.6% on average) compared to the control plant (24.9%).The hemicellulose content at 30 days was superior in the control plant compared to the native grasses (30.5 vs. 23.3 on average for native plants). At 40 and 50 days, the control and Although in the evaluated plants, on average, the maximum forage yield (GFY and DMY) was reached at 50 days of age, their best performance in terms of DM, CP, NDF, ADF, lignin and P nutritional composition variables was at 30 days of age, without any drastic changes until day 50. Even some variables such as ash and Ca content attained their highest values at 30 days.p < 0.05). The pH was higher at 12 h compared with the other measurement times (6.7 vs. 6.6), while acetic and propionic acids were superior at 48 than 24 h .The fermentation parameters of the grasses varied according to the species, measurement time and the species x measurement time interaction. Variables such as pH, acetic and propionic acids were influenced only by the measurement time (p < 0.05). H. amplexicaulis presented the lowest gas volume compared with the other plants ; similarly, the gas volume increased during the evaluated measurement times with values of 124.3, 155.0 and 196.7 mL/ g DM for 12, 24 and 48 h, respectively (p < 0.05). TVFA was higher in L. hexandra compared with the other plants . Additionally, higher TVFA levels were found at 48 than 24 h . A similar result was observed with butyric acid concentration. In this case, L. hexandra presented the highest value , followed by A. zizanioides (4.7 mmol/L), while U. arrecta (control) and H. amplexicaulis were similar (4.3 mmol/L on average). Butyric acid was superior (p < 0.05) at 48 h (7.1 mmol/L) respect to 24 h (2.1 mmol/L).Gas volume, TVFA and butyric acid were affected by the grass species and measurement time independently (U. arrecta (control) presented a superior DMD compared with the native species in all of the evaluated measurement times (p < 0.05), with values of 31.9, 48.8 and 60.6% at 12, 24 and 48 h, respectively. Native grasses showed DMD mean values of 24.4, 37.1 and 49.5% at 12, 24 and 48 h, respectively. The ammonia was higher at 24 and 48 h in the U. arrecta (control) and L. hexandra species (p < 0.05) than the other grasses, with values ranging from 42.2 to 52.6 mg/dL. Methane at 24 and 48 h of fermentation was similar between the studied plants. The only difference observed was at 12 h of fermentation was that H. amplexicaulis presented higher methane concentrations than the L. hexandra species .Finally, DMD, ammonia and methane variables varied by the species x measurement time interaction. Fermentation parameters of the interaction are shown in U. arrecta (control) plant showed an average DMY of 1.0 ton/ha which is a lower value compared with the 1.3 to 1.5 ton/ha found in another study without any type of fertilization [Forage yield varied among the studied grasses with value ranging from 4.4 to 5.3 ton/GFY/ha and 1 to 1.9 ton/DMY/ha. The GFY and DMY values are within the range observed in tropical pastures adapted to the floodplain “banks” ecosystem in the Colombian Orinoquia, and other grasses cultivated in the dry season with 6 to 8 weeks of age ,27. The lization .L. hexandra and A. zizanioides behaved better than the control in terms of biomass production, with values ranging among 5.2 to 5.3 and 1.3 to 1.9 ton/ha for GFY and DMY, respectively. These yield differences could be attributed to several causes. Although the control plant (U. arrecta), L. hexandra and A. zizanioides present a similar growth habit (Stoloniferus), differences in grass height were observed, the control grass being one of the tallest, followed by L. hexandra and A. zizanioides. Likewise, in the case of native grasses a greater forage biomass density was also observed. Thus, the results suggest that native grasses’ height together with a better forage density as a consequence of a proper adaptation to the study area, allowed the observed biomass differences to be explained. Similarly, even though the U. arrecta (control) plant is recognized for growing in swampy and flooded grasslands [Under the lowland conditions of the studied ecosystem, native grasses such as asslands , it seemGrasses showed the greatest GFY and DMY at 50 days of age with values between 4 and 5.4 and 1.1 and 1.5 ton/ha, respectively. These yields are lower than the 4.4 to 8.5 ton/GFY/ha and 1.5 to 2.5 ton/DMY/ha observed in native grasses of “banks” physiography positions present in the same studied ecosystem . The lowU. arrecta. L. hexandra was remarkable in the DM (33.5 to 39.1%) content compared with the other species. Although waterlogged stressed plants are known to reduce the dry matter production [L. hexandra allow it to withstand the adverse flood conditions. Since DM in L. hexandra is high from an early to medium age (30 days onwards), it is expected that the cell content fraction of the DM  predominate over the fiber fraction for a longer period, allowing the pasture digestibility to last longer over time which is useful in livestock systems with long grazing periods [In terms of DM, CP, cellulose, ash, Ca and P content, native grasses performed better or similar than the control plant oduction , it seem33.5 to 3.1% conte periods .A. compresus and P. plicatulum native grasses from the floodplain “banks” ecosystem [A. zizanioides and L. hexandra were superior to the control plant during most of the studied growth phases , which makes them an important protein source for the livestock activity in the study area.The native grasses’ CP content was not significantly affected by the 50-day experimental period, while in the control grass a decreasing behavior was observed. In general, the grasses’ CP concentration is expected to reduce over time as the plants’ metabolic activity also decreases through the age ,33. The cosystem . Likewiscosystem . The grecosystem . In termL. hexandra and A. zizanioides plants , during the studied 50-day experimental period, while the P content was remarkable in H. amplexicaule and A. zizanioides, especially at 40 days of age . The ash content in L. hexandra was superior to the values observed in several Brachiaria accessions [L. hexandra presents a high nutrient extraction capacity. This property allows it to use the greatest nutrients availability reported in the “low” physiographic position [L. hexandra extraction capacity has been used to decontaminate soils with high levels of chromium or weathered oil and to bioestimulate the microbial activity in degraded soils [L. hexandra constitutes a useful resource to feed grazing animals; however, it is important to identify and quantify the mineral component of the ash fraction to ensure safe use in animal nutrition.The mineral content of some native grasses outperformed the control grass. The ash and Ca levels were the highest in cessions , native cessions . This reed soils ,37. The A. zizanioides and H. amplexicaulis are similar to other forages under tropical environments, with values within the optimal range for cattle feeding [The Ca and P content observed in  feeding .H. amplexicaulis and the control grass (U. arrecta), the NDF tended to increase over the experimental period. This is an expected behavior since plant cell wall components  increase as grasses reach maturity [L. hexandra and A. zizanioides, the NDF levels remained stable, ranging from 56.6 to 61.6%, attaining statistically lower values at 40 and 50 days of age compared to the control plant. This result suggests that in L. hexandra and A. zizanioides, the fiber components accumulation occurred at slower rates during the evaluated experimental period. A similar result was obtained with the Axonopus compresus grass under the “bank” physiographic position from the floodable savannah [The grasses’ NDF ranged from 56.6 to 69.6%, consistent with the values reported in grasses grown in the tropical regions of Mexico and Brazil ,38. In gmaturity . In the In the NDF fraction, the control plant showed a higher hemicellulose concentration among the studied species; however, the native grasses presented higher cellulose levels, which is advantageous since cellulose is the main polysaccharide of the primary and secondary cell wall that is potentially degraded by rumen microbes  to produce glucose. In the case of hemicellulose, despite the fact that it is also a potentially degradable polysaccharide, its degradation is less since it is bound to the indigestible lignin fraction which results in a lower digestibility ,40.Brachiaria brizantha (A. Rich.) Stapf, Brachiaria humidicola (Rendle) Schweick and Paspalum notatum Flüggé under tropical conditions [In terms of ADF and lignin content, the values observed in the grasses ranged between 29.8 and 40.6% and 6.5 and 10.4%, respectively, comparable to what was found in other native grasses of the evaluated ecosystem, nditions . DespiteUrochloa lines (Basilik and Cayman) and Megathyrsus maximum cv. Mombaza, where the optimal grazing point in tropical conditions was between 4 and 6 weeks of age, with greater quality loss after 8 weeks [Andropogon gayanus Kunth, cv. Planaltina or Massai grass (Panicum maximum × Panicum infestum cv. Massai) can be managed without losses between 21 and 63 days of age under a Brazilian semi-arid region [Although grasses on average attained their maximum forage yield (GFY and DMY) at 50 days of age, their chemical composition  was stabilized from 30 days of age without drastic changes. This trend is concordant with another report in tropical grasses where the maximum biomass and DMY were reached at 63 days, while the maximum CP and cellular content were observed at 21 days of age . This re 8 weeks . It has d region .Most of the grasses’ fermentation parameters did not change throughout the experimental period. Despite the pH varying according to the measurement time, its value ranged from 6.6 to 6.7 which is within the acceptable range to guarantee the optimal degradation of organic and fibrous components of feeds in in vitro fermentation systems .Urochloa hybrid cv. Cayman, Megathyrsus maximus cv. Mombaza, Urochloa brizantha cv. Toledo and Urochloa decumbens grasses, fermented at ages between 42 and 65 days [L. hexandra and A. zizanioides. Even these two grasses presented a superior butyric acid concentration than the control grass, and in the case of