To investigate the effects of alcohol on serum glycated albumin (GA) levels in Chinese men.  A total of 2314 male subjects from the Jinuo ethnic group in China were enrolled. Of these, 986 subjects drank alcohol frequently and 404 subjects did not. Lifestyle information was gathered by using a questionnaire, and measurements of blood pressure, body mass index, blood glucose level, liver function, and kidney function were collected. GA was measured by using an enzymatic method. Frequent drinking was defined as a history of drinking ethanol > 80 g/d within the past two weeks. Nondrinking was defined as no alcohol consumption in the past three months. Subjects with an alcohol intake between 0 and 80 g/d in the past two weeks were included in the drinking-occasionally group. Analysis of variance (ANOVA), correlation analysis, and linear regression were used to evaluate the effects of drinking on serum GA levels. Decision tree regression (DTR) algorithm was used to evaluate the effect of features (variables) on GA levels. p < 0.05). Spearman's correlation analysis calculated a coefficient of −0.152 between drinking and GA (p < 0.005). Linear regression established that drinking was an independent predictor for GA levels with a standardized regression coefficient of −0.144 (p < 0.05). Decision tree regression showed that the effect of drinking on GA levels (0.0283) is five times higher than that of smoking (0.0057).  We found that male subjects who drank frequently had significantly lower serum GA levels than subjects who did not drink (13.0 ± 1.7 vs. 14.1 ± 3.7,  Frequent alcohol consumption could result in decreased GA levels in men of the Jinuo ethnic group in China. Serum glycated albumin (GA) is a nonenzymatic glycation product of albumin and glucose . Since tPrevious work has demonstrated that GA levels are influenced by various factors , 9. ConcDM and IGR were diagnosed based on the diagnostic WHO 1999 criteria using the results of their 75 g oral glucose tolerance test (OGTT) without medical treatment. Drinking-frequently was defined as a history of drinking, the equivalent > 80 g/d of ethanol within the past two weeks. Nondrinking was defined as no alcohol consumption in the past three months. If the alcohol intake was between 0 and 80 g/d in the past two weeks, the subject was classified as drinking-occasionally. Smoking frequently was defined as smoking at least 1 cigarette per day for the past three months. If a subject had not smoked in the past three months, he was classified as no-smoking. Those who smoked some cigarettes but less than 1 cigarette daily in the past three months were classified as smoking-occasionally. Fibrosis index based on the 4-factor (FIB-4) value was used to evaluate liver fibrosis, FIB-4 = age × AST/PLT × ALT .From January to May 2012, a cross-sectional survey was conducted among men aged 18–75 years old from the Jinuo ethnic group in Yunnan Province, China. The study protocol was followed in accordance with the Helsinki Declaration and was approved by the Shanghai Jiao Tong University Affiliated Sixth People's Hospital's ethics committee. Informed consent was obtained from all participants prior to the survey. All subjects underwent an oral glucose tolerance test (OGTT), GA measurement, serum liver function, and renal function test.Liquid enzymatic determination of GA  was performed using an Olympus AU2700™ Chemistry-Immune Analyzer. GA is represented as % with a detectable range of 3.2. − 68.1%, and the coefficient of variation (CV) was <3.0%.p values were double-tailed, and p < 0.05 was considered as statistically significant. SPSS 21.0 software  was used for all statistical analyses.Analysis of variance (ANOVA) and linear trend tests were used to compare the differences between the groups and subgroups. Spearman's correlation analysis was used to observe the relationship between GA and other variables . Linear regression analysis was used to determine the effect these variables had on GA levels, with GA as the dependent variable and drinking status , smoking status , age, body fat percentage, fasting plasma glucose (FPG), 2h postprandial glucose (2hPG), albumin, glutamic-pyruvic transaminase (ALT), serum uric acid (sUA), FIB-4, and triglycerides (TG) as independent variables. All r = 0.51, p < 0.001). The DTR model selects the tree's node  based on the decision criterion of mean squared error (between the true GAs and the GAs predicted by the model with the selected features). The importance of a feature refers to the  total reduction of the criterion brought about by that feature.Since linear regression cannot represent the nonlinear relationships between the GA and the independent variables, we used a decision tree regression (DTR) algorithm, a feature engineering method, to find the influence of these features/variables on GA levels. The following features such as drinking status, smoking status, age, body fat, 2hPG, albumin, ALT, sUA, and triglycerides were used. The FPG was not used because it has a significant correlation with 2hPG  drank frequently and 404 (17.5%) did not drink; 1710 (73.9%) smoked frequently and 390 (16.9%) did not smoke; 169 (7.3%) neither drank nor smoked; and 825 (35.7%) both drank and smoked frequently.γ-GT), total bilirubin test (TBIL), FIB-4, albumin, and TG levels were all significantly increased in the drinking-frequently group compared to the nondrinking group, while the 2-hour insulin (2hINS) levels were significantly lower (p < 0.05) (p > 0.05).The clinical data showed that systolic blood pressure (SBP), gamma-glutamyl transferase ( < 0.05) . There wp < 0.05). The subjects were further divided into subgroups based on their glucose tolerance status, age, smoking status, and body fat. GA levels of each subgroup of the drinking-frequently group, except the smoking-occasionally group, were significantly lower than those in the nondrinking group (p < 0.05). The results of the ANOVA analysis showed that there was a statistically significant difference in serum GA concentrations among the subgroups with different drinking statuses and that the serum GA concentrations in the no-drinking group, the drinking-occasional group, and the drinking-frequently group had a decreasing trend . Analysis of smoking status showed that there was also no linear trend in serum GA levels .The average serum GA levels for the whole study population were 13.2 ± 2.4%. The serum GA concentrations in the drinking-frequently group were significantly lower than those in the no-drinking group  and −0.152 (p < 0.001), respectively. After adjusting for age, FPG, 2hPG, BMI, body fat percentage, γ-GT, TBIL, albumin, and TG, the partial correlation coefficient between GA and drinking status was −0.149 (p < 0.001), and −0.049 between GA and smoking status (p=0.020)  .p < 0.001), −0.167 in model 2 (p < 0.001), and −0.144 in model 3 (p < 0.001) with increasing independent variables. The linear regression models also showed that age, FPG, 2hPG, body fat, albumin, FIB-4, and TGs were all independent predictors of GA levels  compared to subjects who were not drinking. This decrease was independent of blood glucose and body fat content and should be noted when making a clinical interpretation. Classifying patients by drinking statuses such as no-drinking, drinking-occasionally, and drinking-frequently is convenient to apply, and it could effectively account for the effect of drinking on GA levels in clinical practice.It is well known that both a high-fat diet and alcohol can induce hepatic steatosis, inflammation, and fibrosis and can also change gut microbiota . Further


This cross-sectional study aims to analyze the physical demands of elite beach handball players during an official competition. Nine elite female  beach handball players of the Spanish National Team were recruited for this study. A Global Positioning System was incorporated on each player’s back to analyze their movement patterns. Speed and distance were recorded at a sampling frequency of 15 Hz, whereas acceleration was recorded at 100 Hz by means of a built-in triaxial accelerometer. The main finding of the study is that 53% of the distance travelled is done at speeds between 1.5 and 5 km/h and 30% of the distance is between 9 and 13 km/h , which shows the intermittent efforts that beach handball involves at high intensity, as reflected in the analysis of the internal load with 62.82 ± 14.73% of the game time above 80% of the maximum heart rate. These data help to orientate training objectives to the physical demands required by the competition in order to optimize the players’ performance. Beach handball is a discipline that was born on the beaches of Italy in 1992 with the intention of promoting court handball during the summer period , decisioThus, the study of physical demands during sports practice has become a fundamental aspect in the design of training sessions and the assessment of physical load during competition ,9,14. FoGPS is a satellite radio navigation system with technology that allows for the monitoring of the three-dimensional movement of a subject or group in different outdoor environments ,17,18. T−1 during competition in elite players, with moderate–high intensity movements. On the other hand, Zapardiel and Asín-Izquierdo [The use of this technology in beach handball for monitoring physical demands during competition is limited. Previous studies only analyzed the distance travelled and the analysis of the internal load of the player using heart rate monitoring devices ,9. TheseA cross-sectional study was carried out analyzing physical and physiological parameters of elite women’s beach handball players. This study was conducted during the Spanish Beach Handball Tour .Nine elite female  beach handball players of the Spanish National Team were recruited for this study. All the beach handball players were previously informed about the objectives of the study, methods, and risks of the research and they provided informed written consent to be part of the research. The Ethics Committee at the European University of Madrid (Spain) approved the methodology of the study according to the Declaration of Helsinki.The study was conducted during the Spanish Beach Handball Tour held in Orihuela-Alicante (region of south-east Spain). A maximum of six matches per team were played in two days. A total of 46 observations were included in the