The increased use of a new rice cultivar is the result of increasing consumer demands for healthier choices. In this study, physicochemical, thermal, pasting, and functional properties of flour from RD43 rice, a new rice variety, and its food application were investigated. RD43 rice flour demonstrated an irregular and polyhedral shape with a volume mean diameter of 103 ± 0.15 µm. In addition, the amylose content of RD43 rice and Hom Mali rice flour was 19.04% and 16.38%, respectively. The X-ray diffraction (XRD) and Fourier Transforms Infrared (FTIR) confirmed the presence of a V-type crystalline structure and less crystallinity in RD43 rice flour, which resulted in a significant reduction of the water absorption index (WAI), swelling power (SP), water solubility index (WSI), gelatinization temperature, and pasting properties. Comparing with Hom Mali rice flour, RD43 rice flour had greater ability to disrupt cholesterol micellization and bind bile acid. Furthermore, it had lower starch digestibility, with a lower percentage of rapidly digestible starch (RDS) and higher percentage of undigestible starch than Hom Mali rice flour. Moreover, steamed muffins based on RD43 rice flour had lower starch digestibility than Hom Mali steamed muffins. The sensory analysis showed no significant differences between Hom Mali and RD43 steamed muffins. The findings suggest that RD43 rice flour could be an alternative ingredient for lowering the glycemic index of food products. Oryza sativa, L.), one of the most important crops in the world, is a principal staple food in many regions of the world’s human population, especially in Asia [Rice  and Hom Mali 105 rice (Oryza sativa L.) were obtained from the Rice Department, Bangkok, Thailand. Pepsin from porcine gastric mucosa powder (250 U/mg), α-amylase Type VI-B from porcine pancreas (15.8 U/mg), pancreatin from porcine pancreas (4×U.S. Pharmacopeia (USP) specifications), pure amylose from potatoes, phosphatidylcholine, oleic acid, taurocholic acid, glycodeoxycholic acid, taurodeoxycholic acid, taurocholic acid, and porcine cholesterol esterase (35 U/mg) were purchased from Sigma-Aldrich, Inc. . Amyloglucosidase  was purchased from Megazyme International Ireland Ltd. . Glucose oxidase kit (Glucose liquicolor) and Cholesterol test kits (Cholesterol liquicolor) were purchased from Human . The total bile acid kit was purchased from BIOBASE .RD43 rice  with a voltage of 50 Hz and a speed of 4800 rpm. Then the ground rice was passed through 150 µm (mesh) for removing large particles. The flour was stored in sealed plastic bags at room temperature until further analysis. To determine moisture content, each flour (5 g) was heated at 105 °C for 10 min. Moisture content was measured using the infrared moisture determination balances . The moisture content of the RD43 and Hom Mali rice flours were 11.5% and 11.0%, respectively. Total dietary fiber was carried out using the Association of Official Analytical Chemists (AOAC) method . The totThe granular morphology of rice flour was observed using a scanning electron microscope (SEM) . The sample was mounted on an aluminum specimen holder using double-sided scotch tape and coated with gold at an accelerating voltage of 10 kV. The microstructure of the flour particles was scanned and photographed at various magnifications (100× and 1500×).4,3D), average particle size (50D), and equivalent diameters at 10% and 90% cumulative volume . Three replicates were performed for each analysis.The particle size distributions (PSDs) of flour were analyzed using a laser particle size analyzer  with dry dispersion. The PSD was calculated using the Mastersizer 3000 software and reported as volume mean diameter . Before measurements, a standard black glass and white tile were used for instrument  calibration. The The amylose content in rice flour was determined according to the previous study . In brieX-ray diffraction patterns of rice flour were performed with an X-Ray Diffractometer  equipped with a copper tube operating at 40 kV and 45 mA, and the spectra scanned over a diffraction angle (2θ) range of 5–40° at a rate of 0.02° 2θ/second. The percentage of crystallinity was calculated using this equation :% RelatiThe FTIR of the rice flour was determined according to a previous study . The ricWi) (0.05 g) was added into 1 mL of DW, then the mixture was heated at 90 °C for 10 min in the heat block. After that, the sample was cooled in an ice water bath for 10 min and centrifuged at 3000× g and 4 °C for 10 min. The supernatant was separated and evaporated at 105 °C until it reached constant weight (Ws). The residue (Wr) was weighed. The results were calculated according the equation below:The hydration properties of rice flour was determined following the previous method . The iniTo), peak temperature (Tp), conclusion temperature (Tc), and gelatinization enthalpy (ΔH), were recorded.The thermal properties of rice flour were determined using differential scanning calorimeter (DSC) . An empty aluminum pan was used as the reference. The rice flour sample  was mixed with 10 μL of deionized distilled water and put into the sample pan. Then, the pan was sealed and allowed to stand at room temperature for 1 h before heating from 25 °C to 100 °C, with a heating rate of 10 °C/min. The DSC parameters, including onset temperature . In brief, the sample (3 g) was suspended in 25 g DW. Then, the suspension was continuously heated from room temperature to 95 °C at a rate of 12 °C/min. After that, the sample was held at 95 °C for 2–3 min, and finally cooled to 50 °C at a rate of 12 °C/min. Pasting temperature, peak time, peak viscosity, trough, breakdown, final viscosity, and setback were obtained from the pasting curves.Starch digestibility of rice flour were performed according to a previous method, with minor modifications . In brieThe bile acid binding assay was determined according to a previous method . The bilThe inhibition of cholesterol micellization formation was performed according to a previously established method . The mixRice flour (100 g) was mixed with refined sugar (70 g), yeast (1 g), and water (160 mL) for 5 min and left at room temperature for 90 min. Finally, baking powder (3 g) was added into the mixture for 2 min. Thereafter, the muffin batter was poured into molds, which were then steamed over boiling water for 15 min. Then, the muffins were cooled at the room temperature and removed from the molds.The sensory evaluation of the steamed muffins was performed using untrained panelists to evaluate the acceptability for the two steamed muffins . The 30 t-test. The significance of difference was considered at p < 0.01. The analysis was performed by using an SPSS package .The results are reported as mean ± standard error of the mean. The mean comparison was carried out by independent The photographs and scanning electron micrographs of RD43 and Hom Mali rice flour are demonstrated in In addition, the photographs presented the aggregation of starch granules into particles with a smooth cutting surface. The obtained results of this study are consistent with the previous study following the same method of flour preparation . This miThe curve of particle size distribution of rice flour is presented in 4,3D, 10D, and 50D of RD43 rice flour was significantly lower than that of Hom Mali rice flour, suggesting that RD43 rice flour had smaller average particle size. The finer particle size of flour resulted from less grain strength [As shown in strength . The preL*) and higher redness (a*) and yellowness (b*) when compared to Hom Mali rice flour. The rice flour with a greater degree of yellowness and less intensity in lightness might result from different rice cultivars, storage time, and the exposure to high temperatures during the milling process [As shown in  process ,23. In tAs demonstrated in As presented in According to the calculation of the X-ray diffraction pattern, the degree of crystallinity in RD43 rice flour (24.55%) was significantly lower than observed in Hom Mali rice flour (26.8%). This result suggests that the lower degree of relative crystallinity in RD43 rice flour results from the higher presence of amorphous area .