L. hexandra a higher TVFA level was also observed.The control grass showed the highest DMD among the grasses with percentages between 31.9 and 60.6% from 12 to 48 h of fermentation. These results are comparable with the DMD observed in  65 days ,45. Alth 65 days ; however2, CH4, microbial cells, etc.); however, the gas volume produced is mainly related to the VFA formation [L. hexandra native grass is more advantageous than the control grass, since the TVFA production is encouraged with a higher proportion of butyric acid. The latter effect was also observed in the A. zizanioides native plant. TVFAs  are important because they constitute the main energy source for the animal. The butyric acid is the principal energy source of epithelial cells in ruminants and promote cell proliferation that leads to improved feed degradation and utilization [L. hexandra and A. zizanioides grasses.In the gas production technique, several fermentation products are formed  and 48 h  of evaluation. These results are similar or even higher than the values obtained in temperate (Cenchrus clandestinus (Hochst. ex Chiov.) Morrone or tropical (Megathyrsus maximus (Jacq.) B.K.Simon and S.W.L.Jacobs, Urochloa humidicola (Rendle) Morrone and Zuloaga) grasses, fermented during 24 and 48 h at ages from 30 to 42 days [L. hexandra grasses are a consequence of the fact that these grasses presented the highest CP levels at 30 days . CP is degraded to form ammonia, which is used as a source of nitrogen by ruminal microorganisms and is therefore a determining factor in the synthesis of microbial cells [L. hexandra or introduced species such as the control grass (U. arrecta) constitute a useful forage resource to supply the dietary protein in grazing animals, one of the most limiting nutrients in different livestock scenarios under lowland tropics conditions.The highest ammonia production was found in the control and  42 days ,51. The al cells . These rMost of the studied variables were influenced by the measurement time. The gas production increased through the time. Additionally, TVFA, acetic, propionic and butyric acids were higher at 48 than 24 h. This is a recurrent result found in temperate and tropical grasses ,54,55 siU. arrecta and L. hexandra) promote greater DMD, ammonia and TVFA. This is an expected behavior since dietary protein is rapidly degraded by ruminal microorganisms, increasing the ammonia concentration in the medium, and favoring the microbial cells synthesis and growth, which promote the substrate degradation efficiency and the TVFA production [oduction ,56.L. hexandra and A. zizanioides. This result suggests that possibly the indigestible component of the fiber fraction varied among the native grasses and that further studies must be performed to identify the real NDF digestibility and nutrient availability.Urochloa hybrid cv. Cayman, Mega-thyrsus maximus cv. Mombaza, Urochloa brizantha cv. Toledo and Urochloa decumbens grasses, harvested between 42 and 65 days and fermented at 24 and 48 h [Megathyrsus maximus (Jacq.) B.K.Simon and S.W.L.Jacobs, Urochloa brizantha (A. Rich.) R.D. Webster, Urochloa decumbens (Stapf) R.D. Webster, Andropodon gayanus Kunth, Urochloa riziziensis (R.Germ. and C.M. Evrard) Crins and Pennisetum purpureum Schumach grasses fermented during 24 h [Andropogon gayanus Kunth, Urochloa decumbens (Stapf) R.D. Webster, Urochloa mutica (Forssk.) T.Q.Nguyen, Digitaria milanjiana (Rendle) Stapf, Megathyrsus maximus (Jacq.) B.K.Simon and S.W.L.Jacobs, Pennisetum purpureum Schumach and Setaria sphacelata var. splendida harvested between 35 and 39 days of age [In general, the fermentation parameters of the experimental pastures were comparable with other improved grasses commonly used under different livestock systems in tropical grazing conditions. Native grasses DMD was similar to and 48 h ,45. Totaing 24 h ,60. TVFAs of age . The aces of age ,61; simis of age ,62. ThesL. hexandra and A. zizanioides presented higher biomass production, CP, ash, cellulose and Ca levels than the control plant U. arrecta, an introduced species commonly used for grazing animals given its ability to withstand waterlogging conditions. The evaluated grasses can be harvested from 30 to 50 days of age to feed grazing animals without significant changes in nutrient availability.In the “low” physiographic position of the Orinoquia floodplain savannahs, the studied grasses performed similar to other tropical forages in terms of biomass production or chemical composition. Remarkable native plants such as L. hexandra native grass due to the increase in TVFA and butyric acid concentrations. The latter effect was also observed in A. zizanioides grass. Likewise, the highest ammonia concentration was obtained with the L. hexandra native plant, with optimal values to ensure microbial cell synthesis on low digestibility tropical forages.In terms of fermentation characteristics, the studied grasses were also within the ranges reported in tropical pastures. The energy production efficiency was improved by the L. hexandra and A. zizanioides constitute a valuable alternative forage resource during the flooding times of the studied ecosystem. Further research to evaluate fertilization responses and animal acceptability and productivity is still required.Native grasses such as


Background and objectiveVideo games have become a popular source of entertainment among children and adolescents, not only targeting the young generation but also increasingly popular among older demographics as well. This study aimed to assess the association between video game addiction and depression, anxiety, and attention deficit hyperactivity disorder (ADHD) among children and adolescents in Saudi Arabia.Materials and methodsThis was a cross-sectional study involving Saudi adolescents aged 12-16 years. A self-administered online survey was distributed on social media . The survey addressed sociodemographic characteristics . Arabic versions of the Patient Health Questionnaire-9 (PHQ-9), the seven-item Generalized Anxiety Disorder Scale (PHQ-GAD7), the ADHD Rating Scale-IV (ADHD-RS), and the seven-item Game Addiction Scale (GAS-7) questionnaires were distributed as well, including Arabic terms for depression, anxiety, and ADHD.ResultsA total of 393 surveys were completed . Of note, 63.1% of the respondents reported playing video games every day with an average of more than five hours of gaming time per day (25.2%). The prevalence of video game addiction was 62.1%. Independent risk factors of video game addiction included being young in age, playing video games every day, playing more than three hours per day, and playing multiplayer games. Furthermore, our findings suggest that a higher video game addiction score is correlated with higher scores in inattention, anxiety, and depression.ConclusionThis study found a high prevalence of video game addiction among Saudi children and adolescents. Excessive video game playing negatively influences mental health and leads to issues including anxiety, depression, and ADHD. Also, younger males with an increased rate of regular playtime were more likely to exhibit video game addiction in comparison to the rest of the population. Further research is required to more comprehensively assess the prevalence rate of video game addiction and its effect on the mental health of the younger generation within the region. Video games, whether online or offline, can be a source of addiction for adolescents. Studies by the American Psychiatric Association (APA) and the World Health Organization (WHO) have shown that they are related to mental health conditions in children and adolescents. The WHO labels it as "gaming disorder" and has defined it as impaired control over gaming, increasing priority given to gaming over other activities to the extent that gaming takes precedence over other interests and daily activities, and continuation or escalation of gaming time despite the occurrence of negative consequences. Current estimates suggest that video game addiction ranges between 1 and 15% in Europe and 3 and 8.5% in the US, while it is as high as approximately 14% in the UK and Korea and 17% in Iran; of note, 11.4% of the adolescents in Saudi Arabia are considered to be under a high level of stress ,3,4.It has been recognized that there is some correlation between gaming addiction and poor mental health conditions, mainly anxiety, depression, and inattention issues. Stress and anxiety affect the life of a child or adolescent in several ways; they affect their health, familial relationships, educational performance, social life, and ability to learn new things and make new friends. While there is some data on anxiety and stress among Saudi adolescents, scarce data is available on video game addiction. A study was conducted in Al-Qassim with a view to shed light on video game addiction and perceived stress among Saudi adolescents. Gaming addiction was measured with the Game Addiction Scale (GAS), while stress was measured with the Perceived Stress Scale (PSS). The researchers found that nearly 75% of the participants experienced stress, including 11.4% who had high levels of stress. Around 5% of the participants were addicted to gaming. The study also reported that gaming addiction is associated with higher levels of perceived stress in Saudi adolescents, which raises concerns about the mental health of Saudi youth [Another study was conducted to observe the impact of video game addiction and psychological distress among expatriate adolescents. It indicated that non-Saudi adolescents who live in Saudi Arabia may have an increased risk for video game addiction, which is strongly associated with greater distress. The study recommended that public health authorities and researchers prioritize examining the effects of video game use and addiction in the community. Future research should aim to gain a more comprehensive understanding of video game addiction among adolescents in Saudi Arabia for the development of programs to manage this problem and its consequences . The prevalence of depression has been on the rise in the last decade and has become a leading cause of disability in recent years globally. In the US, childhood depression is associated with a significant economic burden on the healthcare system. The prevalence of depression among