study. Some matches reached the third set (shoot out), but this additional set was not considered in the analysis.A global positioning system  was used on each player’s back to analyze their physical demands during the match, in stable atmospheric and signal reception conditions. Positional variables (distance and speed) were registered at a sampling frequency of 15 Hz. On the other hand, acceleration was recorded at 100 Hz through a triaxial accelerometer. A heart rate (HR) band was also used by each player . A standardized warm-up was applied before the test with running, mobility, and high-intensity activities .This system consists of three segments: (a) satellite signal reception and location system ; (b) Earth calibration station (control); and (c) GPS receiver (user). The space segment is made up of a network of at least 24 satellites in Medium Earth Orbit (MEO) that emit the signals , of which at least four are required for three-dimensional navigation, which includes latitude, longitude and altitude  . This naThe user segment corresponds to the GPS receivers that receive and decode the signals transmitted by the satellites . All of −1; zone 2: 0.5–4 km·h−1; zone 3: 4.1–7 km·h−1; zone 4: 7.1–13 km·h−1; and zone 5: >13.1 km·h−1. Speed zone thresholds were considered according to previous studies conducted in beach handball players [MEAN) and maximum (VMAX) speed according to the positional parameters  [The external load was evaluated according to the distance covered in five speed zones: zone 1: 0–0.4 km·h players . In addis  ,41. MAX) of each player was calculated using the formula proposed by Tanaka et al. [MAX of each player: zone 1: <60% HRMAX; zone 2: 60–70% HRMAX; zone 3: 70–80% HRMAX; zone 4: 80–90% HRMAX; zone 5: 90–95% HRMAX; and zone 6: >95% HRMAX. The average of the HR (HRMEAN) was also included in the analysis. Data were evaluated using the software provided by the GPS manufacturer . Internal load was monitored by means of Heart Rate (HR) data. The maximum HR  was used to obtain the jump height. Beach handball players kept their hands on their hips to avoid the effect of arm movement on jump performance. Every participant performed two different jumps before and after the matches with two minutes of recovery between jumps. The best jump was used for the analysis of the results . Upper-body strength was evaluated using a hand dynamometer with adjustable grip . Beach handball players closed their hands in a 2-s maximum repetition with the dominant hand and the arm totally extended. Two repetitions were performed before and after the matches and the best result (in kg) was included in the statistical analysis .p < 0.05.Data are presented as means ± standard deviations. The Kolmogorov–Smirnov distribution test was performed to confirm a normal distribution of the variables. Firstly, physical demand variables were compared between the first set and second set Student’s t-test, using the set (first and second set) as an independent variable and the movement patterns and physislogical parameters as depent variables. Secondly, physical performance test results were compared between the basal situation and the six matches by one-way analysis of variance (one-way ANOVA), using the match as the independent variable and the results of the physical performance tests as the dependent variable. Effect sizes  were calculated and defined as follows: trivial, <0.19; small, 0.2–0.49; medium, 0.5–0.79; and large, >0.8. All data were statistically analyzed using SPSS V24.0 for Windows . The level of significance was set at −1. The distance travelled in the different speed ranges showed a greater distance in zone 2 (554.50 ± 151.64 m) and a sprint distance (zone 5) of 28.28 ± 19.27 m  in the second set . Finally, there were no significant decreases in jumping ability and handgrip during the tournament in beach handball players , the distances covered in zone 3 , and zone 4  as well as the V®). For this analysis, GPS devices have been used [This study is the first investigation that analyses the physical demands of beach handball players during an official competition . Previous studies show a similar distribution in relation to the accumulated time in the different intensity zones, highlighting the intermittent nature of this sport [The influence of the playing period on the performance of the players has been studied in other sports, such as futsal ,48, baskis sport .p > 0.01) [Edwards et al. , define  > 0.01) . In othe > 0.01) . The exp > 0.01)  explaine > 0.01) , since uMAX. Regarding the difference between sets, the distance travelled in zone 4 stands out, with this difference being significant in defining the kinematic demand of the discipline. The results of this study demonstrate that a congested period of matches does not alter the jumping ability or upper-body strength of beach handball players. The dynamics of the game itself, as well as the unlimited possibility of substitutions, are the key elements to maintaining performance during the competition. This aspect will allow for better training planning with greater specificity and improvement of beach handball players.The main finding of the present manuscript is that 53% of the distance travelled is done at speeds between 0.5 and 4 km/h, and 30% of the distance is between 7.1 and 13 km/h, adding up to 83% of the total distance covered, which shows the intermittence of efforts that beach handball involves at high intensity, as reflected in the analysis of the internal load with 62.82 ± 14.73% of the time above 80% of the HR


We encountered a case of marginal ulcer in the jejunum after distal gastrectomy with jejunal pouch interposition. However, it has not been reported and not confirmed the treatment. We chose truncal vagotomy, considering reduced morbidity and postoperative complications.A case was a 69-year-old woman who was admitted to our hospital with melena. She had received curative distal gastrectomy with a 15-cm jejunal pouch reconstruction for early gastric cancer. Marginal ulcer in the jejunal pouch was detected by upper gastrointestinal endoscopy. She was given medication; however, she repeated hospitalization for melena and abdominal pain. Therefore, we decided to perform surgery, and truncal vagotomy was performed. The patient’s postoperative course was uneventful and was discharged on the 22nd postoperative day. Symptoms such as abdominal pain and melena were improved after truncal vagotomy.We presented a case with a complicated peptic ulcer after distal gastrectomy with reconstruction by jejunal pouch interposition, which was successfully treated by truncal vagotomy, a surgical acid-reducing procedure which does not require resection of remnant stomach. Over the last half-century, the epidemiology of both gastric and duodenal ulcer disease has changed markedly . The disRecently, because of the increasing number of early gastric cancer and improvements in its survival rates, greater attention has been directed towards the quality of life and the nutritional status of patients with gastric cancer after surgery. However, conventional reconstructions, namely Billroth I, Billroth II, and Roux-en-Y are known to exhibit certain limitations, such as a small reservoir and food stasis. Also, all known procedures are inevitably accompanied by postgastrectomy syndrome, which may involve weight loss caused by eating restrictions, heartburn, loss of appetite, and changes in daily activity, all of which are consistent with a decline in physical strength. Jejunal pouch interposition between the remnant stomach and duodenum has been introduced for conventionally open distal gastrectomy, not only to substitute for the small reservoir but also to maintain a physiologic pathway for ingested foods and to alleviate postgastrectomy syndrome. The usefulness of jejunal interposition for gastric substitution has since been reported by several authors –9. HowevWe encountered a case of marginal ulcer in the jejunum after distal gastrectomy with jejunal pouch interposition. However, to our knowledge, complications of this reconstruction, especially the development of an ulcer in the jejunal pouch, have not been reported. Although several surgical options such as conversion from jejunal pouch interposition to Roux-en-Y or Billroth I reconstruction were present, we underwent truncal vagotomy for reducing morbidity and postoperative complications –12.A 69-year-old woman was admitted to our hospital with melena, who received curative open distal gastrectomy with a 15-cm jejunal pouch reconstruction for early gastric cancer 5 years ago. Histopathological results showed that the depth of cancer was within the mucosal layer with the peptic ulcer and no lymph node metastasis of forty-two resected lymph nodes. Marginal ulcer in the jejunal pouch was detected by upper gastrointestinal endoscopy Fig. . SimultaTruncal vagotomy was performed as follows: upper-middle laparotomy was performed with severe adhesion between the abdominal wall and upper abdominal organs. The lateral segment of the liver was retracted cranially, and the subphrenic abdominal esophagus was appeared with care, not to injure the liver and the remnant stomach. The abdominal esophagus was gradually exposed, and EGJ (esophagogastric junction) was pulled vertically and caudally by a tape to separate the anterior and posterior vagal trunk Fig. . Both vaThe patient’s postoperative course was uneventful and was discharged on the 22nd postoperative day. A 24-h pH monitoring after surgery demonstrated an improvement of the acidic phase in the remnant stomach Table . SymptomThe most popular reconstruction method of distal gastrectomy has been either Billroth I or Roux-en-Y . HoweverIn this case, vagus nerve-preserving distal gastrectomy with the reconstruction of jejunal pouch interposition was performed. Marginal ulcers are relatively rare complications after gastrectomy if the truncal vagotomy is incorporated . In distElective surgery for peptic ulcer disease has recently become obscured from the medical world because of the significant advances in medical treatment over the past three decades –18. ThisWe presented a case with a complicated peptic ulcer after distal gastrectomy with reconstruction by jejunal pouch interposition, which was successfully treated by truncal vagotomy, a surgical acid-reducing procedure which does not require resections.