−1 represent the sensitive change in the starch structure. The observed peaks at 1081 and 1022 cm−1 indicate stretching of the anhydroglucose C–O ring, which related to polysaccharide molecules [−1 was assigned to polysaccharides with α-1,4 glycosidic linkage [−1 are the crystalline region of starch, whereas a band at 1022 cm−1 is characterized as an amorphous region of starch [-1 could refer the association between amount crystallinity and amorphous structure [−1, indicating the lower degree of double helices and ordered structure [−1 especially is interpreted as a high amount of amylose with a low amylopectin content [The FTIR spectra of Hom Mali rice and RD43 rice flour are illustrated in olecules . In addi linkage . Severalf starch ,31. In ptructure . Comparitructure . The low content . This reThe WSI represents the ability of components to dissolve with water under an excess water condition. As demonstrated in pT) and conclusion temperature (cT) than Hom Mali rice flour. There was no significant difference in onset gelatinization temperature (oT) and gelatinization enthalpy (ΔH) between RD43 rice flour and Hom Mali rice flour. The thermal properties are normally affected by the chemical composition, the origin of starch, moisture, the presence of other biomaterials, and processing and pretreatment conditions [pT, cT, and crystallinity in rice flour [The differential scanning calorimetry (DSC) curves and values are shown in nditions . Previouce flour . The grece flour . TherefoThe pasting curves of RD43 rice and Hom Mali rice flour are presented in The distinct pasting behavior of rice flour is influenced by cultivars, resulting in variation of the undigestible starch and amylose content ,38. HighThe starch digestibility of RD43 and Hom Mali rice flour is demonstrated in The percentage of bile acid binding and the inhibition of cholesterol micellization of RD43 rice and Hom Mali rice flour are demonstrated in The steamed muffins of Hom Mali and RD43 rice flour are presented in RD43 rice flour presented a higher amylose content with a smaller particle size than Hom Mali rice flour. The results from X-ray diffraction indicated the presence of amylose–lipid complexes in RD43 rice flour. This structure influences hydration, thermal properties, and pasting properties of RD43 rice flour. Furthermore, RD43 rice flour had a greater ability to decrease starch digestibility, bind bile acid, and disrupt the formation of cholesterol micellization than Hom Mali rice flour. Steamed muffin made from RD43 flour had lower starch digestibility without affecting the sensory acceptability. Based on our findings, RD43 rice flour may be used as an alternative ingredient for lowering the glycemic index of food products.


The second domain which comprises the ribosomal binding site in a putative regulatory helix, however, has withdrawn from detection of ligand-induced structural modulation thus far, and therefore, the identity of the cognate ligand and the regulation mechanism have remained unclear. Here, we report crystal structures of both riboswitch domains, each bound to NAD+. Furthermore, we demonstrate that ligand binding to domain 2 requires significantly higher concentrations of NAD+  compared to domain 1. Using a fluorescence spectroscopic approach, we further shed light on the structural features which are responsible for the different ligand affinities, and describe the Mg2+-dependent, distinct folding and pre-organization of their binding pockets. Finally, we speculate about possible scenarios for nadA RNA gene regulation as a putative two-concentration sensor module for a time-controlled signal that is primed and stalled by the gene regulation machinery at low ligand concentrations (domain 1), and finally triggers repression of translation as soon as high ligand concentrations are reached in the cell (domain 2).Riboswitches are important gene regulatory elements frequently encountered in bacterial mRNAs. The recently discovered  Riboswitches are gene regulatory elements that are frequently found in the 5′-untranslated regions of bacterial mRNAs . They arS-adenosylmethionine or tetrahydrofolate. Only very recently, the nadA RNA motif has been assigned to function as a riboswitch which regulates the expression of genes involved in biosynthesis of the enzyme cofactor nicotinamide adenine dinucleotide (NAD+)  . AlthougμM range . The binμM range , consist analogs . Interes analogs , and the+ riboswitch, both individually bound to NAD+. We furthermore demonstrate that ligand binding to the second domain requires significantly higher concentrations of the ligand. We also shed light on the structural matters that are responsible for the different ligand affinities of the two domains as well as determine the Mg2+-dependent, distinct folding and pre-organization of their binding pockets. Finally, we reflect on possible scenarios for the gene regulation mechanism of this intriguing riboswitch.Here, we report crystal structures of the first, as well as the second domain of the NADnadA RNA constructs, GNRA tetra-loop motifs and/or the U1A-protein recognition loop were introduced as apical variable loop of stem P1b of domain 1 and domain 2, respectively . The product RNA was visualized using ultraviolet light at a wavelength of 254 nm, excised and soaked in 0.5 × Tris-acetate-EDTA (TAE) buffer at 4°C. The leach solution was precipitated with iso-propanol and washed by 80% ethanol. The lyophilized RNA was dissolved in diethyl pyrocarbonate treated, double-distilled water for further experiments.To facilitate crystallization of ectively . The seqcription . In vitr10-SUMO-tag and an ubiquitin-like protease (ULP1) cleavage site was expressed in E. coli (BL21-Codon Plus®) at 18°C overnight. The harvested cell pellets were suspended in buffer A  and lysed with a French press. After high-speed centrifugation, the supernatant was loaded on a Ni2+ column. The His10-SUMO-tag recombinant U1A protein was eluted using buffer A supplemented with 500 mM imidazole and then cleaved with ULP1, which was followed by a second Ni2+ column to remove the cleaved His10-SUMO-tag. The U1A protein was further purified with ion-exchange  and gel-filtration  chromatography methods, and finally concentrated to 20 mg/ml for further experiments. The selenomethionine (Se-Met) derivative of the U1A protein was expressed in cells grown in M9 medium supplemented with Se-Met and other amino acids. The purification process of Se-Met U1A was the same as for the native protein.The U1A protein  with an N-terminal His+, NADH, adenosine 5'-diphosphate ADP and adenosine 5'-triphosphate (ATP) were purchased from Yuanye Bio-Technology Co., Ltd (Shanghai). Nicotine mononucleotide (NMN) was purchased from Bide Pharmatech Ltd (Shanghai).The ligands NAD+ riboswitch RNA in the buffer containing 50 mM HEPES, pH 7.0, 50 mM NaCl, 5 mM MgCl2 was annealed at 65°C for 5 min and then incubated on ice for half an hour. NAD+ was added to the RNA with a ratio of RNA to NAD+ 1:15 and kept on ice for one more hour. For the constructs containing the U1A-recognition loop, U1A protein was added with the ratio of RNA to U1A protein 1:1.2. Crystallization experiments were carried out by mixing 0.16 µl of the RNA-ligand complex with reservoir solution at an equimolar ratio using sitting drop vapour diffusion at 16°C. Well-diffracting crystals of domain 1 construct 17delU1A in complex with NAD+ grew from the buffer solutions containing 0.2 M Mg(OAc)2, 0.1 M NaAsO2, pH 6.5, 30% MPD after 3 days, while the crystals of the domain 1 construct 18GAAA in complex with NAD+, ADP and ATP, grew from buffer solutions containing 0.1 M MES pH 6.5, 1.6 M MgSO4 after 1 or 2 days. The crystals of domain 2 construct 832GAAA-NAD+ grew from buffer solutions containing 0.2 M NaCl, 0.1 M CHES pH 9.5, 50% PEG400 after two weeks. All the crystals were flash-frozen with liquid nitrogen before data collection.A final concentration of 0.4 mM NAD+ was solved based on the anomalous diffraction signal collected from the co-crystallized Se-Met modified U1A protein by the single-wavelength anomalous diffraction (SAD) method using the program Autosol in the Phenix software suite (+ and 18GAAA with different ligands were solved by molecular replacement (MR) using the Phaser program in the CCP4 software suite with the structure