children aged 3-17 years was 4.9% across the US in 2012. In Saudi Arabia, there is limited data to help estimate the extent of depression among children. A study conducted in 2019 in Riyadh focused on adolescents aged between 12-19 years and showed that out of 960 students, 32.4% had moderate to severe depression [Attention deficit hyperactivity disorder (ADHD) is a neurodevelopmental disorder characterized by a lack of attention, aggressiveness, and/or hyperactivity, and is primarily diagnosed in childhood . The preThis was a cross-sectional study conducted to evaluate the association of gaming addiction with depression, anxiety, and inattention among children and adolescents in Saudi Arabia. We employed a validated Arabic version of the Patient Health Questionnaire-9 (PHQ-9) ,9 to meaStatistical analysisData were summarized using numbers, percentages, means, and standard deviation (SD). The relationship between the level of video game addiction and sociodemographic characteristics and the frequency of playing games was analyzed using the Chi-square test. A multivariate regression model was used to determine the independent significant factors associated with game addiction with the corresponding odds ratio (OR) as well as a 95% confidence interval (CI). The Pearson correlation coefficient was also used to determine the correlation between the score of video game addiction and ADHD, anxiety, and depression. A p-value of 0.05 was considered statistically significant, while a p-value of 0.01 was considered highly statistically significant. All statistical analyses were carried out using IBM SPSS Statistics for Windows, Version 26.0 .A total of 393 children and adolescents completed the survey. As seen in Table As shown in Table As depicted in Figure As summarized in Table As presented in Table When assessing the relationship between game addiction and the sociodemographic characteristics and the frequency of playing video games Table , the preAs per the multivariate regression model performed Table , the chaThis study investigated links between video game addiction and depression, anxiety, and attention deficit among adolescents (aged between 12-16 years) in Saudi Arabia. The findings of this study suggest that 62.1% of the subjects are addicted to playing video games with 63.1% reported playing video games daily for a minimum of five hours or more (25.2%). The addiction to video games in our population can be compared to that among American adolescents . AccordiLiterature suggests that children with inattention are more susceptible to gaming addiction due to neuropsychological issues . AccordiOur findings suggest that age, gender, playing games on PlayStation, playing extended periods daily, playing multiplayer games regularly, playing shooter-type games, playing alone, and children or adolescents mimicking games in real life were the important variables in terms of video game addiction; however, in our multivariable regression analysis, only age, playing for an extended period of time, and playing multiplayer games regularly remained significant with regard to video game addiction. In a study by Schou Andreassen et al. , age wasParent supervision while children are playing video games is necessary to monitor what types of games they are playing and the time spent on this activity. In our study, although nearly half of the parents (48.9%) reported supervising their children during video game playing, 59.3% of children reported playing alone in the room, and 26.2% of them stated that they tried to mimic the game in real life, such as imitating the actions of the character they used in the games as well as copying what the character said in the game, including using age-inappropriate language. The findings of a recent literature review  showed tLimitations of the studyOne limitation of this study is that we used screening scales rather than clinical interviews to diagnose depression, anxiety, and inattention. This may have resulted in clinical bias. Another limitation is that we did not address the duration of gaming addiction and its association with depression, anxiety, and inattention.Our findings reveal that there is a high prevalence of video game addiction among children and adolescents in Saudi Arabia. Excessive video game playing can negatively influence mental health, leading to issues such as anxiety, depression, and inattention. Furthermore, younger males who engage in gaming for prolonged periods of time regularly are more likely to exhibit video game addiction when compared to the rest of the population. The findings of this study highlight the need for educational interventions to curb excessive video game playing and its potentially harmful effects on children's and adolescents' mental and physical health. Further research is needed to gain deeper insights into the prevalence of video game addiction and its effect on the mental health of young people in the region.


Video gaming is an extremely popular leisure activity, with over two billion users worldwide .Nevertheless, excessive video game playing exposes to potential dangers. The WHO reminds us that “every gamer should be aware of the time spent on games, especially if their daily activities are affected, as well as any physical or psychological, social and health changes that could be attributed to gaming behaviour.”The aim of our study is to analyse the frequency of video game use, and to determine its relationship with psychological and social functioning and academic performance.A cross-sectional study was conducted in the child psychiatry department at Arrazi Hospital in Salé among adolescents aged between 10 and 17 years.A hetero-questionnaire on socio-demographic characteristics, reasons for gambling, type of gambling, satisfaction, self-esteem, ability to make friends and degree of social support, and school resultsThe DSM 5 criteria proposed in the appendix for the researchThe K-SADSFor this, we used:Our study was carried out on a sample of 57 adolescents aged between 10 and 17 years, with an average age of 13.47.There was a predominance of males:Socio-demographic dataBoys: 87.5 , Girls: 17.5Primary 26.3Middle school 59.6High school 14 %All the adolescents are in school:54.4% reported having both real and virtual friends, 29.8% specified that all their friends are virtual, And 15.8% noted that they have no friendsAcademic decline was noted by the parents of 61.1% of adolescents Psychological functioning68.4% reported low self-esteem31.6% reported being dissatisfied with their lives0.5% met the diagnostic criteria for video game addictionSocial functioning and academic performanceOur study finds that problematic video game use is related to male gender, low academic performance, difficulties with social interactions and also low self-esteem and satisfaction.Screening for psychiatric co-morbidities and vulnerability factors is essential for the management of this type of pathology.None Declared


A commonplace sight is seeing other people walk. Our visual system specializes in processing such actions. Notably, we are not only quick to recognize actions, but also quick to judge how elegantly (or not) people walk. What movements appear appealing, and why do we have such aesthetic experiences? Do aesthetic preferences for body movements arise simply from perceiving others’ positive emotions? To answer these questions, we showed observers different point-light walkers who expressed neutral, happy, angry, or sad emotions through their movements and measured the observers’ impressions of aesthetic appeal, emotion positivity, and naturalness of these movements. Three experiments were conducted. People showed consensus in aesthetic impressions even after controlling for emotion positivity, finding prototypical walks more aesthetically pleasing than atypical walks. This aesthetic prototype effect could be accounted for by a computational model in which walking actions are treated as a single category (as opposed to multiple emotion categories). The aesthetic impressions were affected both directly by the objective prototypicality of the movements, and indirectly through the mediation of perceived naturalness. These findings extend the boundary of category learning, and hint at possible functions for action aesthetics.The online version contains supplementary material available at 10.1186/s41235-023-00510-0. Living in a world full of objects and events, there is one kind of stimuli that captivates most of us: other people. Our visual systems specialize in processing sights related to other people, including their eyes Emery, , faces , bodies How do these aesthetic impressions of other people arise? Extensive research has uncovered various perceptual factors that determine facial and body attractiveness, including shape averageness, symmetry, and sexual dysmorphism  impact of appearances on our personal and professional lives, it is critical to first understand what kind of aesthetic experiences can arise upon seeing a person. This study aimed to fill the gap between the vast scientific explorations on static stimuli and dynamic real-world experiences. Second, knowledge of action aesthetics will aid the design and development of the virtual world, such as those in animations, avatars in virtual reality, and robotics. Third, being able to evaluate human movements plays critical roles in medical diagnosis and rehabilitation  from the University of California, Los Angeles (UCLA) community completed an individual 30-min experimental session online in exchange for course credit. A total of 40 additional observers participated  but were removed based on predetermined criteria (see details in the Observer exclusions section below). The sample size was predetermined arbitrarily, preregistered, and fixed throughout all experiments. The study was approved by the UCLA Institutional Review Board.Because the stimuli were rendered on observers’ own web browsers, viewing distance, screen size, and display resolutions could vary depending on computer monitors used by observers; hence, we report visual stimulus dimensions using pixel (px) values.From the motion capture dataset , they were asked to maximize their window size, informed about their task, and quizzed about their understanding of the instructions, and then, they provided their consent. During