Takifugu rubripes in Liaoning Province, northern China, Uronema marinum, a scuticociliate, was identified. In this study, using Illumina MiSeq next-generation sequencing, the mitochondrial genome of U. marinum was assembled and analysed phylogenetically using mitochondrial genomes of other scuticociliates. The complete U. marinum mitochondrial genome was 39,845 bp; it contained two rRNAs, six tRNAs, and 39 protein-coding genes (PCGs). From the 39 PCGs, 15 PCGs were located on the heavy strand, and 24 PCGs on the light strand of U. marinum mitogenome. The phylogenetic tree showed that there were two main clades, Oligohymenophorea and Spirotrichea. Nine ciliate species were clustered together within Oligohymenophorea; Uronema marinum was a separate cluster sharing a relatively close ancestry with Hymenostomatida. The results of this study will help advance the systematics, and studies of evolution and molecular epidemiology of scuticociliates.Scuticociliates are dangerous parasitic pathogens for in worldwide mariculture. Scuticociliates cause high mortality to marine fish. After an outbreak of scuticociliatosis in  Uronema marinum is a scuticocilaite that belongs to order Shieldophilus, family Sphingidae, and genus Uronema. It causes scuticociliatosis, a disease with a high mortality in marine fish , we isolated a pure pathogenic ciliate from the skin ulcers of ill T. rubripes and identified it as the scuticociliate U. marinum. Although there are many scuticociliate species reported, such as Pseudocohnilembus persalinus . The sequence of U. marinum mitochondrial genome was deposited in GenBank (accession number MG272262). The whole mitogenome of U. marinum was 39,845 bp. Its nucleotide composition was as follows: A, 39.83%; T, 41.17%; C, 8.94%; and G, 10.06%. There were two rRNAs, six tRNAs, and 39 protein-coding genes (PCGs). Furthermore, the genes were coded on both the heavy strand and the light strand. From the 39 PCGs of U. marinum mitogenome, 15 PCGs were located on the heavy strand, and 24 PCGs on the light strand. Six genes  used the start codon ATT. Seventeen genes  used the start codon ATG. Eight genes  used the start codon ATA. Two genes  used the start codon GTG. Four genes  used the start codon TTA. The genes orf149 and ymf66 used the start codon TTG and ATC, respectively. The stop codon TAA was present in 38 PCGs. Only the gene ymf56 used the stop codon TAG. The G + C content of PCGs was 18.4%, very close to the 19% average G + C content of U. marinum mitogenome. The overall number of codon occurrences was 10,345. The most frequent amino-acid encoding codons were Leu (13.71%), Phe (11.69%), Asn (9.98%), Ile (9.73%), Lys (8.08%), Tyr (7.51%), Ser (6.83%), and Val (5.43%). Two rRNA genes  were located on the heavy strand of U. marinum mitogenome. The G + C content of rRNA genes was 23.38% for rnl, and 24.78% for rns, which was higher than the average 19% G + C content of the U. marinum mitogenome. There were six tRNAs on the mitogenome of U. marinum. tRNA-Glu, tRNA-Met, and tRNA-Trp were on the heavy strand, and tRNA-Tyr, tRNA-Phe, and tRNA-His were on the light strand. These six tRNA genes had the predicted typical cloverleaf secondary structure.In this study, the total DNA of Uronema marinum was a separate cluster, sharing a relatively close ancestry with Hymenostomatida.Phylogenetic relationships between 12 ciliates in the Oligohymenophorea taxa were analysed according to the nucleotide and amino-acid sequences of 13 PCGs from GenBank using BI and ML methods. The resulting phylogenetic tree showed that there were two main clades, Oligohymenophorea and Spirotrichea . Nine ci


DEC-205 (CD205), a member of the macrophage mannose receptor protein family, is the prototypic endocytic receptor of dendritic cells, whose ligands include phosphorothioated cytosine–guanosine oligonucleotides, a motif often seen in bacterial or viral DNA. However, despite growing biological and clinical significance, little is known about the structural arrangement of this receptor or any of its family members. Here, we describe the 3.2 Å cryo-EM structure of human DEC-205, thereby illuminating the structure of the mannose receptor protein family. The DEC-205 monomer forms a compact structure comprising two intercalated rings of C-type lectin-like domains, where the N-terminal cysteine-rich and fibronectin domains reside at the central intersection. We establish a pH-dependent oligomerization pathway forming tetrameric DEC-205 using solution-based techniques and ultimately solved the 4.9 Å cryo-EM structure of the DEC-205 tetramer to identify the unfurling of the second lectin ring which enables tetramer formation. Furthermore, we suggest the relevance of this oligomerization pathway within a cellular setting, whereby cytosine–guanosine binding appeared to disrupt this cell-surface oligomer. Accordingly, we provide insight into the structure and oligomeric assembly of the DEC-205 receptor. The MR  (PLA2R) . Althoug+ DCs are involved in the sensing of necrotic and apoptotic cells in a manner thought to promote peripheral tolerance  (DEC-205 (LY75 or CD205) is predominantly found on dendritic cells (DCs) , 6, specolerance . Indeed,olerance  with DECll death , Despitenditions , 15, 16,nditions . Indeed,nditions , 15. To nditions , 19, a c, 9.6 Å) , which ain vitro as well as the cryo-EM structure of the DEC-205 tetramer at 4.9 Å resolution. The tetrameric form of DEC-205 showed an extensive remodeling of the secondary CTLD ring and suggested a role for the β2-3 loop of CTLD5 in mediating this transition state. The potential physiological relevance of the DEC-205 tetramer was suggested using molecular imaging techniques in the absence and presence of CpG ligand. Our structural analysis of DEC-205 provides a fundamental advance in our understanding of this promiscuous endocytic receptor and the mannose-receptor family more broadly.Here, we present the cryo-EM structure of the human DEC-205 monomer at 3.2 Å resolution, revealing a compact lemniscate structure formed of two concatenated rings of CTLDs. Further, we describe a pH-dependent and ligand-dependent oligomerization of DEC-205 We produced and purified the soluble DEC-205 ECD  from mammalian expression to yield protein that was well behaved and suitable for structural studies. Given that DEC-205 adopts a compact conformation at low pH , we prepA) and N-linked glycans .The structure of the DEC-205 ECD was determined to a resolution of 3.2 Å . The cryFig. S2A–B). The centrally located and globular CysR and FNII domains lead into a compact 60 Å ring with a central cavity of ∼15 Å comprised of a counter-clockwise arrangement of CTLD1 through to CTLD4 (C). The second and slightly larger ∼65 Å ring comprises of a clockwise arrangement of CTLD5 through to CTLD8. Here, the central cavity of the second ring was occupied by an extended loop of CTLD5 (D). Although the reconstruction was of high quality, surprisingly CTLD9 and CTLD10 were lacking in the final reconstruction, although Western blot targeting the C-terminus confirmed intact protein, and no signs of degradation post imaging (data not shown). Inspection of the 2D class averages ..13).A), comprising of 12 antiparallel β-sheets with a loose two-fold internal symmetry (A). Each of the tripartite nodes includes four short β-sheets β1-4, β5-8, and β9-12, respectively, that are stabilized by three anchoring disulfide bonds akin to the CysR domain of the MR (A). However, although the β11 to 12 loop of the DEC-205 CysR formed a similar confirmation to that in the MR, the deletion of the ligand coordinating N102 found in the MR would likely render DEC-205 unable to coordinate sulfated ligands, corroborating previous findings . Overall, the domain closely resembled that of the recently solved MR FNII notwithstanding subtle movements within the connecting loops (B) (B) . Aoops (B)  has beenoops (B) B (20).C–J). Each of the CTLDs adopted a compact β2-3 loop architecture with the exclusion of CTLD5 that stood in stark contrast with a fully extended β2-3 loop that played a role in coordinating the secondary CTLD ring (G) and thus in stabilizing the DEC-205 ECD. The first CTLD ring of the DEC-205 ECD had a 15 Å cavity with no loop plugging the core to serve a similar coordinating role. Owing to divergent amino acid sequences, the 10 contiguous CTLDs of DEC-205 are thought to be a structural scaffolds for ligand binding and are not predicted to bind calcium ions which facilitate oligosaccharide binding in other CTLDs . Ind. IndC–J)er CTLDs . NotablyA–B). Similarly, the FNII domain moves 9° and translates 5 Å in the DEC-205 ECD, whereas the CTLD1 and 2 rotate ∼6° and move 5 and 2 Å, respectively, although the individual domains are relatively similar (C–F).Structural comparison to the structure of a truncated N-terminal fragment of the MR showed a high amount of structural conservation with ∼2.2 Å root mean squared deviation when aligning to the corresponding CysR-CTLD2 of the DEC-205. Within the respective structures, there are subtle domain movements, most notably in the CysR that rotates 8° and moves 2 Å in the context of the DEC-205 ECD relative to the truncated MR structure , A–B. Si similar , C–F.FigIn short, the cryo-EM structure of the DEC-205 ECD is of high quality, revealing the atomic architecture of the mannose-receptor family in new light and will serve as a platform for further characterization of DEC-205E). Further, W69 of the β4 sheet of the CysR accommodates F743 of CTLD4, which projects up from the first CTLD ring (E). The neighboring Q745 of CTLD4 contacts Q1168-D1170 of the retracted β2-3 loop of CTLD7 to stabilize the interface around H73 (E). Although a much more compact domain, the FNII made peripheral contact to CTLD7 and 8 but mainly interacts with the α1 and face of the β1, driven by a salt bridge from R167 (FNII) to E242 (CTLD1). In addition to the extensive contacts from the CysR and the FNII, CTLD1 made extensive contact to the terminal CTLDs of the two rings, namely CTLD4 and 8, which serves to stabilize the first CTLD ring. Instead of calcium-binding, the CTLD β2-3 loops seem to have been repurposed toward stabilizing the overall arrangement of the DEC-205 ECD. In fact, around ∼50% of the total interdomain buried surface area (BSA) of the DEC-205 ECD is accounted for by β2-3 loop-mediated interactions (F). Most striking was the extended β2-3 loop of CTLD5, which acted as a “recruitment hub”, whereby it co-ordinated several domains within the secondary ring including CTLDs 6, 7, and 8. Intriguingly, the β2-3 loop of CTLD5 contained an apical charged cluster of, W893, H894, and R895 that interacted with CTLD7. Here, R895 seemed to tether the loop apex via sidechain interaction with a neutral pocket composed of L1172, N1173, and Q1193 within CTLD7 with peripheral interaction between W893 and H1183, which further stabilized the extended long-loop conformation (G).The compact DEC-205 ECD was stabilized by an extensive network of interdomain interactions. Given the central position of the CysR domain, it made numerous contacts to CTLD4 and 7 and the neighboring FNII. The contacts from the CysR includes Q72 and H73 of the β4-5 loop that projects toward the CTLD rings and are buried within Q1168-D1170 of the retracted β2-3 loop of CTLD7 E. FurtheTLD ring E. The neound H73 E. Althouractions F. Most sormation G.H and The extensive interdomain BSA and interactions of the DEC-205 ECD included major contributions from the central domains of the lemniscate, namely CysR, CTLD1, CTLD4, and CTLD5 and 8 . Notably, these biophysical measurements further validated that the protein being produced was intact and simply mobile within the cryo-EM reconstruction. Next, we employed sedimentation velocity analytical ultracentrifugation (SV-AUC) to further investigate the oligomeric state of the DEC-205 ECD at acidic pH. Similarly, DEC-205 existed as a monomer at pH 6.0  with a small amount of dimer evident (B). Building on this finding, we observed a large change in the elution profile of DEC-205 at pH 8.0, eluting with a mass four times that of the monomer, suggesting that in basic conditions DEC-205 formed a stable tetramer (C). Further SV-AUC experiments confirmed the presence of a stable 730 kDa DEC-205 tetramer ,) with a small proportion of 1020 kDa hexamer  (D). Further, we investigated the monomeric-oligomeric state of DEC-205 at neutral pH and found that the vast majority was monomeric with a consistent yet reproducible component of tetramer. Together, these data show that DEC-205 can oligomerize in vitro from a stable monomeric form into a tetramer in response to a change in environmental pH.Utilizing size-exclusion chromatography coupled to multiangle light scattering, purified DEC-205 ECD at acidic conditions (pH 6.0) reproducibly formed a monomer with a mass in agreement with the predicted mass (192 kDa as derived from amino acid sequence) A. Notabl evident B. Builditetramer C. Furthe/fo 1.5) D. FurtheE–G) and was ultimately resolved to 4.9 Å . Here, the DEC-205 tetramer formed a 115 × 200 × 100 Å toroid shaped particle with a large central cavity , E–G andto 4.9 Å .A) that enabled four protomers to assemble into a prolate toroid with clear D2 symmetry in the 2D class averages and final reconstruction (B). Here, the Cys-R, FNII, and CTLD1-3 remained globally unchanged within the first CTLD ring and stacked against the corresponding ring of another protomer such that the CysR and FNII domains of the respective protomers were exposed (C). Notably in this confirmation, the hydrophobic