of 17delU1A as the initial model (X-ray diffraction data was collected at Shanghai Synchrotron Radiation Facility (SSRF) and processed with HKL3000 (HKL Research). The phase problem of 17delU1A-NADre suite . The modre suite  with Phere suite . The stral model .2+ soaking experiments, the crystals were transferred into the crystallization solution supplemented with 500 mM MnCl2 at 4°C for 1 h for 18GAAA-NAD+ or with 25 mM MnCl2 at 4°C for 1.5 h for 832GAAA-NAD+.For MnAll X-ray data collection and crystallographic refinement statistics are listed in 2. RNA was refolded by annealing at 65°C for 5 min and followed by incubating on ice for 30 min after dilution to a concentration of 0.1 mM. All ligands were dissolved in the dialysis buffer and diluted to a concentration of 2 mM before titration into the RNA in the sample well (200 μl). The titration experiments were performed at 25°C and initiated with an injection of 0.4 μl, followed by 18 serial injections of 2 μl of the ligand each, with a 120 s interval between each injection and a reference power of 5 μcal s−1.All isothermal titration calorimetry (ITC) experiments during this study were performed on a Microcal PEAQ-ITC instrumentation at the National Center for Protein Science·Shanghai (NCPSS). The RNA samples were dialyzed at 4°C overnight against a buffer containing 50 mM HEPES pH 7.0, 50 mM NaCl, 10 mM MgClKd) was calculated based on the ‘one set of sites’ binding model. All the binding constants and thermodynamic values are listed in The titration data were integrated and analyzed using MicroCal PEAQ-ITC analysis software. The apparent dissociation constant  that was pre-equilibrated with the buffer . Data was processed in Microsoft Excel and Origin software.A final concentration of 0.3 mM RNA in 50 mM HEPES, pH 7.0, 50 mM NaCl, 5 mM MgClAll steady-state fluorescence spectroscopic experiments were measured on a Cary Eclipse spectrometer  equipped with a peltier block, a magnetic stirring device and a RX2000 stopped-flow apparatus . The data obtained were processed with OriginPro 2018 software .2). The samples were heated to 90°C for 2 min, allowed to cool to room temperature, transferred to quartz cuvettes equipped with a small stir bar and held at 20°C in the peltier controlled sample holder. Then, ligands were manually pipetted in a way not to exceed a total volume increase of 3%. The solution was stirred during each titration step and allowed to equilibrate for at least 15 min before data collection. Spectra were recorded from 330 to 500 nm using the following instrumental parameters: excitation wavelength, 308 nm; increments, 1 nm; scan rate, 120 nm/min; slit widths, 10 nm. The apparent binding constants Kd were determined by following the increase in fluorescence after each titration step via integration of the area between 330 and 500 nm. Changes in fluorescence (F − F0) were normalized to the maximum fluorescence measured at the maximum concentration of ligand. The measurement for each titration step was repeated at least three times and the mean of the normalized fluorescence intensity and the corresponding error bars for each value were plotted against the ligand concentration. Data were fit using a two-parametric (Kd and δ) quadratic equation implying 1:1 stoichiometry, according to reference  was obtained at a resolution of 2.80 Å. The space group was P6122, in which each asymmetric unit contained one molecule. The phase problem of 17delU1A-D1/NAD+ was solved by the SAD method using the anomalous signal of selenium in selenomethionine (Se-Met)-derivatized U1A protein. The second structure with the GAAA tetra-loop replacement in domain 1 from an environmental sequence of the nadA RNA motif  was solved at a resolution of 2.40 Å in space group I222. The phase problem of 18GAAA-D1/NAD+ was mastered by MR using the trimmed RNA structure of 17delU1A-D1/NAD+ as the initial model. In addition, we solved the crystal structures of 18GAAA-D1 in complex with adenosine 5′-diphosphate (ADP) and adenosine 5′-triphosphate (ATP) at a resolution of 2.60 Å and 2.80 Å, respectively.In co-crystallization with the U1A protein, the crystal structure of the NAD+ riboswitch, we used the sequence information from Acidobateriaceae bacterium KBS83  that we solved independently at slightly lower resolution is illustrated in + and 17delU1A-D1/NAD+ shows high compatibility  mediates between A10/A11 of one molecule and C15′/G16′/U17′ of the other  and RNA 17delU1A-D1 (without/with NAD+). Clearly, under the conditions used, both RNAs existed as monomers in solution without any indication for dimer formation  and ATP (Kd = 90.2 μM) while NADH (Kd = 305 μM) gave a 3-fold decrease in binding affinity compared to NAD+. NMN does not bind to domain 1 of the NAD+ riboswitch  aligning the binding pocket  forms inner-sphere coordination with its nucleobase  interactions with pA7, pC14 and pC15  is key for mediating the interaction of the negatively charged pyrophosphate of ligand and selected phosphates of the aptamer, thereby creating a crucial recognition feature. Of additional note, the three Mg2+ cations were also observed in the binding pocket of ADP- and ATP-bound structures of domain 1 of the NAD+ riboswitch , diffracting at a resolution of 2.1 Å. The structure is shown schematically in Figure + riboswitch domain 2—like domain 1—adopts a ‘Y-shape’ overall topology in complex with NAD+ without forming the characteristic long-distance J1-J2 pairing interaction that we see in domain 1  and domain 2 (832GAAA-D2/NAD+) of the NAD+ riboswitch reveals the same alignment of stem P1, J1 and the bound ADP-moiety of the ligand NAD+. The long double helical arrangement of stem P1a and P1b, however, shows clear distinctions originating from the altered nucleoside composition of J2 in 832GAAA-D2 that comprises only four instead of the original 6 nt  via its N7 atom  construct.The NAD7 Figure –H. It is7 Figure . Likewisd Figure . Furtherm Figure –M is cru+. The single point mutations G36C and G38C, respectively, and the double mutation G36C/G38C impair the interactions between J1 and J2, and all three mutants do not bind NAD+ under otherwise the same conditions. Additionally, we analyzed the crucial J1/J2 interaction for a possible tolerance of compensatory base pair mutations, such as C14G/G36C, C13G/G38C or C13U/C14U/G36A/G38A. However, these mutants have no affinity to NAD+ under otherwise the same conditions  addition , the required conformation of the binding pocket does not properly pre-fold to enable the ‘lock and key’-type recognition as observed for the WT domain 1. Furthermore, we note that the open binding pocket is rather untypical for riboswitches which usually encapsulate their ligands through relatively slow ligand-induced and RNA-adaptive recognition processes. Ligand recognition of the NAD+ riboswitch was estimated to be in the order of one per second  riboswitch variant Figures  and 6A. e Figure  for domag Figure . Importas Figure  were fitM Figure . In prins Figure  were fitM Figure . We weret Figure . Indeed,n Figure . We concd Figure  which is2+ ion concentrations. This is obvious from the marginal fluorescence increase of the U11Ap mutant of domain 2 in response to Mg2+ . This finding is consistent with the fact that the G36A/G38A domain 1 mutant is essentially domain 2, lacking the long-range interaction but leaving the conserved nucleotides in J1 and their stacking identities absolutely unchanged.Taken together, the fluorescence spectroscopic studies reveal that the J1/J2 long-range interaction through Watson–Crick base pairing is the crucial determinant to tune the ligand binding affinity. Although we already knew from ITC measurements that the compensatory base-pair mutations C13U/C14U/G36A/G38A in domain 1 resulted in a loss of affinity to NADce assay . Moreove+ riboswitch domain 1 from Acidobacterium capsulatum and from an environmental sequence of the nadA RNA motif are consistent with the recent structure of domain 1 from Candidatus koribacter versatilis  moiety, including NAD+ in bacterial cells, which have been reported to be 2.6 mM or even higher  that sequesters the ribosomal binding site and therefore has been proposed as the determinant which is responsible for providing the ON versus OFF