the instructions, they were shown 4 videos from different walkers and different emotional expressions, to help them gauge the range of experiences they would have. They then performed one practice trial to get familiar with the rating scale.Observers were directed to a website where stimulus presentation and data collection were controlled via custom software written in HTML, CSS, JavaScript, JQuery, and PHP. Observers were not allowed to participate using phones or tablets. After completing a CAPTCHA task . For Experiment 3, instead of aesthetic ratings, the observers provided ratings to indicate their subjective impression of each walking style’s prototypicality. To avoid jargon, we did not use the word prototypicality directly in the instructions, but asked the observers to rate the walking styles’ naturalness . Again, they used a 6-point scale with naturalness labels .In the second part of all three Experiments, the observers rated the emotion positivity of the walkers (Experiment 1 and 3) or the creatures (Experiment 2) in the videos , using a 6-point scale with emotion positivity labels .The videos displayed intact walkers in Experiment 1 and 3, and spatially scrambled walkers in Experiment 2. Each of the 80 videos was displayed once in each block in different random orders. Whether the original or the mirrored version were shown was randomly decided for each video and each observer but kept the same across the two rating tasks.At the end of the experiment, observers answered a series of debriefing questions to ensure they had completed the experiment without any issues.In addition to the 150 observers whose data were analyzed, 40 observers  participated and were excluded based on criteria decided before data collection began, with some observers triggering more than one criterion. For Experiment 1, five observers reported that they did not follow the instructions or did not take the experiment seriously; one observer failed the instruction quiz more than once; one observer spent less than 0.5 s on at least one page of the instructions; one observer had a browser viewport smaller than 800px × 600px; one observer had at least one trial with the video not fully in view during the rating task; one observer gave the same rating to more than 15 consecutive trials; six observers hid the experiment browser tab more than three times during the trials; and four observers took too long to complete the experiment .For Experiment 2, seven observers reported that they did not follow the instructions or did not take the experiment seriously; one observer spent less than 0.5 s on at least one page of the instructions; three observers had a browser viewport smaller than 800px × 600px; one observer had more than four trials with response times longer than two minutes in at least one block; two observers hid the experiment browser tab more than three times during the trials; one observer provided a non-sensical answer to one of the debriefing questions; and three observers took too long to complete the experiment.For Experiment 3, one observer reported technical issues, three observers reported that they did not understand the instructions or did not take the experiment seriously; one observer spent less than 0.5 s on at least one page of the instructions; one observer had at least one trial with the video not fully in view during the rating task; three observers hid the experiment browser tab more than three times during the trials; and two observers took too long to complete the experiment.Each video was preprocessed separately following these steps. (a) We first subtracted the head’s x position in each frame from all joints’ x positions to remove the global horizontal movement. (b) The mean x and y positions for each joint across all the frames were calculated and subtracted from the joint’s x and y positions in each frame. This way, all joints’ trajectories ended up centering at coordinate . (c) The video was segmented manually into multiple action clips based on the type of movements the walker was performing—walking from left to right, turning around on the right, walking from right to left, or turning around on the left. The number of action clips differed between videos and ranged from 1 to 5 clips.Since the same type of movements in different action clips was performed in each walker’s own walking speed and rhythm. Comparing different clips required a method to map the corresponding frames in the gait cycle across walkers. For example, the frame where Walker A raised their right foot in Clip A should be compared with the frame where Walker B raised their right foot at a different time point in Clip B. We used dynamic time warping algorithms (DTW) to measure dissimilarity between walking sequences. DTW was performed by “warping” the temporal sequences of coordinates nonlinearly in time to find the optimal  correspondence between two sequences. We use a simple example to illustrate this algorithm: Considering computing dissimilarity between two sequences of 2D coordinates—A sequence:  and B sequence: , the algorithm would correspond A1  and A2 (the second frame of A sequence) both to B1 (the first frame of B sequence), and A3 to B2, and finally A4 to B3, B4, and B5. With this correspondence mapping over time, the algorithm can minimize the total dissimilarity, calculated by the distance between matched coordinates (0 + 0 + 1 + 0 + 0 + 1 = 2). This procedure allowed us to map corresponding frames between two clips in an automatic and data-driven manner.To model prototypicality, we performed pairwise comparison between all 80 videos. For each action clip in each video, we applied DTW algorithm multiple times to find the best matching sequence in all other 79 videos following these steps: (a) We measured the clip length in frame number and dropped any action clip that had less than six frames (200ms) as they contained too little information for meaningful mapping. (b) For each of the rest of the action clips, we found the best matching sequences within the full length of each of the other 79 videos. We did so by defining the max and min clip length for the possible matching sequence as ± 12 frames from its own clip length, with the constraint that the length should be no longer than 150 frames (the full video) and no shorter than seven frames. (c) We looped through all allowed clip lengths for the possible matching sequences. For each clip length, there will be multiple possible matching sequences in each of the other 79 videos. For example, a clip length of 5 frames includes sequences like Frame 1–5, Frame 2–6, Frame 3–7, and so on, from another video. We performed DTW between the action clip and each of these possible matching sequences to find the one that had the smallest dissimilarity across clip length. Thus, we found the best corresponding sequence and its dissimilarity to the action clip for each of the other 79 videos. Note that the DTW was performed considering all 15 joints’ coordinates at the same time.The total dissimilarity between two videos was then calculated by summing over the dissimilarity of each action clip in one video and their best corresponding sequences in the other video. This dissimilarity score was then normalized to the standard video length of 150 frames (since a few action clips might be dropped from finding a corresponding sequence because of its short clip length). Note that this process yielded asymmetrical dissimilarity scores when we find the matching sequences from Video A to Video B, and Video B to Video A. We simply took the smaller value to represent two videos’ dissimilarity. A video’s prototypicality score was calculated by one divided by its average dissimilarity score with all other videos in a category . Hence, higher prototypicality score of a video indicates that the video is more representative of a category as it shows higher average similarity to all exemplars in this category.z-scores of the ratings and the residuals after regressing out emotion positivity ratings, we followed these steps: (a) We first calculated the predicted aesthetic rating for each video according to a linear regression model including both modeled prototypicality and naturalness rating as predictors. (b) The aesthetic ratings’ residuals from these predicted values were calculated. (c) We calculated the predicted naturalness rating for each video according to a linear regression model including only modeled prototypicality as the predictor. (d) The naturalness ratings’ residuals from these predicted values were calculated. (e) We iterated the following steps (f-j) for 10,000 times. (f) The residuals calculated from step b were permutated and led to a permutated set of aesthetic ratings. (g) The same permutation was applied to naturalness residuals and led to a permutated set of naturalness ratings. (h) Using a linear regression model with model prototypicality as the predictor to predict permutated naturalness ratings, we found the permutated slope for prototypicality (denoted by a*). (i) Using a linear regression model with both modeled prototypicality and the original naturalness rating as predictors to predict permutated aesthetic ratings, we found the permutated slope for naturalness (denoted by b*). (j) We then multiplied a* and b* to get the permutated indirect effect. (k) After 10,000 iterations, we got a distribution of a* x b* values and found the confidence interval based on the 250th and the 9751st values after sorting.To understand the causal path between objective prototypicality and aesthetic impression, we conducted a mediation analysis to separate direct effect and indirect effect through subjective prototypicality. Beside the standard Sobel test, we performed the “permutation confidence interval for ab” method . This pattern indicates substantial consensus across observers and systematic variations in aesthetic impressions across different walks.To gauge the consensus on aesthetic impressions, we used data from Experiment 1 and calculated each observer’s “aesthetic taste typicality”  = 0.662, p < 0.001; Scrambled creatures: r(78) = 0.627, p < 0.001; all tests reported were two-tailed tests), and further supported by a one-sample t-test of comparing correlations between the two ratings at the individual level to zero , t(49) = 12.18, p < 0.001, Cohen’s d = 1.72; with 