groove within the FNII domain was no longer occluded by the CTLD7-8 linker.The overall architecture of the DEC-205 tetramer revealed a major rearrangement of the second CTLD ring A that entruction B. Here,  exposed C. NotablD). Within this extended confirmation, CTLD8 from one protomer contacted CTLD4 of the opposing protomer and vice versa. The cryo-EM reconstruction was unambiguous for the placement of the 10 (CysR to CTLD8) domains except for CTLD5. Here, the cryo-EM reconstruction suggests that CTLD5 is highly mobile within the tetramer, and the domain could act as an allosteric center for DEC-205’s pH-dependent rearrangements. Following localized refinement of the reconstruction, it enabled overall domain placement of CTLD5, but it was not possible to determine the conformation of the β2-3 loop (B). As observed in the DEC-205 monomer, CTLD9 and 10 were absent from the final reconstruction, solidifying the notion that they are a highly dynamic part of the protein. The BSA for the tetramer was much more extensive, and the modest resolution did not allow atomic analysis of the interfaces. Broadly the BSA of the protomer 1 and 2 head–head interaction accounted for ∼2300 Å2, whereas the protomer 1 and 3 tail—tail interaction was ∼1500 Å . Further, the specificity of interaction was shown to be specific for the phosphorothioate of the CpG ligand, whereby a nonthioated CpG control showed no cellular staining.We first sought to establish whether cell surface DEC-205 could bind to the CpG oligonucleotide 1668 (5′TCC ATG ACG TTC CTG ATG CT), a previously established DEC-205 ligand . Cells eFig. 6gfp, we directly quantified the oligomeric state of DEC-205 on a cell surface. Confocal scanning microscopy showed cell surface expression of FL-DEC-205 with B values consistent with the presence of monomers, dimers, and oligomers potentially corresponding to tetrameric DEC-205 (B). Higher-order species were also observed and are thought to correspond to a small observation of a hexameric form of FL-DEC-205 that was observed during the cryo-EM analysis of the DEC-205 tetramer (B). Notably, a control cell line expressing full length CD80gfp exhibited similar average intensities; however, the predominating CD80 cell surface species was monomeric, in agreement with the well-documented monomeric nature of this receptor (B). Thus, our N&B analysis served to support the observation that FL-DEC-205 existed in both monomers and larger oligomers on a cell surface and provided context for our structural work.We next sought to determine the impact of DEC-205 oligomeric state on the cell surface using number and brightness (N&B) fluctuation spectroscopy analysis. N&B analysis is a method that measures both the mean and variance of fluorescence intensity distributions per pixel from a stack of images to deconvolute the number of molecules (N) and their brightness (B), where B directly relates to the oligomeric state . Conduct DEC-205 B. Highertetramer B. Notablreceptor B. Thus, gfp+ cells shifted from an equal abundance of monomers, dimers, and oligomers  to a predominantly monomeric cell surface form (∼73%) in the presence of CpG (B). Thus, these microscopy data suggest that FL-DEC-205 resides on a cell surface in a state of structural equilibrium comprising of DEC-205 monomers and oligomers. Thus, these observations on the cell-surface DEC-205 support the structural work on the DEC-205 ECD. Furthermore, such observations provide a possible biological context for the monomer and tetrameric arrangements of DEC-205 identified in our biophysical and structural data.To begin to address the notion of whether DEC-205 oligomerization had a functional role in ligand recognition, we next conducted N&B analysis in the presence of CpG oligonucleotide. Here, the addition of CpG induced a switch to mostly monomeric DEC-205 with a notable decrease in cell surface tetramers . Indeed,e of CpG B. Thus, in vitro. SV-AUC analysis of the DEC-205 ECD at pH 7.4 showed a largely monomeric species with a minor component of higher-order species somewhat consistent with the cell surface FL-DEC-205(C). A subtle movement of the pH to 7.7 skewed the distribution toward an equal monomer–tetramer equilibrium; however, upon the addition of CpG, this shifted the DEC-205 ECD to a monomer (D). These data suggest that the monomeric form of DEC-205 is the ligand binding component. To decipher the impact of pH on the ligand-binding ability of DEC-205, we next conducted an ELISA assay at differing pH. Immobilized soluble DEC-205 ECD was then incubated with CpG at a pH 6.0, 7.4, and 8.0. DEC-205 binding to CpG was enhanced at acidic pH relative and neutral pH but diminished notably in alkaline conditions (E). Taken together, these data suggest that monomeric DEC-205 is the configuration that binds to CpG and that low pH is thus conducive to CpG binding because of disruption of the DEC205 oligomer.Given how the oligomeric state of FL-DEC-205 was impacted by ligand binding investigated the impact of CpG oligonucleotide binding on DEC-205 oligomerization L-DEC-205C. A subt monomer D. These nditions E. Taken The high-resolution structure of the DEC-205 ECD has provided insight into the atomic structure of the macrophage mannose-receptor family. In addition, characterization of the pH-dependent oligomerization of DEC-205 and the solving of the tetrameric DEC-205 structure showed a complex conformational remodeling occurs during oligomerization.e.g., the insulin-like growth factor 2 receptor  CTLD ring that facilitated a head–head and tail–tail protomeric arrangement in the tetramer. Such structural rearrangement was directly resultant from an increase in pH, a trait observed in studies of the MR. Similar pH-dependent rearrangements were observed for another receptor containing multiple-repeat domains, receptor . Notablyreceptor . In partreceptor , 30. In receptor , 32, 33 receptor . Similarrization .in vitro. We speculate that the oligomeric status of these endocytic receptors is either prerequisite for ligand binding or ligand dissociation within the endosomes. Considering DEC-205 is structurally pH-labile and that both CpG and keratin binding is pH-dependent , the protein was loaded onto a FLAG-resin column overnight at 4 °C. Following column washing, DEC-205 was eluted with FLAG peptide and peak fractions collated and concentrated before a final clean-up over SE in either TBS, HBS (10 mM HEPES pH at 7.4 and 150 mM NaCl), or BIS-TRIS buffer (10 mM BIS-TRIS at pH 6.0 and 150 mM NaCl).The cDNA encoding the human DEC-205 ECD  in-frame with a C-terminal FLAG tag (sequence DYKDDDK) and biotinylation consensus sequence (NSGLHHILDAQKMVWNHR) was synthesized by GeneArt/ThermoFisher. The DNA was codon-optimized for expression in human cells and cloned into the pcDNA3.1+ vector (ThermoFisher). Secreted protein was expressed by polyethylenime transient transfection of human embryonic kidney (HEK) 293S cells totaling four 1900 cm−1 was loaded onto a Superdex 200 5/150 column  in either TBS, HBS, or BIS-TRIS buffer at a flow rate of 0.3 ml min−1. The system comprises of a DGU-20A5 degasser, LC-20AD liquid chromatography, SIL-20ACHT autosampler, CBM-20A communications bus module, SPD-20A UV-visual detector, and CTO-20AC column oven (Shimadzu) coupled with a DAWN HELIOS-II light scattering detector and Optilab T-rEX refractive index detector (Wyatt). Detector number 12 was substituted for a WyattQELS detector installed at a 90° angle. The system was controlled using LC solutions (Shimadzu), and data collection and analysis were performed in ASTRA6 (Wyatt Technology Corp).50 μl of DEC-205 ECD at 1 mg ml−1 in either TBS buffer, HBS, or BIS-TRIS buffer was loaded into a dual-compartment cell next to 400 μl reference solution. All experiments were performed using an 8-hole rotor at 37,000 RPM at 20 °C, and the sedimentation velocity profiles were collected at wavelengths ranging from 230 to 310 nm. The collected data were analyzed in SEDFIT with a c(s) distribution model with a maximum entropy regularization of p = 0.68. The buffer density (1.0047 g ml−1) and viscosity (0.01002 cP) as well as sample frictional ratio (f/f0) were calculated from the SEDNTERP program using the primary amino acid sequence.Oligomerization state and molecular weight assessments of DEC-205 ECD in solution were assessed via sedimentation velocity experiments using an Optima analytical ultracentrifuge (Beckman Coulter). DEC-205 at 0.2 mg ml−1. Using a Vitribot Mark IV, sample was applied to the grids at 4 °C and after ∼20s were blotted for 2s at −2 blot force before immediate vitrification in liquid ethane. Grids for the DEC-205 tetramer were prepared by applying 3 μl of DEC-205 ECD in TBS buffer at a concentration of 0.1 mg ml−1 and blotted as described above.Cryo-EM grids of purified DEC-205 ECD monomers were prepared by applying 3 μl of DEC-205 in BIS-TRIS buffer to copper 200 mesh Quantifoil R1.2/1.3 holey-carbon grids (ProSciTech) at a concentration of 0.3 mg ml2. Similarly for the DEC-205 tetramer, 7.2 s exposures through a defocus range of −0.8 to −3.5 μM were dose fractioned into 18-frame movies collected at 130,000 × magnification, corresponding to a pixel size of 1.06 Å with a total dose of 63 electrons per Å2.Frozen grids were transferred to a Titan Krios transmission electron microscope (FEI) operated at 300 kV, equipped with a Quantum energy filter (Gatan) and Summit K2 (Gatan) detector. Automatic data acquisition was performed using EPU (FEI). Briefly, for the DEC-205 monomer, 12 s exposures through a defocus range of −0.8 to −2.8 μM were dose fractioned into 25 frame movies collected in energy filtered mode using a slit width of 20 eV at a nominal magnification of 130,000 × (energy filtered transmission electron microscopy), corresponding to a super resolution pixel size of 0.53 Å with a total dose of 53 electrons per Åab initio initial model was generated. The well-defined particles from clean 2D classifications were used for Initial 3D classification followed by initial 3D refinement. Following particle polishing, 3D classification with an increased Tau factor, T=(4), further separated the higher-resolution particles leading to a final refined and sharpened reconstruction at 3.2 Å. For the DEC-205 tetramer, the processing proceeded via the same workflow until 2D classification, and here, the high-resolution classes displayed an obvious D2 symmetry. The symmetry axes were aligned using Relion 3.0 . The final DEC-205-monomer was then utilized for domain placements for the DEC-205 tetramer followed by rigid-body real-space refinement in Phenix.Upon finalization of the respective cryo-EM maps, crystal structures of the previously solved Cys-R (PDB 1DQO) , FN PDB, and CTLCoot , the domCoot , 46. RegCoot  and PDB gfp+ HEK293T cells were stained with Hoechst 33342 (1 μg ml−1) DNA marker, then incubated with 5 μM of either CpG or a nonthioated CpG control for 5 min before imaging. The following phosphorothioated 3′ labeled Atto647N 1668 CpG oligonucleotides were purchased from Geneworks: 5′TCC ATG ACG TTC CTG ATG CT as well as a nonthioated control. Images were acquired using a Zeiss LSM 980 Airyscan 2  with a C-PlanApo 63x/1.4NA oil objective and an environmental (37 °C with 5% CO2 supplied) chamber.For confocal microscopy experiments, HEK293T cells were plated out onto a 35 mm FluoroDish  and cultured in DMEM supplemented with 10% (v/v) fetal bovine serum (FBS). Cells were transfected using lipofectamine 3000 and 14 μg of DNA of either full-length DEC-205  or CD80  cloned into pcDNA-3.1+ in frame with a C-terminal eGFP. Before imaging, the media was switched to OPTI-MEM–phenol-free media supplemented with 2% (v/v) FBS. For the CpG co-localization study, DEC2052 supplied) chamber (Olympus) equipped with a 488 nm laser and 60×/1.2NA water immersion lens. Images for N&B analysis were captured with the 488 nm laser attenuated to 0.1% laser power, PMT detector set to photon-counting mode, scan speed 12.5 μs/pixel, scan area 256 × 256 pixels (50 nM pixel size), and 100 frames were captured per image series  cultured in DMEM supplemented with 10% (v/v) FBS and transfected as above. Before imaging, the media was switched to OPTI-MEM–phenol-free media supplemented with 2% (v/v) FBS. Confocal images were collected 16 h after transfection using a Fluoview1000 CLSM  equipped with a 488 nm laser and 60× NA 1.2 water immersion lens in combination with an environmental (37 °C and 5% COe series .N) of diffusing particles within the focal spot and the intrinsic brightness (B) of each particle (B) of monomeric eGFP (Bmonomer = 1.3), being imaged under the same conditions as DEC-205–EGFP and CD80-eGFP; Sfactor for the experimental setup was determined to be 1.3. The brightness of the eGFP was used to indicate the monomers (1.3 ± 0.15), dimers (1.6 ± 0.15), and tetramers (2.2 ± 0.15) on the Bmap and for further comparative analysis of oligomeric states  to determine the number (particle . Solublec states .−1 of MMRI7 anti-DEC-205 mAb (BD Biosciences). Unbound mAb has washed away  before proteins  were added . Captured protein was exposed to various biotinylated CpG oligonucleotide at specified concentrations , detected with streptavidin-horseradish peroxidase   and colorimetric reaction visualized using ABTS . Each titration point was performed in duplicate.ELISA plates were coated overnight at 4 °C with 10 μg mlPDB 7JPT and 7JPU and EMDB codes 22422 and 22423 for the monomer and tetramer, respectively.The authors declare that they have no conflicts of interest with the contents of this article.