signal for translation. Considering the very high concentrations of NADn higher ,31, a sce Figure . If so, s Figure .ydaO motif RNA which was reported to weakly bind ATP  and appear to be weak. To this end, we also note that all our efforts to crystallize the full-length (two-domain) riboswitch failed. Finally, what makes it difficult to narrow down possible mechanistic scenarios of this riboswitch further is that—despite intensive efforts—we were unsuccessful to establish a robust cellular system in E. coli to analyze the gene response of the nadA WT RNA motif and diverse mutants, which was mainly due to the high concentrations of NAD+ needed in the broth medium that negatively affected cell growth.Although a two-concentration sensing riboswitch might be advantageous for the time course of regulation of high-abundant metabolite biosynthesis, we should be cautious with ultimate final conclusions. At this time, it cannot be ruled out that a distinct, thus far undiscovered high-affinity ligand might exist for domain 2. We remind of the bind ATP , and latbind ATP . It also+ of this riboswitch comes from, we mention that the ligand assignment has been merely based on the genomic context which finds the tandem nadA motif never associated with genes unrelated to NAD+ biosynthesis, and also not in combination with genes related to ADP or ATP biosynthesis , 7D7W (18GAAA-NAD+ structure), 7D7X (18GAAA-ADP structure), 7D7Y (18GAAA-ATP structure), 7D7Z (18GAAA-NAD-Mn2+ soaking structure), 7D81 (832GAAA-NAD+ structure) and 7D82 (832GAAA-NAD+-Mn2+ soaking structure).Atomic coordinates and structure factors for the reported crystal structures have been deposited with the Protein Data Bank under accession numbers 7D7V (17delU1A-NADgkaa1029_Supplemental_FilesClick here for additional data file.


Web applications can implement procedures for studying the speed of mental processes  and can be administered via web browsers on most commodity desktops, laptops, smartphones, and tablets. This approach to conducting mental chronometry offers various opportunities, such as increased scale, ease of data collection, and access to specific samples. However, validity and reliability may be threatened by less accurate timing than specialized software and hardware can offer. We examined how accurately web applications time stimuli and register response times (RTs) on commodity touchscreen and keyboard devices running a range of popular web browsers. Additionally, we explored the accuracy of a range of technical innovations for timing stimuli, presenting stimuli, and estimating stimulus duration. The results offer some guidelines as to what methods may be most accurate and what mental chronometry paradigms may suitably be administered via web applications. In controlled circumstances, as can be realized in a lab setting, very accurate stimulus timing and moderately accurate RT measurements could be achieved on both touchscreen and keyboard devices, though RTs were consistently overestimated. In uncontrolled circumstances, such as researchers may encounter online, stimulus presentation may be less accurate, especially when brief durations are requested (of up to 100 ms). Differences in RT overestimation between devices might not substantially affect the reliability with which group differences can be found, but they may affect reliability for individual differences. In the latter case, measurement via absolute RTs can be more affected than measurement via relative RTs . During the past decade, touchscreen devices  have surpassed keyboard devices  to become the most frequently used devices for browsing on the internet  opacity or (2) background-color Cascading Style Sheet (CSS) properties of a Hypertext Markup Language (HTML) element  and the accuracy of RT measurement (Exp. 2) via external chronometry . Accuracy was examined on ten combinations of devices and browsers, formed by two touchscreen and two keyboard devices, each running a different OS, and two or three browsers per device. Technical capabilities were evaluated for two research settings: a lab setting, in which the device and browser could be controlled, and a web setting, in which they could not. For the former setting, the most accurate devices and browsers were evaluated, and for the latter, we evaluated variation across devices and browsers. In Experiment 1, the accuracy of each of the presentation and timing methods listed above was first assessed on the basis of the proportion of trials in which the realized stimulus duration was exactly the number of frames that was requested. Next, the most accurate method was further examined in terms of the distribution of frame differences, reporting on different methods if they produced notably different patterns of results. We compared the accuracy with which stimuli were presented for both brief and long durations—across devices and browsers—in order to assess the degree to which mental chronometry paradigms may be affected that require very brief or precise stimulus durations.In Experiment 2 we assessed how accurately web applications can measure RTs across devices and browsers. RT overestimations were expected to vary substantially, both within and between devices and browsers. To assess how well RT overestimations represented simulation assumptions, we examined distributions across devices, paying particular attention to the presence of any quantization. As in Experiment 1, only the most accurate method of timing and presenting stimuli was considered for further investigation; we have reported on different methods if they produced notably different patterns of results. Informed by the findings of Experiment 2, we conducted a set of simulations to quantify the impact of the accuracy of RT measurements on measurement reliability. In contrast with prior modeling, our simulation did not examine the reliability with which differences between groups or conditions can be detected, but the reliability with which individual differences can be measured.Summarizing, the present study describes how accurately web applications on keyboard and touchscreen devices can present stimuli and register the responses. For stimulus presentation, we examined the presence and magnitude of timing errors in terms of frame differences. For RT measurement, we examined the accuracy with which RTs are measured, the distribution of RT overestimations, and how these might affect the reliability with which individual differences can be measured. Exploratively, we examined a set of methods for improving timing accuracy based on different approaches to timing stimuli, presenting them, and measuring internal chronometry, so as to assess the most accurate method on offer for modern devices and browsers.Table During the stimulus presentation experiment, stimuli were presented for intervals of 16.67, 50, 100, 250, and 500 ms. As each device had a refresh rate of 60 Hz, these intervals corresponded to 1, 3, 6, 15, and 30 frames. The stimulus was a white square on a black background. The presentation experiment consisted of 120 sequences, in which each of the five combinations of timing and presentation methods was first prepared by adding the relevant HTML element to the web page, followed by one trial with each of the five intervals, followed by removing the HTML element. Each sequence was administered in one unique order, out of the 120 possible orders in which the five intervals could be arranged. Each sequence and interval was demarcated by 400 ms of background, followed by 400 ms of an intertrial stimulus presented via a separate HTML element, followed by 400 ms of background. Because the intertrial stimulus was reliably presented for at least 200 ms across all devices, it could be used to match the individual trials of each sequence in terms of both internal and external measures. During the RT experiment, a stimulus was presented until a response had been registered. For each of the five presentation methods, a sequence of 300 RT intervals was generated, consisting of each whole number of ms in the range 150 to 449 in a pseudorandom order. Each interval was distinguished by 1,200 ms of background and each sequence was demarcated by 5,000 ms of background.Three presentation methods were compared, in