48/50 observers showing positive correlations, p < 0.001; Scrambled creatures: M = 0.186, SD = 0.174, Range = ), t(49) = 7.59, p < 0.001, Cohen’s d = 1.07; with 42/50 observers showing positive correlations, p < 0.001).For both Experiment 1  and Experiment 2 (scrambled creatures), we depict each video’s mean aesthetic and emotion positivity ratings in Fig. Mhappy = 3.5 (0.6), Mneutral = 3.9 (0.5), Mangry = 2.9 (0.6), Msad = 3.1 (0.6); and showed a significant main effect of emotion category on aesthetic ratings  = 62.0, p < 0.001, η2p = 0.559; all post hoc comparison with neutral walkers: ts > 4.4, ps < 0.001 after Bonferroni correction; see Additional file Mhappy = 4.0 (0.4), Mneutral = 3.7 (0.4), Mangry = 3.4 (0.5), Msad = 2.6 (0.4); one-way ANOVA main effect with z-scores: F = 185, p < 0.001, η2p = 0.791; all post hoc comparison with happy walkers: ts > 5.9, ps < 0.001 after Bonferroni correction). Furthermore, the effect of neutral walkers having higher aesthetic ratings than other emotion categories of walkers remained to be significant after regressing out the emotion positivity ratings from the aesthetic ratings  = 41.0, p < 0.001, η2p = 0.455; all post hoc comparison with neutral walkers: ts > 5.8, ps < 0.001 after Bonferroni correction). In Experiment 2, when body configuration was removed by spatial scrambling, this effect was not observed , Mneutral = 3.6 (0.4), Mangry = 3.8 (0.6), Msad = 3.3 (0.5); main effect with z-scores: F = 18.7, p < 0.001, η2p = 0.276).If the effect of emotion positivity was unidimensional (differing only with respect to how positive the expressions appeared), the relationship between aesthetic and emotion positivity ratings described above would predict that happy walkers/creatures would yield most positive aesthetic impressions compared to neutral, angry, and sad walkers/creatures. However, we found a different patterns of results: In Experiment 1 with intact walkers, when different emotional categories were examined Fig. b, the neWhy do we aesthetically prefer neutral walkers? If categorical representations based on prototypes exist for human walkers, it is possible that neutral walkers appeared the most prototypical, and thus, the preference is a result of an aesthetic prototype effect. To test this idea, we used a computational model to quantify objective prototypicality in walking stimuli, and in Experiment 3 we measured observers’ subjective prototypicality.z-scores average: r(78) = 0.566, p < 0.001, aesthetic residuals average: semipartial correlation r(78) = 0.546, p < 0.001). Critically, the objective prototypicality index revealed that the neutral walkers indeed were the most prototypical  = 6.91, p < 0.001, all post hoc comparison with neutral walkers: ts > 2.9, ps < 0.05 after Bonferroni correction). Thus, the computational model provides an account for the aesthetic prototype effect, supporting the hypothesis that dynamic events involve representations of a category.We first constructed a single category model: Using the dynamic time warping algorithm (DTW) to compute the similarity of joint movements from two actions  = 0.382, p < 0.001). However, this correlation from the emotion category model was weaker than that from the single category model (emotion category model: r(78) = 0.382, single category model: r(78) = 0.566; comparison: p = 0.012; all comparisons between correlations were conducted with cocor, Diedenhofen & Musch, r(78) = 0.440, p < 0.001) numerically  = 0. 546; comparison: p = 0.138). Thus, the additional emotion-based categories worsened the model predictions of aesthetic ratings for the walkers, suggesting that the aesthetic judgments are better explained by assuming that human walks form a single category.Walking stimuli in our study might be represented as exemplars of a single category , or as exemplars of multiple categories depending on the different emotions the walkers were expressing . Would a model with more refined emotional categories more accurately predict human aesthetic judgments relative to the parsimonious model with a single category of walking? To address this question, we tested the emotion category model: For each walk, instead of computing the overall average similarity to the other 79 walks, we computed average similarity only within the same expression .After observing the strong correlation between prototypicality and aesthetic impressions, we next ask: what is the causal relationship between these variables? In general, correlational data afford multiple causal interpretations. However, in this case an additional constraint is apparent: model-derived prototypicality is an objective statistical measure that solely depends on the distribution of stimulus exemplars, whereas aesthetic impressions were measured by subjective human judgments. It seems logically impossible for a subjective measure to have a causal impact on an objective statistical measure on stimuli. A remaining question, however, is whether the causal path from objective prototypicality to aesthetic impression operates by a direct causal path, indirect path via some other variables , or both.While it is often possible in general that a third factor could cause both objective prototypicality and aesthetic experiences, it is paradoxical in this specific case: the third factor would need to influence the objective prototypicality, i.e., causing the estimate to be higher or lower, yet not itself be part of the objective prototypicality, i.e., not changing the estimate. For example, perhaps actors find it easier to perform neutral walks compared to happy, angry, and sad emotional walks, which led to differences in naturalness in the movements. However, this could not be considered as a third factor that could cause both the differences in objective prototypicality and aesthetic experiences, as the differences in naturalness manifest in the movements themselves and thus, would be part of the measure of objective prototypicality, rather than acting as a third factor that could influence the objective prototypicality.r(78) = 0.556, p < 0.001; with naturalness residuals after regressing out the emotion positivity: semipartial correlation r(78) = 0.540, p < 0.001), suggesting that typical movements were indeed associated with more natural impressions. This result was further supported by the similar patterns found with naturalness ratings , Mneutral = 4.0 (0.5), Mangry = 3.1 (0.5), Msad = 3.5 (0.5); one-way ANOVA main effect with z-scores: F = 55.6, p < 0.001, η2p = 0.531; all post hoc comparison with neutral walker: ts > 6.0, ps < 0.001 after Bonferroni correction). Moreover, the effect persisted after regressing out the emotion positivity ratings from the naturalness ratings  = 58.4, p < 0.001, η2p = 0.544; all post hoc comparison with neutral walkers: ts > 6.4, ps < 0.001 after Bonferroni correction). For emotion positivity ratings, we replicated the finding in Experiment 1 that happy walks were perceived as the most positive (Mhappy = 3.9 (0.4), Mneutral = 3.5 (0.4), Mangry = 3.4 (0.5), Msad = 2.5 (0.4); one-way ANOVA main effect with z-scores: F = 227, p < 0.001, η2p = 0.823; all post hoc comparison with happy walkers: ts > 9.0, ps < 0.001 after Bonferroni correction).We first examined the relationship between objective prototypicality derived from the single category model and subjective prototypicality  provided by observers from Experiment 3. Each video’s mean naturalness ratings and model-derived prototypicality are depicted in Fig. ngs Fig. f and modt(77) = 4.84 p < 0.001; the permutation CI = , which did not include zero; residual analyses: the Sobel test, t(77) = 5.18, p < 0.001; the permutation CI = , which did not include zero). The gross direct relationship between the objective prototypicality and the aesthetic impression  was substantially weakened after removing the indirect effect of the subjective impression of prototypicality .Next, we asked if a movement’s model-derived prototypicality exerts its effect on aesthetic experience through a subjective impression of prototypicality. We conducted mediation analyses to examine both the direct effect , and the indirect effect . Because the same stimuli were used in Experiments 1 and 3, this analysis included both aesthetic judgments (from Experiment 1) and naturalness judgments (from Experiment 3). With a Sobel test and the method of permutation confidence interval  People share substantial consensus on how aesthetically pleasing a walk looks. (2) Human walks look more aesthetically pleasing when they expressed positive emotions; this preference depends on holistic processing and explicit recognition of human body configurations. (3) Aesthetic prototype effects can be observed in human actions: People find prototypical walks more aesthetically pleasing than atypical walks. (4) This effect was caused both directly by the prototypicality of the walk itself and indirectly through the mediation of the subjective impression of prototypicality.The observed prototype effects indicate that human walks expressing different emotion states form a single category within a representational space, which opens a new dimension in the exploration of categorical processing. Beyond static objects and animals, dynamic events can form categories and afford the same kind of representational structures that lead to prototype effects. These categories likely form at the basic level (Rosch et al., At the same time, the demonstration of an aesthetic prototype effect in human walks also constitutes a unique approach to understanding human action aesthetics. In contrast to the focus on domain-specific explanations for aesthetic experiences based on dance movements (Cross et al., These discoveries inform several practical fields. The discovered clear consensus of how good a walk looks suggests a new source of bias to navigate in our personal and professional lives. At the same time, the prototype effect introduces a design principle for depicting animated humans, developing virtual agents, and creating robots. The computational models used in this study can also be used for machine assistance in medical diagnosis, rehabilitations, and prosthetic limbs design Pitkin, , such asAdditional file 1. Posthoc tests for all main effects of emotion category across three experiments and the modeling results were reported here.