Speed climbing involves an optimization of the velocity of the ascent and the trajectory path during performance. Consequently, any amount of energy spent in the two other directions than vertical, namely the lateral direction and the direction perpendicular to the wall plane, is a potential loss of performance. To assess this principle, we present a study on 3D motion analysis and its 3D visualization for a subject during a speed climbing performance. The fundamentals of geometrical measurement in 3D require to integrate multiple 2D cues, at least two, in order to extract 3D information. First results with two drones following an athlete's ascent show that a 3D velocity profile can be provided from the tracking of a marker on the harness, pointing critical phases in the ascent where the vertical speed is not dominant any more. We further investigate 3D motion of full body using markerless video-based tracking. Our approach is based on a full body 3D avatar model of the climber, represented as a 3D mesh. This model and its deformation are learned in a laboratory studio. The learning needs to be done only once. Result is a manifold embedding of the 3D mesh and its deformations, which can be used afterwards to perform registration onto video of performance of speed climbing. The results of the tracking is an inference of the 3D mesh aligned onto videos of speed climbing performance. From this 3D mesh, we deduce an estimation of the center of mass (COM). We show that this estimation from 3D mesh differs from the usual approximation of the COM as a marker on the harness. In particular, the 3D mesh COM takes into account the whole body movement such as the influence of the limbs which is not detected by a marker on the harness. Video analysis is now regularly used by high-level athletes and coaches to address performance optimization. Sequences are usually acquired through portable devices or fixed environment, allowing to quickly visualize a current trial and providing an instant feedback on the performance. The quantification of benefit of such video feedback in self-control condition has been reported in Basketball shot for example  as depicted in the literature review by Orth et al. . Among fWe describe here a method to capture 3D information about the motion of a speed climbing athlete from video acquired by several points of view, fixed, or possibly moving such as drones. The key aspect of our method is to be based on a 3D avatar of the athlete. This avatar is first learned in a laboratory studio. Granting the morphology of the athlete is not significantly changing, this learning needs to be done only once. Afterwards, the learned 3D avatar is automatically registered onto any video of the performance of the athlete, without the need for any markers. The end result of the approach is an animated 3D representation of the performance which can be interactively explored by changing viewpoint. In addition, we show that a prediction of the 3D trajectory of the COM can be derived from this 3D representation, providing an estimation more reliable than a marker on the harness.In video recording of climbing performance, viewpoints from the ground classically introduce artifacts such as bottom view distortion which impedes the efficiency of visual inspection and automatic processing. For this experiment, we have thus used two drones as they provide high-quality video capabilities and can be easily monitored to follow the ascent of the athlete. As each drone is moving with respect to the environment, motion recorded in the video mixes both the motion of the athlete and the motion of the drone. This is resolved by performing an auto-calibration of the drones 3D position and orientation at each frame with respect to reference frame linked to the wall. Drones usually embed inertial and GPS sensor to monitor their position. Such sensors turned out to be not precise enough to compute an accurate estimation of their relative position and orientation—accuracy goal is to be <1 cm and 1 degree per frame). We used instead a geometrical approach, based on a prior 3D scanning of the wall. At each frame, a prior 3D model of the wall is registered onto the video view by aligning salient features of the holds. Registration is performed by optimizing the 3D location and orientation of the drone with respect to a metric on wall features. Like for traditional markers-based system, the quality of such a calibration can be assessed through back projection. Results showed that the required accuracy can be achieved.As a first result following calibration and as a matter of validation, we implemented a 3D reconstruction of the trajectory of a marker on the harness. While not exactly the COM (as detailed later), such a location is close enough to COM to be worth noticing and to be considered as a good representative of the overall body 3D location. The 2D location of the marker is tracked on each view using normalized cross correlation. As the 3D location and position of each drone is known at each frame, the 3D location of the marker can be derived using Direct Linear Transform approach (DLT). Drones are calibrated with respect to a fixed reference frame related to the wall, hence the extracted 3D location of the marker. By derivating this 3D location using finite difference and knowing the video frame rate, the athlete's speed can be estimated in metric units. This constitutes a first visualization of a motion quantity in 3D, with a possibility to identify key moment where the vertical speed is decreasing or when the climber is getting too much away from the wall, inducing a loss of performance for the goal of reaching the top in minimal time.kg/dm3. This experimental data provides the required information to learn the manifold of all the deformations of the athlete's 3D body surface. This learned model can subsequently be used for tracking live motion from video during a performance on the wall.To go beyond the 3D trajectory of an isolated marker, we focus now on 3D visualization of the body as a whole based on a 3D mesh representation. This 3D representation will be used to implement a markerless video tracking approach (next section). To build this 3D model of the athlete, we used a laboratory facility consisting in a studio equipped with 68 video cameras. This studio allows to compute, for each video frame, a full 3D reconstruction of the body surface in motion using a convex hull approach . The two drones have been temporally synchronized at the frame level with a common light signal triggered at the beginning of the performance. The procedures  were approved by the French Federation of Mountaineering and Climbing (FFME), and conformed to the declaration of Helsinki. It has been approved by the University of Lyon ethic committee as not invasive because it is limited to the video recording of a regular practice without any contact with the subject's body. Piloting of the drones was performed under the supervision of a certified pilot (drone certificate MAVIC-53) in a closed non-public environment. Flying was limited to a vertical ascent of <20 m.One female climber performed a set of maximal speed ascents on the official route. We selected the best trial (ascent time = 7.9 s) for this analysis. The performance has been filmed with two drones (DJI Mavic pro) with resolution 3,840 Figure 2). The 3D animated scene can also be explored from 3D viewpoint, different from the original drone video viewpoint, following a subjective camera principle . Lastly, the 3D view can be augmented with information such as the 3D trajectory of the COM or velocity cues. Hints on time of grasping of the holds can also be visualized by computing 3D velocity of mesh vertices at limbs extremities.We assemble all the results into a final 3D scene. First, the 3D scan of the wall used for calibration can be directly imported. Dedicated texturing can be added to augment the quality of the rendering. The 3D mesh representation of the athlete in motion is integrated, with also a possible texturing for aesthetics consideration. It is worth noticing that such texturing needs to be done only once off-line as the topology of the mesh remains constant. Only the 3D location of the vertices is updated by the automatic tracking phase. For the sake of validation of the process, the original video of the drones can be first augmented with the projection of the 3D animated scene  and the gravity vertical axis (axe Y), with the origin at the bottom of the wall . TrianguWe report on On bottom of The velocity ratio clearly outline moments when the vertical ascent is less dominant. It typically corresponds to “dyno” transition from hold 8 to 9 and hold 16 to 17. During these periods, the wall-orthogonal Z-axis component becomes dominant, corresponding to a posture which is getting farther from the wall. On hold 7 to 8 and hold 13 to 14, the velocity components ratios profiles show that the lateral X-axis becomes important with respect to the vertical Y-axis. They correspond to a required change of route but also show a drop in the vertical component. The visualization of these 3D cues provide first insights on speed climbing performance.The previous steps validated the experimental infrastructure to extract 3D information from video in terms of the trajectory of a single marker. We report here the extension to full body analysis through its 3D visualization. The official 15 m high speed climbing wall can obviously not be replicated into the laboratory as the volume which can be captured at the laboratory facility is limited to 5 × 5 × 3 m. Consequently, the athlete has been asked to perform a mimicry of the speed climbing ascent as the route is standardized and completely memorized. Protocol for the simulation of movement has been left to the expertise of the climber who is a word-level athlete. We used this sequence to learn the manifold of 3D shape variation of the athlete's 3D surface appearance. We used a Gaussian Process Latent Variables Model as described in Duveau et al. . SimulatWe examined here the approximation of considering the COM as a fixed marker on the harness near the hips vs. a prediction as the COM of 3D mesh. The prediction from 3D mesh was automatically computed from the 3D mesh as the barycenter of the enclosed 3D volume with the hypothesis of uniform density. We compared this trajectory to the trajectory of the marker on the harness with the result of this prediction. Results show that the mean distance between the marker on the harness and the COM from 3D mesh are, respectively in the three directions: 7.3 ± 5.9 cm for the X direction , 8.7 ± 4.6 cm for the Y direction , and 24.1 ± 4.3 cm for the Z direction . The biggest difference is on the Z direction as the marker is attached on the back while the 3D mesh COM is more likely also influenced by limbs projection toward the wall. The 3D scene can be visualized from different angles and as such represent a valuable enhancement of standard video. In addition, visual cues can be added onto the 3D scene such as velocity. In Results show that the trajectories of a marker on the harness and the 3D mesh COM differ. Although no exact measurement of the true COM exists for validation, its estimation from the 3D mesh follows some rational insight and tends to prove it is more reliable. Typically, unlike the marker, the 3D mesh method accounts for the projection of the limbs toward the wall or the flexion of the legs. Therefore, we compared the velocity computed from the marker with the one computed from the COM, following the same scheme as cm/s to identify grasping from velocity magnitude of limbs extremity vertices . In particular, our finding is that they are some noticeable discrepancies between the 3D trajectory of a marker on the harness, approximating the COM, and an estimation of the COM of the 3D model of the athlete registered on videos. The estimation of the 3D trajectory of the COM from our video-based 3D mesh tracking tends to follow rational insights that a marker-based approach does not allow.Future works will explore more precisely dynamics in speed climbing. The goal will be to extend our previous experience in this domain, obtained in a laboratory set-up, to similarly address the context of athletic speed climbing  for the publication of any potentially identifiable images or data included in this article.LR developed the techniques, performed the experiment to collect the data to build the athlete's 3D model, performed the 3D video analysis, computed the 3D model kinematics, and wrote the paper. SC provided the sport supervision. PL performed the experiment during the speed climbing tests. FQ and PL participated in the writing of the paper. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Reducing consumption of drinks which contain high levels of sugar and/or alcohol may improve population health. There is increasing interest in health behaviour change approaches which work by changing cues in physical environments (“nudges”). Glassware represents a modifiable cue in the drinking environment that may influence how much we drink. Here, we report three laboratory experiments measuring consumption of soft drinks served in different glasses (straight-sided vs. outward-sloped), using distinct paradigms to measure drinking. In Study  Sugary drink consumption, in particular, is linked with a number of health conditions, including Type 2 diabetes, cardiovascular disease, and others6. Developing novel and effective interventions to change drinking behaviour is thus an important goal of research and policy. There is increasing interest in approaches that work by changing cues in physical environments—known also as “nudging”10. Broadly speaking, these interventions are thought to engage automatic (rather than reflective) processes, requiring relatively less active engagement or high-level cognitive processing to elicit a change in behaviour than other types of behaviour change techniques11. One aspect of the drinking environment that has the potential to influence drinking behaviour—possibly outside of awareness—is the glassware in which drinks are served.Overconsumption of drinks containing excess sugars and alcohol is a major threat to population health globally12—with some evidence that the use of larger wine glasses increases wine consumed15. The shape of a glass—in particular, whether it is outward-sloped or straight-sided—may also influence consumption. Two studies have explored the impact of glass shape on the total time spent drinking. One study found slower consumption from straight-sided glasses for beer served in beer glasses, though no evidence that soft drink consumption differed16. These authors argued that the effects may be stronger for alcohol vs. soft drinks generally. The second study did find slower consumption from straight-sided glasses for a soft drink served in tumblers17. Further research is required to determine whether soft drink consumption differs, whether straight-sided glasses also reduce the amount consumed , and what the underlying mechanisms might be.There is a growing evidence base for the effect of glass size and shape on drinking behaviours. Wine glass size has increased over the past 300 years—in particular in the last 30 years18. Gaining the much needed insight into the mechanisms of drinking behaviour will foster the development of more targeted, effective, and possibly less expensive approaches to reducing intake of unhealthy drinks (including soft drinks). Several mechanisms might contribute to the effects of glassware design on consumption. One concerns biases in visual perception. Specifically, when estimating the volume remaining in a drink, people may use height as a cue to volume19. For example, the true midpoint of drinks presented in outward-sloped glasses is underestimated, as compared to straight-sided ones20. This midpoint bias reflects an inability to make accurate visual judgments about the volume remaining in the glass, which might in turn influence drinking speed and amount consumed.Characterising ‘mechanisms of action’ is a key goal of behaviour change intervention research21. Decelerated drinking is characterised by a greater amount being consumed in the first half of the drinking episode. Characterising these trajectories may help understand the mechanisms that drive the effects of glass shape on consumption.Another possible mechanism concerns the physical characteristics of the container such that glasses of certain shapes cue or ‘afford’ larger or smaller sip sizes and/or other micro-drinking behaviours affecting consumption. For example, there is some evidence that glasses of different shapes lead to different drinking trajectories, with more decelerated drinking found from short, wide glasses than from tall, narrow ones22. Activity in these muscles has been found to distinguish subtle differences in embouchures of musicians playing brass and wind instruments25. To our knowledge, only one study has investigated the impact of the receptacle from which a drink is consumed on activity in the orbicularis oris muscle26. These researchers measured lip muscle activity using facial electromyography and found higher levels of muscle activity—indicative of a more pursed embouchure—when sips were taken through a straw, as compared to those taken from a cup or a spoon. However, there is an absence of evidence characterising the embouchures associated with drinking from differently-shaped glasses and the relationship between embouchure and sip size.One way that affordance of glass shape on drinking may operate is via the position of the lips—‘embouchures’—and in particular, the extent to which they are pursed during sipping. The orbicularis oris muscle is responsible for compressing the lips and protruding them forward into a puckerThe present set of studies aimed to estimate the effects of glass shape—straight-sided vs. outward-sloped—on several drinking behaviours during consumption of soft drinks, measured in laboratory studies using distinct paradigms to measure drinking, including direct observation and physiological measures.17, and drinking trajectory21. Study 2 extended Study 1 by exploring the effect of glass shape on the volume consumed, using more extreme differences in glass shape—comprising straight-sided wine flutes vs. outward-sloped martini coupes . The bogus taste test has been validated and correlates with other measures of intake for alcohol29 and food30. Study 3 tested a novel potential mechanism for decreased consumption from straight-sided glasses, namely, the extent the lips are pursed during sipping—lip embouchure. We used facial electromyography (EMG) to detect the muscle activity of the upper and lower lips during sipping of soft drinks from straight-sided wine flutes and outward-sloped martini coupes . Analyses were conducted in RStudio. For a summary of key outcome measures split by condition, see Table All studies  were pre-registered on the Open Science Framework, where data and code is also available —predominantly students and staff at the University of Cambridge, England—to take part in the study between May and July 2018. For more information on sample size determination and eligibility, see SI Appendix. Due to a video-recording malfunction, data from two participants  were excluded from the analysis, leaving a total sample of N = 198. Baseline demographic and study-relevant characteristics are given in SI Appendix Table 17), and the primary outcome was total drinking time. Video recordings were taken during the experiment and subsequently coded for all drinking behaviours—total drinking time, drinking trajectory i.e., drinking pattern over time, mean sip size, mean interval duration, and mean sip duration. After finishing the drink, participants completed a midpoint estimation task by physically filling or emptying drinks from the same shape glass they had previously drunk from until the glass was perceived to be half-full . Midpoint bias reflected estimations which deviated from the drink’s true halfway point .In a between-subjects design, participants were randomised (stratified by gender) to receive a soft drink in one of two glasses (straight-sided or outward-sloped tumbler). Participants consumed the drink at their own pace while watching a nature documentary .Visual inspection of distributions indicated a positive skew for total drinking time. This was transformed using a log10 function to satisfy regression modelling assumptions. Back-transformed geometric means (geomeans) with 95% CIs are thus reported. Adjusting for pre-specified covariates—gender, thirst, and BMI—there was no evidence that total drinking time differed between glass shapes , the volumes remaining in the glass were not recorded after every sip taken. This was due to not placing the glass on the table in-between sips, and/or holding the glass and occluding the liquid. These factors prevented the accurate measurement of heights of the liquid and glass to determine volume remaining after each sip. To deal with missing data, we excluded participants who had incomplete drinking trajectory data—leaving Subset A: participants for whom volume remaining was recorded after every sip (n = 16), and Subset B: participants for whom volume remaining was recorded after at least 50% of sips (n = 94). As a result, one limitation of the trajectory data is that the tendency of participants in these subsets to put down their glass may represent a particular style of drinking behaviour. For participant characteristics and outcome measures split by subset, see SI Appendix Table S3.21), for both outward and straight conditions, and thus was the best fit of the drinking trajectory data. Using Subset A, the cubic model predicted that at 50% time, 66.0% had been consumed from outward glasses , while 50.3% had been consumed from straight glasses , see Fig. For both subsets, a cubic (S-shaped) model had the lowest AIC, as compared to quadratic models (used to represent the drinking trajectory data in a previous paper—seet(8.264) = 3.60, p = 0.0067) and 6.3% larger for the outward-sloped glasses for Subset B  = 2.32, p = 0.023), see SI Appendix Table S4 for average AUCs, by condition, for both subsets.The area under the drinking curves (AUC)—a proxy for drinking trajectory—was also calculated. Larger AUCs  are indicative of a more decelerated pattern . AUCs were 24.6% larger for the outward-sloped glasses for Subset A , sips were 11.1% larger from the outward-sloped glass than the straight glass, however the data were also consistent with smaller sips , p = 0.123). Larger sips were associated with faster total drinking times, r(198) = -0.50, p < 0.0001). Gender did not significantly predict mean sip duration or mean interval duration, so was not included in these models. Sip durations did not differ between glass shapes . Interval durations did not differ between glass shapes , p = 0.312).Adjusting for the effects of gender on log mean sip size (Bt(196) = 6.1, p < 0.0001). Midpoint bias was not associated with total drinking time, r(198) = -0.09, p = 0.196).Midpoints were underestimated from both glasses, consistent with under-filling the glass when estimating the halfway point. Individuals under-estimated the midpoints of outward-sloped glasses to a greater degree , 17), though there was some evidence that drinking trajectories differed, with more decelerated drinking from outward-sloped glasses—characterised by a greater amount consumed in the first half of the drinking episode—and more linear drinking from straight-sided glasses. While this indicates that straight-sided glasses may lead to a different pace of drinking, it remains to be seen whether glass shape influences the amount that is consumed. As in previous research, there was evidence of midpoint bias, with midpoints underestimated more from outward-sloped glasses20. However, there was no evidence that this bias in perception of drink midpoints was associated with drinking time.There was no evidence that total drinking time differed depending on the glass being drunk from  