which stimulus onset and offset were realized by (1) manipulation of the opacity CSS attribute of a DIV element by changing it from 0 to 1 or from 1 to 0; (2) manipulation of the background position of a DIV element by having a picture shift position such that a white or black part is presented; or (3) drawing a white or black rectangle on a canvas element. Two timing methods were compared, in which the stimulus onset and offset were (1) timed via CSS animations that manipulated the appropriate CSS properties, or (2) timed by having a call from rAF manipulate the appropriate CSS properties or draw to a canvas element, after a fixed number of frames. All manipulations were programmed via JavaScript, using the jQuery 3.31 library. The HTML, CSS, and JavaScript were configured according to the best practices recommended in previous timing research . The signals were recorded via a dedicated computer running in-house-developed software , followed by an intertrial stimulus of about 400 ms. See the analysis scripts at the accompanying OSF repository for more details.In Experiment 2, an Arduino Leonardo microcontroller received a signal on stimulus onset, waited the number of milliseconds specified by each RT interval, and then sent a signal to trigger a solenoid. The solenoid was aimed at a touch-sensitive HTML element positioned at the bottom right of the screen of touchscreen devices and at the Q key of keyboard devices. After triggering, the solenoid consistently took 11 ms to go down and trigger a second optical sensor. The second sensor was positioned just above the point at which the solenoid would come in touch with the touchscreen or keyboard. The time between detecting a stimulus via the stimulus sensor and the solenoid touching the touchscreen or keyboard was considered the actual RT. Each interval between detecting stimulus onset and triggering the solenoid was reduced by 11 ms to correct for the solenoid delay.For each combination of device, browser, and experiment, the device was first prepared by installing updates to the OSs and web browsers. The OSs of the MacOS and iOS devices were not updated, as their owners expressed concerns with the stability of the most recent OS versions at the time of the study. Screen brightness was set to maximum, and screensavers and notifications were disabled. Next, for each browser, the browser and experiment software were loaded, a minute was waited , and the experiment was started. Per combination of device and browser, Experiment 1 took about 80 min, and Experiment 2 took about 40 min.z tests on all pairs of percentages collapsed across devices and browsers. A Holm–Bonferroni multiple-comparison correction was applied, with a family-wise alpha of .05, as was the case for all pairwise comparisons reported below. All differences were statistically significant except for rAF background versus rAF canvas; timing via rAF was more accurate than timing via CSS, regardless of the presentation method. When timed via rAF, presentation was more accurate via opacity (81.0%) than via background position (76.3%) or canvas (75.5%). When timed via CSS, presentation was more accurate via background position (23.7%) than via opacity (17.6%).Table z tests. All differences were statistically significant, except for the following: between any of the iOS browsers and Windows Chrome, between any of the Android browsers and Windows Firefox, between any of the MacOS browsers, and between Windows Firefox and either MacOS Firefox or MacOS Safari. Hence, iOS could be considered very accurate, Android moderately accurate, and MacOS not accurate. The accuracy of Windows depended on the browser, with Chrome being very accurate, but Firefox being moderately to not accurate.We selected the most accurate timing and presentation method (rAF opacity) and made pairwise comparisons of all devices and browsers via two-proportion z tests on all pairs of percentages revealed significant differences between rAF-based and animation-based measures, but not within the rAF-based and animation-based measures. Since rAF-based measures were more accurate than animation-based measures, these were selected for further analysis. Because each of the four rAF-based measures was similarly accurate, rAF before created was selected for further analysis.We examined to what degree presentation duration could be estimated via internal chronometry, using four rAF-based measures and two animation-based measures. The distributions of rAF-based measures were strongly quantized, with a resolution of 16.67 ms, whereas the distributions of the animation-based measures were moderately so. To examine how accurately internal chronometry could predict realized duration, we quantized internally estimated durations into frames and calculated the percentage of trials in which the realized duration was exactly the same as that estimated via internal measures . For all devices, browsers, timing, and presentation methods except for MacOS Safari, rAF-based were more accurate than animation-based duration measures. Collapsed across devices and browsers, each of the four rAF-based measures was correct on 75.8% to 76.4% of trials, whereas the animation-based measures were correct on only 34.8% to 34.9%. Two-proportion p values for the z tests on percentages were ≥ .05). When timing via CSS, estimating stimulus durations via internal chronometry was more accurate than CSS animations were at achieving accurate stimulus durations .Table The distributions of frame differences were consistent across presentation methods within the rAF- and animation-based timing methods, but showed pronounced differences between timing methods. Therefore, we report on the most accurate presentation method for timing via rAF (opacity) and for timing via CSS (background color). There was variation in the sizes and signs of frame differences across devices, browsers, presentation methods, and intervals Fig. . Both iOrAF before created was used, and for the response, event created, though the results were similar for other timestamps . RT overestimation was calculated as the difference between the measured and realized RTs. Table t tests between all pairs of devices and browsers. All differences were statistically significant, except between the iOS browsers. Hence, there were substantial differences in mean RT overestimation between devices and browsers, which were particularly low for iOS and particularly high for MacOS Safari.For RT measurements, only trials presenting stimuli via the most accurate timing method (rAF) and presentation method (opacity) were analyzed. As the timestamp for stimulus onset, the internal measure We compared the variance of RT overestimations via Levene’s tests between all pairs of devices and browsers. Variances did not differ significantly between Android, iOS, Windows Firefox, and MacOS Safari. All other variances were statistically significant, expect between MacOS Chrome and MacOS Safari. Hence, Windows Chrome added the least random noise to the RT measurement, MacOS Firefox added the most, and other devices and browsers added moderate levels.SD) values of 60.0 (12.4) and 89.1 (2.4) ms.Inspection of scatterplots of measured RT versus realized RT revealed quantization on iOS with a resolution of 60 Hz. The distributions of RT overestimations for most devices could be described well as having a normal to uniform distribution, with a small number of outliers. However, Android showed some degree of bimodality, whereas MacOS Chrome and MacOS Firefox showed pronounced bimodal distributions Fig. . FittingSD of the Gaussian component was drawn from a uniform distribution between 25 and 75 ms, and the mean of the exponential component was drawn from a uniform distribution between 50 and 100 ms. To model the accuracy with which devices measured RTs, we added two types of device noise. For each participant, a constant RT overestimation was modeled by adding a constant value to RTs drawn from a uniform distribution between 60 and 130. For each participant, a variable bimodal RT overestimation was modeled by adding to their RTs a value drawn from one of two Gaussian distributions with equal probability. Each Gaussian distribution had an SD drawn from a uniform distribution between 2 