BackgroundSurgical ward round documentation, essential for high-quality patient care, is often completed poorly. The advent of electronic medical records offers an opportunity to introduce proformas, aiding junior staff in completing notes both timely and accurately. We aimed to assess whether the introduction of a proforma would improve the quality and speed of ward round documentation.MethodsWe completed a prospective cohort analysis of ward round documentation at a single institution. Analysis was conducted on the documentation of a single surgical team over a 10-week period, comprising five weeks of baseline data collection followed by five weeks with implementation of a proforma. This proforma was based on the "David & Wendy" acronym, encompassing diet, activity, vital signs, investigations/IV therapy, drains/lines, wound assessment, examination findings, nursing concerns, drugs/deep vein thrombosis (DVT) prophylaxis, and barriers to discharge.ResultsA total of 711 ward round notes were analyzed, 349 with proforma and 362 without. Statistically significant improvements were observed in the documentation of diet, activity, investigations/IV therapy, drains/lines, wound assessment, nursing concerns, drugs/DVT prophylaxis, and barriers to discharge (p < 0.05) with proforma use. No significant difference was noted in the documentation of vital signs or examination findings. The time taken to finalize ward round notes was significantly reduced with the proforma .ConclusionThe introduction of the David & Wendy proforma significantly improved the speed and quality of documentation for key surgical ward round information during our study. The daily ward round is a keystone in the modern surgical management of patients in hospitals, and its quality, or lack thereof, has been linked to higher rates of complications and worse outcomes for patients . ContrarSurgery has long utilized checklists to enhance patient care, with the World Health Organisation surgical safety checklist being a prime example of how checklists can revolutionize patient care . SurgicaThe use of memorable acronyms as cognitive tools has been successfully implemented in various medical specialties to reduce errors and standardize practice where solid guidelines exist . For exaThis quality assurance study was conducted at Cairns Hospital. An ethics exemption was granted by the Far North Queensland Human Research Ethics Committee (FNQ HREC Reference: Project EX/2022/QCH/90904). Over a 10-week period (November 2022 to January 2023), ward round notes from the electronic medical record (iEMR) of a surgical team at Cairns Hospital were reviewed. Documentation of the listed parameters was recorded and stored on a secure, password-protected MS Excel™ spreadsheet within the Queensland Health Computer network. No patient demographics or clinical details were included in the study; the data was de-identified and subsequently tabulated.Documentation by a single surgical team was carried out in a standard fashion for five weeks without any intervention; this was then largely up to the interns completing the note and the registrar guiding the ward round as to what was or was not included. Subsequently, a ward round proforma was introduced and utilized for an additional five-week period. The proforma Figure  providedThe study population comprised all patients admitted under the surgical team during the 10-week period. There were no exclusion criteria, and patients may or may not have undergone surgery. Patient demographics and information related to diagnosis, interventions, and outcomes were not collected or analyzed, as these were deemed unrelated to the assessed outcomes. Given the data collection timeframe, it was assumed that these characteristics would be evenly distributed between the two groups, consistent with the nature of an acute surgical unit. The team admitted a range of acute and elective surgical pathologies in a range of general surgical subspecialties; however, the consultants had a predominance for breast, hernia, and hepatobiliary surgeries in their elective work. Ward round notes were categorized as either 'proforma used' or 'proforma not used,' and nominal data were collected on whether the notes specifically mentioned diagnosis, diet, mobility status, vital signs, investigations/IV therapy, drains/lines, wound assessment, examination findings, nursing concerns, drugs/DVT prophylaxis, and barriers to discharge. The start and completion times of the ward round notes  were also recorded.Where appropriate, summary statistics are presented as number (%) for binary and categorical data, and mean (SD) for normally distributed continuous data. Variables were compared using Fisher's exact test. Paired sample t-tests were performed to compare the mean time taken to finalize ward round documentation between the two groups, and effect sizes were determined using the Cohen d method. Stata (version 15.0) was used throughout, and the level of significance was set at p < 0.05.A total of 711 ward round notes were analyzed, 349 where the proforma was used and 362 where the proforma was not used. Documentation of the ward round note parameters for the two groups is presented in Figure There was a significant decrease in mean time taken to finalize ward round notes per patient ; despite significant outliers, the median was also reduced  . Our stuFurther study Previous qualitative reviews of ward round documentation have indicated mixed perceived benefits for nursing staff ,15,19, w The introduction of a ward round proforma based on the David and Wendy acronym can significantly enhance documentation in areas critical to inter-departmental communication and patient-centered outcomes. Our research demonstrated statistically significant improvements in documenting diet, activity, investigations/IV therapy, drains/lines, wound assessment, nursing concerns, drugs/DVT prophylaxis, and barriers to discharge. We also observed a reduction in the time taken to finalize notes. While not every item on this list may be relevant for all patients, nor is it an exhaustive list of all essential aspects of surgical patient care and subspecialties, it serves as a guideline to help both junior and senior staff avoid common pitfalls in the fast-paced surgical environment.


The effect of modern infertility treatment modalities on fetal thymic volume has not been well known.3-dimensional (3D) fetal thymus volumes of 18-24 wk in vitro fertilization (IVF) pregnancies and spontaneous pregnancy cases were compared.135 cases were evaluated in this prospective case-control study. The study was conducted between July 2019 and July 2020 at a university hospital in Trabzon, Turkey. Fetal thymus volume was calculated in the pregnant cases included in the study with the help of the virtual organ computer-assisted analysis system included in the advanced ultrasonography system. The fetal thymus volumes were compared between pregnant women with IVF and spontaneous pregnant women.The fetal thymus size was significantly lower in the IVF pregnancy group than in spontaneous pregnancy cases (p  In light of these results, it may be concluded that small fetal thymus size may be another fetal complication of IVF pregnancies. Assisted reproductive techniques (ART) define the clinical and laboratory methodologies employed in infertile couples to eliminate and rectify causes that prevent them from achieving pregnancy. Each year, more than 200,000 babies are born with ART globally, and today, this number is approximately 5 million .An increased complication rate has been observed , abnormal placental location, preterm birth, and low birth weight) in those who become pregnant with ART (3). Studies comparing babies born from pregnancies obtained with in vitro fertilization (IVF) with the general population show that the rate of congenital anomalies is higher in this group than in the general population .The microinjection technique applied to the oocyte in intracytoplasmic sperm injection cycles and the morphological disorders of the sperm/oocyte affect the development quality of the embryo. Physical damage to the cytoplasm may be possible following a traumatic injection. As a result of these potential risks, it is predicted that zygotes obtained following the intracytoplasmic sperm injection procedure are exposed to nonlethal cell damage and impaired embryonic development (6). Previous studies have shown an increased risk of complications, such as preeclampsia, IUGR, abnormal placental location, and low birth weight in those who become pregnant by using ART .In pregnancy, the development process of the fetus's defense system during the intrauterine period focuses on adapting to the existing environment. The fetal thymus assumes the most important role in this immune system. One study showed that the thymus is smaller in various fetal and neonatal diseases (11). In literature, limited studies are found on fetal thymus volume in IVF pregnancies. This study had significant methodological problems, was a retrospective study, and its results were based partly on simple and subjective fetal thymus measurement . However, considering that the thymus is a 3-dimensional (3D) structure, 2D evaluation may not provide sufficient information. There is no literature study evaluating the use of 3D virtual organ computer-assisted analysis system (VOCAL) measurement for fetal thymus in IVF pregnancies.The study aimed to compare the sonographic 3D fetal thymus size in second-trimester spontaneous and IVF pregnancies.Totally 135 cases (18-24 wk pregnant women without any high-risk pregnancy risk factors) were evaluated in this prospective case-control study. The study was conducted between July 2019 and July 2020 at a university hospital in Trabzon, Turkey.Participants in the study group were divided into 2 groups (n = 37/each): a) conceived with IVF and b) conceived spontaneously. Since it is forbidden in our country to use donated egg or embryo, their own embryo was used in IVF cycles.A total of 74 healthy pregnant cases were included according to the inclusion and exclusion criteria of the study. The main criteria for inclusion in the study were being of reproductive age (between 18-35 yr of age) and not having any high-risk pregnancy factors and applying for routine pregnancy