participants—predominantly students and staff at the University of Cambridge, England—to take part in this study between February and March 2019. This study used an adaptive design with an internal pilot29). Secondary outcome measures were number of sips (determined from video recordings which were subsequently coded for sips), and midpoint bias , receiving four soft drinks to taste and rate each 165 ml), served in either straight-sided wine flutes or outward-sloped martini coupes .Visual inspection indicated no evidence of skew in the primary outcome measure: volume consumed (ml). Adjusting for pre-specified covariates , our model indicated that 72.1 ml less was consumed from straight-sided glasses than outward-sloped glasses , p = 0.58. Number of sips was positively associated with total amount consumed, with fewer sips associated with less being consumed overall, r(71) = 0.48, p < 0.0001. Mean sip size—though not pre-specified—was also explored, to account for differences in amount consumed. This was calculated by dividing total volume consumed by number of sips. Mean sip sizes were 2.7 ml smaller from straight-sided glasses than from outward-sloped glasses , p = 0.017. Mean sip size was also strongly and positively associated with total amount consumed, with smaller mean sips associated with less consumed overall, r(71) = 0.67, p < 0.0001.There was no evidence that total number of sips differed between glass shapes , t(68) = − 0.63, p = 0.531). Midpoint bias was not associated with amount consumed, r(72) = − 0.03, p = 0.827.Drink midpoints were underestimated for both glasses. Though outward-sloped glasses led to lower midpoint estimates than straight-sided glasses, there was no evidence that the difference in midpoint bias was meaningful , Consistent with our predictions, Study 2 demonstrated that, in a bogus taste test paradigm, less was consumed from straight-sided glasses than outward-sloped ones, this time using stemmed glasses rather than tumblers, and using a non-carbonated soft drink. Lower number of sips and smaller average sips size were associated with less consumed overall. Midpoint bias was not associated with amount consumed.In Study We recruited 40 females—predominantly students and staff at Macquarie University—to take part in an experiment on campus at Macquarie University, Australia, between June and July 2019. For more information on sample size determination, see SI Appendix. For demographic and study-relevant information see SI Appendix Table S7.In a within-subjects design, participants were served four soft drinks (165 ml each), in two straight-sided glasses (wine flutes) (A), and two outward-sloped glasses (martini coupes) (B), in the sequence ABAB or BABA, with order randomised. Three sips were taken from each drink. Sip volumes were recorded after each sip, using a concealed weighing scale. The study used an adapted bogus taste test with the addition of surface facial electrodes placed on the upper and lower lips, which measured muscle activity, as a proxy for embouchure. The primary outcome measure was lip muscle activity (co-primary endpoints of upper and lower lip muscle activity) which was transformed and expressed as a percentage of each individual’s maximal voluntary contraction (%MVC). Maximal voluntary contraction was measured by asking participants to protrude their lips as firmly as possible. The secondary outcome measure was sip size (ml).Visual inspection of distributions indicated positive skew for embouchure  and sip size (ml). All three variables were transformed using a log function, improving the shapes of the distributions. Back-transformed geomeans with 95% CIs are reported in Table p = 0.0017, and 20.03% more lower lip muscle , p < 0.0001, than when sipping from outward-sloped glasses. Findings were significant after adjusting for co-primary endpoints . See SI Appendix Table S8 for full models.Two linear mixed effects models were used, predicting the co-primary endpoints of i) upper and ii) lower lip muscle activity (log %MVC) from glass shape, adjusting for standard crossover design variables  as well as sip number  and with participant as a random effect. These models indicated that, when sipping from straight-sided glasses, participants used 8.9% more upper lip muscle activity (95% CI 3.3–14.8%), p < 0.0001) and lower %MVC , respectively. However, there was no evidence that muscle activity predicted sip size in these models: for every 1% increase in upper lip muscle activity used, there was a 0.19% decrease in sip size , p = 0.337, and for every 1% increase in lower lip muscle activity used, there was a 0.36% decrease in sip size , p = 0.251. See Table S9 for full models.Two linear mixed effects models were used, predicting log sip size from upper and lower muscle activity (%MVC), adjusting for the same variables as above, as well as baseline thirst, and maximum oral capacity, and with participant as a random effect. These models indicated that glass shape predicted sip size, with sips that were 17.3% smaller and 16.6% smaller from straight-sided glasses than outward-sloped glasses, when adjusting for upper %MVC  predicted sip size; for every 1% increase in upper lip activity used, there was a 0.27% decrease in sip size , p = 0.191.In exploratory analyses, we removed glass shape from these models to determine whether lip muscle activity predicted sip size for upper and lower %MVC respectively, without adjusting for the effects of glass shape on sip size. In this analysis, lower lip muscle activity (%MVC lower) did predict sip size, such that for every 1% increase in lower lip muscle activity used, there was a 1.13% decrease in sip size , In line with predictions, we found that lips were more pursed when participants sipped from straight-sided glasses, as compared to outward-sloped glasses. Correspondingly, sips were smaller from straight-sided glasses, and there was some evidence of an association between lip muscle activity and sip size .. outward-sloped—on the consumption of soft drinks, using diverse techniques to measure drinking behaviours. In Study 1, though the overall time spent drinking was equivalent for those drinking from straight-sided and outward-sloped glasses (failing to replicate a previous study17), when exploring the micro-drinking behaviours in detail, we found that drinking trajectories differed. In particular, drinking from outward-sloped glasses was characterised by a more decelerated pace, a similar finding to previous research21, which found more decelerated drinking from short-wide glasses than tall-narrow ones. In Study 2, using a bogus taste test paradigm, those tasting and rating drinks served in straight-sided glasses consumed less overall than those drinking from outward-sloped ones. Average sip sizes were also smaller from straight-sided glasses, and there was an association between sip size and total amount consumed. In Study 3, using an adapted bogus taste test paradigm with sips taken in regimented stages to allow for the measurement of lip muscle activity, lips appear to be more pursed when drinking from straight-sided flutes. The same study showed a corresponding effect of glass shape on sip size—with smaller sips taken from straight-sided flutes—and preliminary evidence that embouchure may be associated with sip size—for lower lip embouchure, at least. To our knowledge, this final study is the first to use facial EMG to explore lip embouchures during sipping, a potential mechanism for reduced consumption from straight-sided glasses.The studies reported here investigated the effects of varying glass shape—straight-sided vs20, drink midpoints were underestimated more from outward-sloped than straight-sided tumblers in Study 1, indicative of midpoint bias from outward-sloped glasses. However, there was no evidence of a difference in midpoint bias in Study 2 using flutes and coupes . Importantly, however, there was no evidence that midpoint bias was associated with drinking behaviour, including drinking time (in Study 1) or amount consumed (in Study 2). This suggests that—while sometimes present—biases in visual perceptions of drink volumes may not consistently influence drinking behaviour, and that other factors may better explain the effects of glass shape on drinking time and consumption.The three studies thus report broadly consistent findings that together suggest that glass shape influences drinking behaviours, including volume consumed. As discussed, one potential mechanism that may underlie some of the differences in drinking behaviour is the extent of lip pursing—or embouchure—during sipping. Affordance of micro-drinking behaviours—such as sip size via embouchures—seems a good candidate mechanism for explaining, at least in part, the reduction of intake from straight-sided glasses. An additional mechanism that we explored involved biases in visual perception of drink volumes. As in previous studies, both within- and between-subjects. Regardless of the exact glassware used—i.e., tumblers in Study 1 and stemmed glasses in Study 2 and 3—or type of drink served—i.e., carbonated in Study1, non-carbonated in Studies 2 and 3—the findings suggest that serving drinks in straight-sided glasses is a potentially effective way to reduce consumption of drinks which harm health.The strength of this research lies in its novelty and scientific rigour: it is the first comprehensive set of studies focused on the impact of glass shape on drinking behaviour, measured using different study paradigms. The key findings are largely consistent across study paradigms testing different primary outcomes, including a novel physiological measure, and across different study designs—i.e.29, they do not offer insight into how drinking might be impacted over a longer drinking session when multiple drinks might be fully consumed . Finally, though Study 1 utilised a more granular approach to measure drinking behaviours by measuring cumulative intake over time, based on detecting volume consumed from images (a similar method to21), one limitation relates to attrition of data for this analysis. Given the nature of the task, there was often occlusion of liquid in the images of participants drinking, sometimes due to participants holding the glass at an angle, or covering the liquid with their hands. This occlusion in the images prevented the accurate measurement of the liquid:glass heights and thus the calculation of volumes. As such, there were only sixteen participants for whom all sips were recorded as volumes (Subset A). Excluding participants for whom < 50% of sips were recorded as volumes (Subset B) showed similar patterns of findings, though the effects were smaller.The studies also had a number of limitations worth noting. First, it remains unclear which exact features gave rise to the effects—outward-sloped glasses having wider rim apertures than narrower straight-sided ones. The latter two studies used more ‘extreme’ versions of the Study 1 tumblers, with rim diameters which varied more drastically. Rim diameter may have contributed to the observed effects on drinking behaviours, in addition to, or independently from, the effect of glass wall slope. Second, the study procedures may have failed to reflect real life drinking scenarios, limiting the ecological validity of the findings. Laboratory settings often lack certain characteristics that mark ‘real-life’ drinking environments—for example, the absence of drinking companions and the artificial nature of the tasks. There is also a need to examine drinking behaviours when people consume multiple drinks, to ascertain whether behaviour change persists after consuming a single drink. Study 2 went some way to address this, using a bogus taste test paradigm which involved tasting multiple drinks. However, while bogus taste tests are sensitive to desire to consume, comprising a valid measure of ad libitum consumption34. Relatedly, while developing novel interventions to reduce consumption of health-harming drinks remains important, it is worth investigating whether the converse is