and 12 ms. The mean of the first Gaussian distribution was 0, whereas the mean of the second Gaussian distribution was drawn from a uniform distribution from 0 to 30 ms.To examine how the accuracy of RT measurements may affect studies of individual differences, we conducted a set of simulated experiments with 100 participants each. RTs were drawn from an exponentially modified Gaussian distribution. For each simulated participant, the mean of the Gaussian component represented the trait score. The SDs that varied per experiment. The trait score formed the mean component of that participant’s RTs. The relative-RT experiment consisted of two conditions, with half of the trials belonging to each condition. Two trait scores were drawn from a bivariate Gaussian distribution that was correlated .5 across participants, so both trait scores, as well as their difference, had the same SD. Each trait score formed the mean component of that participant’s RT for one of the two conditions. Each experiment was simulated both with and without device noise, with trait SDs of 15, 25, and 50 ms, and with trial counts ranging from 10 to 300 in steps of 10. For each of these combinations, 1,000 experiments were simulated. We calculated reliability as the squared Pearson correlation between trait score and mean RT for the absolute-RT experiment, and between trait score difference and relative mean RT for the relative-RT experiment.Two types of experiments were simulated. The absolute-RT experiment consisted of one condition, in which the trait score of a participant was drawn from a univariate Gaussian distribution with SDs of 15, 25, and 50 ms, respectively. In the relative-RT experiments, device noise decreased reliability by .012 at most across all trial counts and trait SDs. Without noise, the relative-RT experiments were more reliable than the absolute-RT experiments from 120 to 130 trials and beyond. With noise, the relative-RT experiments were more reliable from 40 to 50 trials and beyond.Figure We examined how accurately web applications on touchscreen and keyboard devices present stimuli for specified durations in Experiment 1, and measured RTs in Experiment 2. In a simulation, we examined how the accuracy of RT measurements affected the reliability with which individual differences could be measured. The results of each experiment are discussed below, followed by a general assessment of the technical capabilities of web applications for mental chronometry.With regard to stimulus presentation, we first compare the results for different methods for timing and presenting stimuli, followed by an assessment of timing accuracy across devices and browsers. Timing via rAF was more accurate at realizing precise stimulus duration than was timing via CSS animations. In part, this was because iOS timed via CSS consistently presented stimuli for one frame longer than requested. In those cases, requesting slightly shorter durations than was done in this study could improve the accuracy of stimuli timed with CSS animations. However, such consistency was not found for all devices and browsers, so overall we recommend using rAF for timing stimuli. We suspect that the inconsistencies in the behavior of CSS animations may be due to the standards for CSS animations still being a working draft , we assessed the accuracy with which keyboard and touchscreen devices can time stimuli. Some devices and browsers, of both touchscreen and keyboard type, achieved near-perfect timing: namely, iOS with Chrome, Firefox, and Safari, as well as Windows with Chrome. Hence, in settings where the device and browser can be controlled, web applications can be as accurate as specialized software. Most devices and browsers achieved most presentation durations within one frame of the requested duration, though MacOS Chrome and Safari tended to present durations of up to six frames (100 ms) too briefly. Hence, when the device and browser cannot be controlled, the reliability and validity of mental chronometry paradigms that require brief presentations may be affected.With regard to the accuracy of RT measurements, different internal measures for RT gave similar results. Quantization of RT into 60 Hz was found on one device, which may be acceptable (Ulricht & Giray, Prior simulations have quantified the impact of the accuracy of RT measurements on the reliability of detecting group differences. As far as we are aware, none have quantified the impact on the reliability of measuring individual differences. Our modeling work indicated that different factors may affect reliability, including the number of trials and the variance of the trait that is measured. The reliability of absolute RT measurements was affected by device noise, but relative RT was hardly affected. This could be because between-device variation was larger than within-device variation. For relative RT, between-device variation is removed due to RTs being subtracted between conditions within participants. A rather striking result was that with higher numbers of trials, relative RT was more reliable than absolute RT, even though traits in the relative RT simulations were correlated .5. The former may appear to go against the commonly held belief that the difference between two positively correlated scores is less reliable than each of these scores individually. Although a comprehensive examination of this result is beyond the scope of this article, here we may offer some explanations. First, a classic result underlying the formerly mentioned belief is based on two observations per participant (Lord & Novick, However, in both group and individual difference research, any confound between device type and study design could affect RT results more severely (Reimers & Stewart, In summary, in controlled settings, web applications may time stimuli quite accurately and may register RTs sufficiently accurately when a constant overestimation of RTs is acceptable. In uncontrolled settings, web applications may time stimuli insufficiently accurately for mental chronometry paradigms that require brief stimulus presentations. Differences in the degree to which devices overestimate RT may more severely affect the reliability with which individual differences are measured via absolute RT than via relative RT.Web applications offer a means to deploy studies both inside and outside of the lab. Frameworks are being developed that make it increasingly easier for researchers to deploy mental chronometry paradigms as web applications (Anwyl-Irvine et al., The results in this study may be representative of web browsers, as the three browsers selected in this study represent a large majority of browsers used online (StatCounter, A study in a wider range of devices, preferably having multiple samples per device, could replicate the systematic differences found in this study. If replicated, the results could be used to correct for timing inaccuracies and RT overestimations by detecting participants’ device and browser. Note that this undertaking would require significant efforts, given its scale. Also, it would need to be repeated for each new generation of devices, as well as for significant OS and web browser updates. The design of the present study, which could assess timing accuracy at the level of individual trials, could be helpful. By making all materials openly accessible online, we hope to facilitate such efforts.A solenoid was used for generating responses (similar to Neath et al., Overall, touchscreen devices seem technically capable of administering a substantial number of mental chronometry paradigms, when taking some limitations and best practices into account. As smartphone ownership and internet connectivity are becoming ubiquitous, this offers various opportunities for administering mental chronometry on large scales and outside of the lab. By implementing RT tasks as web applications, they are based on durable and open standards, allowing a single implementation to be deployed on desktops, laptops, smartphones, and tablets. We hope that this article helps answer doubts about the timing accuracy of such an approach and provides some insight into how the reliability of RT measurements can be affected when millisecond accuracy cannot be achieved.