follow-up. Exclusion criteria for the study were the presence of any systemic disease , additional organ disease  or history of drug use other than routine drugs used during pregnancy, or a history of alcohol use, smoking, or abnormal findings in routine biochemical/hematological test results, or abnormal pregnancy findings , or unwillingness to participate in research processes. In this way, 135 cases were evaluated and 61 of them were not included in the study because they did not meet the above criteria. Demographic data of all cases  and routine fetal ultrasonographic measurement, gestational age determination data, and fetal 3D fetal thymus volume measurement data were recorded. The pregnancy and birth of all pregnant women were performed in our clinic, and pregnancy follow-up, pregnancy complications, and birth information were recorded. Apgar scores at the 1Fetal thymus volume was measured with the technique described in detail in our previous study  (13).Non-reliable fetal status formerly non-reassuring fetal status or fetal distress was defined as persistent or worsening category II or III fetal heart rate pattern.The study procedure was approved by the Ethical Committee of Karadeniz Technical University, Trabzon, Turkey . A written informed consent was obtained from all the participants.t test. Data from both groups were compared with each other. A p-value of less than 0.05 was considered statistically significant. The data were presented as percentages, ratios, and mean All the data were encoded and entered into SPSS software . The statistical analyses were made on the computer using the Chi-square and Student 3, respectively; p A total of 74 cases  were included in the present study. While the average age was 28.62 This study showed that fetal thymus size change, which wss an important part of the fetal defense system, was affected by IVF pregnancies. In this prospective case-control study, fetal thymus size was smaller in IVF pregnancies than in spontaneous pregnancies.The clinical use of fetal thymus measurement is a relatively new prenatal sonographic marker whose importance is not clearly known. Despite the limited number of cases, pregnancy complications between the 2 groups were compared in order to complete this deficiency in the literature, to contribute to the literature in this context, and to reveal the relationship between fetal thymus measurement and increased pregnancy complications in IVF pregnancies.It was shown in previous studies that were conducted on fetal thymus that fetal thymus hypoplasia is associated with fetal IUGR, chorioamnionitis because of preterm membrane rupture, and chromosomal anomaly (22q 11.2 deletions) . As an organ, the fetal thymus is sensitive to age and stress-related involution . Stress-induced involution is characterized by acute loss of CD 4/8 expression in cortical thymocytes and the peripheral absence of natural T lymphocytes. It is associated with infection, sepsis, trauma, malnutrition, acute respiratory distress syndrome, and physical stress . In cases of acute stress due to infection and non-infection, there was a histological reduction in the thymus volume. The thymus is very important for the maturing immune system (22).Although the effects of impaired thymus development on life-long immunological competence in fetuses that have growth restriction were not elucidated completely, one prospective study shows that immune function in later life is programmed in the early period (23). The result of a study suggested that the thymus is a mediator in the relation between atopic, autoimmune, and infectious diseases in infancy and childhood and fetal malnutrition .Although thymus size was previously investigated in naturally conceived patients having maternal diabetes or rheumatic disease, there are no studies in the literature other than that of Nau and co-workers conducted in 2018 with patients who achieved pregnancy with IVF .The study of Nau et al., found that the fetal thymus size was smaller in cases who became pregnant by the IVF method at significant levels when compared to spontaneous pregnancies. Although this study's results support the present study's results, the thymus size was evaluated retrospectively in the abovementioned study, and thymic thoracic ratio was calculated using 2D sonographic measurements (12).Identifying the fetuses with small thymus will allow the clinician to monitor the patient in this respect, hospitalize if necessary, and anticipate increasing pregnancy complications because of small thymus and IVF pregnancy, and follow-up with such patients closely. The necessary patients can be hospitalized, maternal corticosteroid administration can be applied for lung maturation, and subsequent follow-up may be improved. On the other hand, early intervention for neonatal sepsis and respiratory distress syndrome might reduce perinatal mortality and morbidity by detecting patients with small thymus volumes.According to a recent study, it has been reported that a 2D ultrasonographic fetal thymus measurement consisting of thymus diameter measurement in a single plane, does not provide effective information about thymus development and evaluation. A 3D-sonographic fetal thymus evaluation can display the fetal thymus more accurately and effectively than standard 2D evaluation providing information about the actual thymic lobular structure and development (28). In this case, our research may more effectively answer the question of whether the commonly used IVF methods cause thymus volume reduction. This makes this research valuable in terms of a new contribution to the literature.The strong sides of the present study were the design , each sonogram is measured by the same experienced specialist in the same institution. The thymus is measured in 3D in the most realistic way.One of the limitations of our study was the small sampling. The number of patients could not be collected at the planned level because of the decreased number of patients who applied to the clinic due to the coronavirus pandemic, which swept the entire world, and the decreased number of patients who underwent IVF treatment.The present study showed a relationship between IVF pregnancies and fetal sonographic 3D thymus volume. It was found that the fetal thymus size is significantly lower in cases who become pregnant after IVF treatment compared to spontaneous pregnancies.It may be concluded that according to the results of this study, fetal thymus 3D volume measurement may be useful in detecting healthy pregnancies and predicting possible neonatal complications. In IVF pregnancies because pregnancy complications are increased, and fetal thymus volume is lower in IVF pregnancy cases at significant levels when compared to spontaneous pregnancies.The authors declare that there is no conflict of interest.


G is a locally compact Abelian group and G. It is proven that the representation is irreducible, which allows us to define a covariant positive operator-valued measure (covariant POVM) generated by orbits of projective unitary representations of We study a projective unitary representation of the product  X, takes values in the cone of positive operators in a Hilbert space, H, and is normed by the condition that the measure of the entire space is equal to the identity operator. Thus, it defines the measurement quantum channel, that is, a map from the convex set of quantum states to the set of probability distributions on X obtained by taking a trace of the product of a state and a positive operator from the measure (h (G=G^×G 4)Uχ,gUUχ,gUH=L2Proposition 1. The Formula  determinProof. P belonging to X in P. The result follows. □Let ear form<<ρ,σ>>=Tors in H . It is kmorphism . MoreoveUsing the ideas in , let us ows from  thatχ′fρ=∫B|UE on Following the Naimark theorem , there eark form π=∫relation Tχ,gn=PHRemark 2. Note that a set of contractions under a construction.Theorem 1. There exists a complex-valued measurable function f on the group Proof. Taking into account , 6) and and6) anentation  is irredtion  .B runs measurable subsets of Taking into account duality  is 12πdxalculate  appearinProposition 3. Remark 4. Thus, the function Proof. Following from ,Tx,y=12On the other hand, using the representation of the displacement operator in the form where the creation and annihilation operators a†,a  are usedixy2Ux,y . □G is a locally compact Abelian group and E corresponding to our POVM E determines a family of unitary operators In this paper, we investigated a projective unitary representation of a direct product as shown ,14 that


We investigated HCN4 palmitoylation and its functional consequences in engineered human embryonic kidney 293T cells as well as endogenous HCN4 in neonatal rat ventricular myocytes. HCN4 was palmitoylated in all experimental systems investigated. We mapped the HCN4 palmitoylation sites to a pair of cysteines in the HCN4 intracellular amino terminus. A double cysteine-to-alanine mutation CC93A/179AA of full length HCN4 caused a ∼67% reduction in palmitoylation in comparison to wild type HCN4. We used whole-cell patch clamp to evaluate HCN4 current (IHCN4) in stably transfected 293T cells. Removal of the two N-terminal palmitoylation sites did not significantly alter half maximal activation voltage of IHCN4 or the activation slope factor. IHCN4 was significantly larger in cells expressing wild type compared to non-palmitoylated HCN4 across a range of voltages. Phylogenetic analysis revealed that although cysteine 93 is widely conserved across all classes of HCN4 vertebrate orthologs, conservation of cysteine 179 is restricted to placental mammals. Collectively, we provide evidence for functional regulation of HCN4 via palmitoylation of its amino terminus in vertebrates. We suggest that by recruiting the amino terminus to the bilayer, palmitoylation enhances the magnitude of HCN4-mediated currents, but does not significantly affect the kinetics.The sinoatrial node (SAN) and subsidiary pacemakers in the cardiac conduction system generate spontaneous electrical activity which is indispensable for electrical and therefore contractile function of the heart. The hyperpolarisation-activated cyclic nucleotide-gated channel HCN4 is responsible for genesis of the pacemaker “funny” current during diastolic depolarisation. S-palmitoylation, the