true—that is, whether outward-sloped glasses may help to increase consumption of healthy drinks, relative to straight-sided glasses. Thus, future studies could explore the impact of glass shape on water consumption in medical settings where hydration is warranted. Second, research is required to ascertain the parameters of these effects and in particular, whether the effects apply to alcoholic drinks as well as to soft drinks. This is worth exploring given that a previous study found evidence for slower drinking from straight-sided glasses than outward-sloped glasses, but only for beer, and not for a soft drink16. It is therefore possible that the effects found in the present studies could be stronger for alcohol. Third, though this set of studies has explored two potential mechanisms for the effects of glass shape on consumption—via biases in visual perceptions of glass midpoints and via affordance of embouchures and their potential impact on micro-drinking behaviours—future studies might usefully identify additional mechanisms. Understanding the processes through which glass shape influences consumption will advance our overall understanding of the effects and in turn lead to optimising glassware design for more effective, more cost-effective, and more targeted interventions.Future studies might build on this set of findings in a number of ways. First, as discussed, studies are required in real-world drinking environments, which will also enable effect size estimates. This might include studies set in pubs, bars and restaurants, comparing the volume purchased and consumed according to the glasses drinks are served in, as has been investigated for wine glass size;10 for reducing consumption of sugar-sweetened beverages. In combination with other behaviour change strategies, adopting straight-sided glasses may prove to be one intervention contributing to the many needed to reduce consumption of drinks that harm health.This set of studies contributes to a small but growing evidence base of a potentially effective, easily implemented, small-scale physical environment interventionhttps://osf.io/4tx3c/; https://osf.io/j9hqu/; https://osf.io/75b89/). Written informed consent was obtained from the participant for publication of their image , and the Macquarie University Research Ethics Committee (Study 3: 5201954159069). All studies were conducted in accordance with the relevant institutional guidelines and regulations. More information (on eligibility and recruitment for each study), is given in SI Appendix. Pre-registered study protocols are available on the OSF , mean sip duration, mean interval duration, and total number of sips. To determine mean sip size, we divided the total amount consumed—330 ml—by the total number of sips.p < 2.5%).To determine drinking trajectory, we first created models predicting volume from the height of known increments of liquid relative to the height of the glass, for each of the glass shapes. These models allowed us to estimate the volume remaining in the glass when it was placed on the table in the study videos . We then used these estimates to plot each individual’s cumulative intake (%) over time (%). To determine drinking trajectory, we combined the plots and fitted models by condition. Using these models, we calculated amount consumed at 50% time. We also calculated the areas under each individual’s drinking curve, as a proxy for trajectory (higher scores are indicative of more decelerated drinking). Due to the need for glasses to be placed on the table with minimal occlusion to the liquid, trajectories were only able to be calculated for subsets of participants—those who regularly placed their glass down (see SI Appendix Table S3 for characteristics and outcomes of these subsets). Given that analyses were conducted on two subsets of data , we applied the conservative Bonferroni adjustment to the threshold of significance (i.e. 20). The six poured estimates of the midpoint of the drink were averaged to provide a single estimate for each participant. This was then subtracted from 165 ml (the true midpoint), to determine midpoint bias. Negative values reflect underestimation of the true midpoint (pouring too little liquid into the glass), while positive values indicate overestimation of the true midpoint (pouring too much liquid into the glass).Midpoint bias was assessed using a task involving trials of filling or emptying drinks from the allocated glass until the glass was perceived to be half-full .A 330 ml can of chilled Appletiser was served in the appropriate glass (based on randomisation). Participants were asked to consume the drink at their own pace, whilst watching a documentary. Video recordings were taken during this time.Participants then completed two filler tasks (word search and drink ratings). Finally, they estimated the midpoint of their drink. A full portion was placed in front of participants (in the glass they drank from previously), and they were asked to pour estimates of the halfway point of the drink . There were six estimates in total . Participants were invited to use a ‘Reference Glass’ throughout the task to aid accuracy, which contained the full 330 ml portion.Finally, participants were asked what they thought the purpose of the study was, to examine the effectiveness of the cover story in blinding participants to the true nature of the study. Participants were paid £7 for their time and expenses, and told they would receive a full study debrief via email once all participants had taken part.Total amount consumed (ml) —the primary outcome measure—was measured by weighing the four glasses before and after the 10-min taste test, using high precision scales. The weights of the glasses after consumption were subtracted from the initial weights to determine the total volume consumed.Micro-drinking behaviours  were measured by coding the video recordings taken during the drinking sessions . Midpoint bias was measured in the same way as Study 1 (with the true midpoint of these drinks being 82.5 ml).Maximum oral capacity was measured—to adjust for in the primary analysis—and calculated by asking participants to fill their mouth to full capacity from a cup filled with room temperature water, before spitting into an empty jug . This task completed twice , and was disguised as a ‘palate cleanser’. Drink enjoyment was also measured—to adjust for in the primary analysis—from ratings given during the taste test, with scores for ‘tasty’ and ‘pleasant’ (rated from 1–10) averaged for all four drinks.Eligible participants attended a single session to take part in a study on ‘taste preferences’. On arrival, participants completed eligibility screening, and demographic questions, including age, gender, height, weight, and thirst (1–10). Next, participants completed the ‘palate cleanser’—twice filling their mouth with water and spitting into a jug to determine oral capacity.27), for 10 min. As well as completing these ratings, participants were be asked to indicate their preferences from 1(favourite)—4(least favourite), to reinforce the cover story. They were told to drink as much or as little of the drinks as they would like, to assist their ratings.For the taste test, the experimenter prepared four drinks—a total of 660 ml (divided into four 165 ml portions) of Teisseire Passion Fruit Le Sirop , served in either straight-sided stemmed wine flutes, or outward-sloped stemmed martini coupes, depending on randomisation. The drinks were placed on mats labelled A, B, C, and D. Participants were told to taste and rate the drinks according to 10 descriptors . The procedure was the same as for Study 1. Finally, the participant was asked to guess the purpose of the study, using an open-ended question presented on screen. As in Study 1, participants were paid £7 for their time and expenses, and received a full study debrief via email once all participants had taken part. After the participant had left, the experimenter weighed the glasses to determine volume consumed.AcqKnowledge 5.0 software . Raw amplitudes of activity were transformed using a script which generates a rectified and integrated copy of the data over a period of 250 ms and then rescales the channel so that this runs from 0–100%, with an individual’s maximum voluntary contraction representing 100% . The signal with the highest amplitude (from the three maximum lip compression attempts) was selected to be used for standardizing by each participant’s maximum voluntary contraction.The primary outcome measure in this study was embouchure (%MVC). Embouchure was measured using surface electrodes which detected the amplitude of activity in the upper and lower lips. Higher amplitudes of lip muscle activity are a proxy for a more ‘pursed’ embouchure. A two-channel Biopac MP160 system was used for continuous electromyographic (EMG) signal acquisition, and data inputs were recorded and processed using The secondary outcome measure was sip size (ml). Individual sip sizes (ml) were measured in real time, for each of the three sips taken from each of the four glasses. Sip sizes were noted by the experimenter after each sip, when the glass was placed on concealed weighing scales. The glass was weighed before consumption  and subsequently after each sip was taken during the taste test.Maximum oral capacity was also measured—to adjust for in the primary analysis—in the same way as Study 2 .Participants were invited to attend a single study session investigating ‘reactions to drinks served in different containers’. On arrival, participants completed eligibility screening, followed by the ‘palate cleanser’ task. The participants answered baseline demographic questions, and rated their thirst (1–10).35 for recommended placement of electrodes for facial EMG). The ground electrode was placed on the temple. Trailing wires were clipped behind the ear. An Impedance checker was used to assess signal conductivity. If impedance was greater than 30 kO the corresponding electrodes would be re-affixed.To prepare the skin for electrode placement, it was wiped with make-up remover, and brushed with an exfoliative pad and a small amount of abrasive gel (NuPrep). The four surface electrodes were then filled with electrode gel (SignaGel), and affixed to the upper and lower lips on the left side see Fig. . using aParticipants were served a total of 660 ml (divided into four 165 ml portions) of Teisseire Passion Fruit Le Sirop , in two straight-sided stemmed wine flutes, and two outward-sloped stemmed martini coupes, in the order assigned to them.26). The four stages were indicated on the screen, as follows.Participants were instructed to take three sips taken from each drink. Each sip was divided into four stages (adapted from a previous studyStep 1— “Relax”: “Please relax, keeping your lips at rest”.Step 2— “Prepare”: “Please lift the glass and hold it in front of your mouth”.Step 3— “Remove”: “Now raise the glass to your lips and take a sip”.Step 4— “Swallow”: “Swallow the sip in one swallow”.Participants first practised these stages with a glass of water. They would next begin the taste test, taking three sips from each glass. After taking three sips from the drink, the participant rated the drink on the same 10 descriptors as Study 2. They were also asked to rate, from 1–10, how much they enjoyed the experience of consuming their drink from that glass. All of these questions were filler questions, to add to the cover story and disguise the true aim of the study. Once all four drinks had been tasted and rated, the participant was asked to complete the maximal lip compression tasks. They were asked to “squeeze your lips together as hard as you can, so they protrude as far as possible”. They did this 3 times, as prompted by on-screen instructions. After electrodes were removed, participants were asked to describe what they believed to be the purpose of the study. Participants were paid $10 for their time and expenses, and told they would receive a full study debrief via email once all participants had taken part.Supplementary file1.