Tar homologue) ligand binding domain containing chemoreceptors possess pentapeptides, whereas chemoreceptor families with other ligand binding domains are devoid of pentapeptides. The abundance of chemoreceptors with C-terminal pentapeptides correlated negatively with the number of chemoreceptor genes per genome. The consensus sequence reveals a negative net charge for many pentapeptides. Pentapeptide containing chemoreceptors are very abundant in the order Enterobacterales, particularly in the families Pectobacterium and Dickeya, where they represent about 50% of the total number. In contrast, bacteria with primarily free living lifestyles have a reduced number of pentapeptides, such as approximately 1% for Pseudomonadales. It is proposed that pentapeptide function is related to mechanisms that permit host interaction.Chemosensory pathways represent a major prokaryotic signal transduction mechanism that is based on signal sensing by chemoreceptors. An essential feature of chemosensory pathways is the CheR and CheB mediated control of chemoreceptor methylation causing pathway adaptation. At their C-terminal extension the Tar and Tsr model chemoreceptors contain a pentapeptide that acts as an additional CheR and CheB binding site. The relevance of this pentapeptide is poorly understood since pentapeptide removal from Tar/Tsr causes receptor inactivation, whereas many other chemoreceptors do not require this pentapeptide for correct function. We report here a bioinformatic analysis of pentapeptide containing chemoreceptors. These receptors were detected in 11 bacterial phyla and represent approximately 10% of all chemoreceptors. Pentapeptide containing chemoreceptors are mainly found in Gram-negative bacteria, are of low abundance in Gram-positive species and almost absent from archaea. Almost 50% of TarH ( The predominant adaptation mechanism is based on the methylation and demethylation of several glutamyl residues at the chemoreceptor signaling domain, catalysed by the CheR methyltransferase and CheB methylesterase, respectively g domain . Data inE. coli chemoreceptors, the high-abundant Tar and Tsr, possess a C-terminal pentapeptide, NWETF, that is tethered to the C-terminal end of the chemoreceptor signaling domain via an unstructured linker of approximately 35 amino acids in vivo and in vitro and abolished chemotaxis Of the KD = 160 µM) in vivo and in vitroIn contrast to CheR, CheB binds to this pentapeptide with much lower affinity  and cluster II (approx. 250–330 amino acids) 6The prototypal chemoreceptor topology comprises a LBD at the N-terminal part and the so far characterized pentapeptide containing chemoreceptors possess a pentapeptide that is fused to the C-terminal extension of the signaling domain. However, there is a poorly characterized chemoreceptor subfamily that contains an LBD at the C-terminal extension. Inspection of the Pfam database has shown that there are at least 17 different types of LBD that are fused to the C-terminal part of the chemoreceptor . The que7Chemoreceptor containing bacterial strains were submitted to an analysis by the fusionDB database 8Pentapeptide containing chemoreceptors have been detected in 11 different phyla A extendi9S. typhimurium CheR The consensus motif of all pentapeptides identified is shown in 10Data show that the phylogenetic distribution of pentapeptide containing chemoreceptors is wider than initially thought and involve 11 phyla. Multiple and interconnected lines of evidence show that pentapeptide chemoreceptors are particularly abundant in Gram-negative bacteria that inhabit the intestine. Firstly, phylogenetic analyses show a high percentage of pentapeptide containing chemoreceptors in Bacteroidetes and Enterobacterales, two taxonomic categories that are composed by many strains that inhibit the intestine. Secondly, the classification of strains according to lifestyle has revealed a high abundance of pentapeptides in strains that inhibit the human intestine. Thirdly, there is a very low abundance of pentapeptide containing chemoreceptors in Pseudomonads that are to a large degree free living environmental bacteria. Experimental research is now required to assess the potential role of pentapeptide containing chemoreceptors in host interaction.11Retrieval of chemoreceptor sequences and identification of chemoreceptors that contain a C-terminal pentapeptide: Chemoreceptors are defined as proteins that contain a MCPsignal domain (Pfam PF00015) Classification of chemoreceptors according to cellular compartment and type of LBD: Proteins that contained at least one transmembrane region, as annotated in the UniProt Topology section, were classified as transmembrane proteins whereas the remaining receptors formed the group of soluble chemoreceptors. The complete set of chemoreceptors as well as members of the 5Pset were analysed for the presence of at least one of the following LBDs: TarH (PF02203), CHASE3 (PF05227), 4HB_MCP_1 (PF12729), PAS_9 (PF13426), PAS_4 (PF08448), PAS_3 (PF08447), sCACHE_2 (PF17200), sCACHE_3_3 (PF17202), protoglobin (PF11563), CZB (PF13682), GAF (PF01590), Pilj (PF13675), dCACHE_2 (PF08269), dCACHE_3 (PF14827), HBM (PF16591), NIT (PF08376), Cache_3-Cache_2 (PF17201) and dCache_1 (PF02743). These domains have been identified as the most abundant LBDs in chemoreceptors Taxonomic classification of chemoreceptors: The taxonomic category of chemoreceptor containing strains was extracted from Names and Taxonomy section of UniProt. A Python dictionary was created adding the frequency of the pentapeptide containing sequences to each of the taxonomic categories. The abundance of pentapeptide containing chemoreceptors with respect to the total number of chemoreceptors was calculated.http://www.hmmer.org) and the generation of a Sequence Logo with Skylign [47].Generation of a pentapeptide motif consensus sequence: The pentapeptide motif sequence was extracted from each of the sequences from the 5Pset entries and a fasta file was created containing the pentapeptide sequence list. This file was used for multiple sequence alignment by MUSCLE Identification of the bacterial habitat: Bacterial strains that contain C-terminal pentapeptides were submitted to an analysis by the fusionDB database Display of three dimensional structures: Images of protein structures were produced by the PyMOL Molecular Graphics System, Version 2.2.0 Schrödinger, LLC.Álvaro Ortega: Methodology, Software, Data curation. Tino Krell: Conceptualization, Writing - review & editing.The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.