reversible conjugation of the fatty acid palmitate to protein cysteine sulfhydryls, regulates the activity of key cardiac Na The intrinsic automaticity of the sinoatrial node (SAN) is fundamental for pacemaking in mammalian hearts. In cells of the SAN (and elsewhere in the cardiac conduction system) slow depolarisation during diastole is the basis of this automaticity, distinguishing these specialised cells from the working myocardium . Multiplf) during diastolic depolarisation  property of being activated by hyperpolarisation (+ and K+) inward current on membrane hyperpolarisation over the diastolic range of voltages, facilitate the channel’s contribution to the early diastolic depolarisation  Act 1986. Rabbit hearts were excised following humane killing in accordance with UK Home Office legislation.Cysteine-to-alanine substitution reactions were performed using Q5-site directed mutagenesis kit (New England Biolabs). The primers for the mutagenesis reactions were designed using the manufacturer’s online primer design tool.The InFusion cloning system was used to subclone full length HCN4. The amino (1-266) terminus and the carboxyl (518-1203) terminus were amplified from the human full length HCN4 in pcDNA 3 and inserted into pEYFP-C1 (Clontech) to express as fusions to the YFP C-terminus. Full-length HCN4 was amplified and inserted in pcDNA5/FRT/TO (Invitrogen).Cell lines stably expressing tetracycline-inducible wild type and mutant HCN4 channels were generated using the Flp-In™ T-Rex™ System. All transient transfections of plasmid DNA were carried out in a 6 or 12 multi-well plate seeded with a high cell density using Invitrogen Lipofectamine 2000 as instructed by the manufacturer. The cells were harvested 18–24 h following transfection.2PO4, 5.3mM KCl, 0.4 mM MgSO4, 5 mM glucose; pH 7.4). Excess blood and tissue were removed from hearts using a sterile scalpel. For analysis of atrial tissue only, the atria were identified and separated from the ventricular tissue.Neonatal rat whole hearts were obtained from male and female Sprague Dawley rats aged 1–4 days old. Briefly, animals were euthanised with a lethal dose of Euthatal before severing the femoral artery. Hearts were quickly excised and placed into a solution of ice-cold excision buffer  . BrieflyIntact cardiac tissue was homogenised using ceramic beads (BeadBug) in blocking buffer for 4 h at 40°C. Any unhomogenised tissue was discarded before the precipitation step.+/K+ ATPase α1 subunit . Secondary antibodies were anti-rabbit conjugated to HRP  and anti-mouse conjugated to HRP .This investigation used antibodies raised to HCN4 (1:5000-1:10000 dilution Alomone labs #APC-052), caveolin-3 , flotillin-2 , GFP  and NaImages of western immunoblots were acquired using a BioRad Chemidoc XRS imaging system and analysed using QuantityOne (BioRad) and ImageLab (BioRad) software.Surface membrane proteins were biotinylated by incubating cells in 1 mg/mL sulfo-NHS-SS-biotin in PBS for 30min at 37°C. Cells were lysed in 1% Triton X-100, 0.1% SDS in PBS supplemented with protease inhibitor cocktail. Biotinylated proteins were captured using streptavidin sepharose . Only su2, 5 EGTA, 5 MgATP and 10 HEPES (titrated to pH 7.2 with KOH) and extracellular Tyrode solution comprised of (in mmol/L): 140 NaCl, 4 KCl, 2.5 CaCl2, 1 MgCl2, 10 glucose, and 5 HEPES (titrated to pH 7.4 with NaOH). Patch pipettes  were pulled on a dual stage glass micropipette puller (Narishige PC-10) and heat-polished  to obtain a final resistance of 2–3 MΩ. The cells on coverslips were transferred to a bath chamber mounted on the stage of an inverted microscope and continually superfused with Tyrode solution preheated to 37°C. Whole cell patch clamp recordings of IHCN4 were made using: 1) an Axopatch 1D amplifier (Axon Instruments) and a CV-4 headstage, with a Digidata 1200B or 1440A A-D interface (Molecular Devices) and Clampex software 10.7 (Molecular Devices); or 2) an Axopatch 200B amplifier (Axon instruments), digitization using an ITC-18 computer interface (Instrutech corporation) and HEKA Pulse software. Series resistance was compensated by 70%–75% . The IHCN4 currents recorded were filtered at 1–2 kHz and digitized at 10 kHz. Data acquired were analysed primarily using Clampfit 10.7 (Molecular Devices), Microsoft Excel 2204, and OriginPro 2021b. Cells with leak current of more than 100 pA at the −40 mV holding potential were not included in the analysis.Stably transfected Flp-In™ 293 T-REx cells (Thermo) were plated on 10 mm glass coverslips in a 35 mm culture dish and HCN4 expression induced with tetracycline (1 µg/mL) for at least 24 h prior to being used for electrophysiology recordings. Whole cell patch clamp recordings employed an internal solution comprised of (in mmol/L): 130 KCl, 1 MgClSequences were acquired from the National Centre for Biotechnology Information (NCBI) GenBank (accessed June 2020). Only HCN4 ortholog sequences with the complete whole channel sequence were selected and partial sequences generating an incomplete transcript were not included in the phylogenetic analysis. To minimise misalignment in the analysis, the full-length sequences were trimmed to amino acids 1 to 300 which included the full N-terminus and the start of the S1 domain which exhibits high sequence identity within the sequences used. Sequence alignments of the trimmed sequences and phylogenetic trees were generated using Clustal omega. Sequences that displayed little homology were removed from the alignment.post hoc multiple comparison tests. A Welch’s ANOVA was used to compare experimental groups with unequal standard deviations. Differences between experimental groups with p-values < 0.05 were considered statistically significant and accordingly denoted by * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001.Statistical analysis was performed using GraphPad Prism software. All quantitative data are presented as mean ± standard error of the mean (SEM). The statistical significance difference between the experimental groups were analysed using one-way analysis of variance (ANOVA) with Tukey’s We investigated palmitoylation of HCN4 using resin-assisted capture of acylated proteins (acyl-RAC), which purifies acylated proteins (but not those interacting with them) under strongly denaturing conditions. All assays were analysed by immunoblotting for HCN4 and a constitutively palmitoylated protein: caveolin-3 from muscle, and flotillin-2 from cultured cells . HCN4 wavia the dominant site. It is only when the dominant site has already been removed that the impact of mutating the secondary site can be seen. We conclude from these experiments that both cysteines in the fusion protein between YFP and the HCN4 amino terminus are palmitoylated, and that C179 may be the principal palmitoylation site.We next set out to identify the palmitoylated cysteines in HCN4. Human HCN4 has seven intracellular cysteine residues: two in the amino terminus before the first transmembrane domain and five in the carboxyl tail after the sixth transmembrane domain . In ordeTo confirm the identity of the HCN4 palmitoylation sites in the full-length protein we generated 293T cells stably expressing tetracycline inducible wild type (WT), C93A, C179A and CC93/179AA HCN4. This system enables consistent expression levels in all cells investigated so is, in principle, considerably superior to transient transfection. Mutagenesis of C179 caused the greatest reduction in the amount of HCN4 captured using acyl-RAC, and mutagenesis of both amino terminal cysteines further reduced the amount of palmitoylated HCN4 . A small+/K+ ATPase α1 subunit in the same samples  in stably transfected 293T cells in which HCN4 expression was induced with tetracycline. Endogenous HCN4 is not expressed in HEK293 cells, but this expression system is well-validated to interrogate IHCN4 (HCN4. Depolarising to 5 mV for 0.5 s following the hyperpolarising test pulse elicited outward tail currents which inactivated in a voltage-dependent manner. These WT currents are similar to those reported in prior studies of HCN4 expressed in HEK cells (0.5) between the WT and mutant channels , the single channel conductance (G), and the channel open probability (Po). We found no obvious influence of palmitoylation in the HCN4 amino terminus on steady state abundance of HCN4 protein at the cell surface membrane. We hence rule out any significant influence of palmitoylation on N. Since the HCN4 amino terminus is relatively far from the channel’s pore  negatively regulates channel activity or that in the palmitoylated state the amino terminus is a channel facilitator.The typical consequence of protein palmitoylation is for the palmitoylated region of a protein to be anchored to the membrane . In the AlphaFold predicts the amino terminus of HCN4 to be extensively disordered, and no secondary structure was observed in this region of the protein in a recent HCN4 cryoEM structure . We acknHCN4 when only ∼33% of HCN4 is palmitoylated. Mature HCN4 channels are tetrameric  and carboxyl (716-1023) termini. Those cysteines in the C terminus that are resolved  are not positioned near membrane, making them unlikely palmitoylation sites [juxtamembrane cysteines are typically palmitoylated because the active site of the palmitoylating enzymes lies at the membrane/cytosol interface ]. We canHCN4 magnitude. The differences in IHCN4 amplitudes observed here were significant at relatively negative membrane voltages. In order to determine the consequences of the loss of HCN4 amino terminal palmitoylation on the channel activity over diastolic depolarization voltages and function of the intact SAN, the future use of appropriately genetically modified mice is now warranted. This would allow effects of HCN4 modification by palmitoylation to be ascertained for the intact native SAN in the absence of whole cell dialysis. The present study provides a strong foundation from which such work can be undertaken.The data in this study collectively indicate that native HCN4 protein in cardiac tissue is palmitoylated and reveal the importance of palmitoylation sites in the amino terminus of HCN4 channels. Further work, likely including measurements at the single channel level, is required to elucidate the mechanism(s) by which the loss of palmitoylation reduces I