In this paper, we present the design, simulation, fabrication and characterization of a microfluidic relative permittivity sensor in which the fluid flows through an interdigitated electrode structure. Sensor fabrication is based on an silicon on insulator (SOI) wafer where the fluidic inlet and outlet are etched through the handle layer and the interdigitated electrodes are made in the device layer. An impedance analyzer was used to measure the impedance between the interdigitated electrodes for various non-conducting fluids with a relative permittivity ranging from 1 to 41. The sensor shows good linearity over this range of relative permittivity and can be integrated with other microfluidic sensors in a multiparameter chip. Microfluidic relative permittivity sensors have great potential for fluid discrimination and composition determination ,2. AppliTo measure relative permittivity, an electric field or electromagnetic wave needs to be applied. Quite often, this is done by using a planar electrode structure, with two electrodes next to each other  or with In this paper, we present a device where the fluid is flowing in between interdigitated electrodes, allowing for both a large channel diameter and large capacitance. The sensor can, for instance, be bonded above a fluid inlet or outlet of an existing sensor, such as a micro Coriolis flow sensor . As suchTo measure the impedance, the sensor is driven by an AC voltage With the reactance k would be equal to the ratio between the surface area of the electrodes and the distance between them, however, because of the complex geometry of the device, a parallel plate approximation cannot be used. Therefore, finite element simulations were done with COMSOL Multiphysics® to estimate the capacitance and the constant k in Equation  and andZ′ anquations . We can Hence, each curve in Equation  to the mThis is confirmed by A micro relative permittivity sensor that allows fluid to flow through the sensing element has been designed, simulated and fabricated. In tests with different fluids with relative permittivity ranging from 1 to 42, the sensor shows excellent linearity with maximum fit error of 0.9% of the full scale. In comparison with conventional designs, the sensor is very small but still provides a high capacitance value that allows electrical measurement to be able to determine small changes in the relative permittivity of the fluid. Future research will focus on measurements with aqueous solutions, design of new sensors that allow on-chip four point capacitance measurement and multiparameter sensor integration.


The syntaxin Habc domain has previously been found to be structurally very similar to the GAT domain present in GGA family members and related proteins that are otherwise completely unrelated to syntaxins. Because the GAT domain has been found to be a ubiquitin binding domain we hypothesized that the Habc domain of syntaxins may also bind to ubiquitin. Here, we report that the Habc domain of syntaxin 3 (Stx3) indeed binds to monomeric ubiquitin with low affinity. This domain binds efficiently to K63-linked poly-ubiquitin chains within a narrow range of chain lengths but not to K48-linked poly-ubiquitin chains. Other syntaxin family members also bind to K63-linked poly-ubiquitin chains but with different chain length specificities. Molecular modeling suggests that residues of the GGA3-GAT domain known to be important for ionic and hydrophobic interactions with ubiquitin may have equivalent, conserved residues within the Habc domain of Stx3. We conclude that the syntaxin Habc domain and the GAT domain are both structurally and functionally related, and likely share a common ancestry despite sequence divergence. Binding of Ubiquitin to the Habc domain may regulate the function of syntaxins in membrane fusion or may suggest additional functions of this protein family.Syntaxins are a family of membrane-anchored SNARE proteins that are essential components required for membrane fusion in eukaryotic intracellular membrane trafficking pathways. Syntaxins contain an N-terminal regulatory domain, termed the H N-ethylmaleimide-sensitive factor attachment protein receptor) proteins are the indispensable mediators of membrane fusion reactions within the endomembrane system of eukaryotic cells5. The SNARE superfamily consists of several sub-families whose members contain one or two SNARE domains of ~ 60 residues in length6. Members of the syntaxin family of SNAREs are central to the formation of SNARE complexes. They contain a C-terminal transmembrane anchor, preceded by the SNARE domain. The latter engages in interactions with cognate SNAREs to form a SNARE complex in a 4-helix bundle arrangement that ultimately leads to membrane fusion9.SNARE  whose Habc domains fold into essentially superimposable three-helix bundles despite limited or absent sequence similarity. The Habc domains of various syntaxins have been found to be binding sites to proteins that regulate SNARE function including those of the munc18, munc13 and synaptotagmin families16. In addition, in some—but not all—syntaxins the Habc domains have the ability to engage in an intramolecular interaction with the SNARE domain resulting in a tetrameric helical bundle. This “closed” conformation generally inhibits the formation of complexes with cognate SNARE proteins and thereby inhibits membrane fusion17. These findings clearly indicate that the Habc domains of syntaxins play a critical role in the regulation of membrane fusion and that this function depends on the conserved three-dimensional structure of these domains.Syntaxins also contain an N-terminal regulatory domain that consists of three α helices and has been termed the Habc domain. The GAT (GGAs and TOM) domain of GGA1 was found to be nearly superimposable with the Habc domains of Stx1A and Stx6 despite their lack of sequence similarity18. GGA proteins are Golgi- and endosome-associated clathrin adaptor proteins involved in cargo recruitment in membrane trafficking pathways. At the time of the discovery of the structural similarity with the syntaxin Habc domain, relatively little was known about the function of the GAT domain. Subsequently, however, the GAT domains of GGA proteins were found to be ubiquitin binding domains24 which helped to explain their function in recruiting ubiquitinated membrane proteins for targeting to multivesicular bodies (MVBs)25.It was found that a conserved domain in a very different family of proteins shares the same fold with the syntaxin H26. In the case of membrane proteins, reversible ubiquitination serves as a signal for targeting to endosomes, and then to intraluminal vesicles of MVBs. MVBs can subsequently either fuse with lysosomes leading to degradation27 or they can fuse with the plasma membrane leading to extracellular secretion of membrane proteins in the form of exosomes28. Ubiquitin itself may be ubiquitinated at any of its seven lysine residues leading to target proteins being tagged with a chain of polyubiquitin molecules29. K48-linked polyubiquitin chains are a signal for proteasomal degradation whereas mono-ubiquitin and K63-linked polyubiquitin chains are another signal for trafficking of membrane proteins to the endosomal pathway, and especially into the MVB pathway32. The GAT domain of GGA proteins has been shown to be required for the sorting of membrane proteins tagged with K63-polyubiquitin chains into the MVB pathway32.Ubiquitin, an 8 kDa protein, is covalently attached to lysine residues of target proteins via E3 ligases19. A large number of ubiquitin-binding proteins have been found to also be ubiquitinated themselves. The reasons for this are not always completely clear but it is thought that concurrent ubiquitin-binding and ubiquitination of sorting proteins aids in the establishment of protein networks to create sorting domains33. We have recently reported that syntaxin 3, a SNARE involved in membrane fusion at the apical plasma membrane of polarized epithelial cells, undergoes mono-ubiquitination at lysine residues adjacent to its transmembrane domain34. Ubiquitination of Stx3 leads to endocytosis from the basolateral plasma membrane, direction into the endosomal/MVB pathway and eventually excretion with exosomes34. Functional studies using a non-ubiquitinatable Stx3 mutant suggested that Stx3 may function to sort specific cargo proteins into the MVB/exosomal pathway34. Such a function is unexpected for a protein thought to be involved in membrane fusion.GGA proteins themselves are also mono-ubiquitinated in a manner dependent on the binding of ubiquitin to their GAT domainabc domain of syntaxins and the GAT domain suggests a common ancestry and related function. Given this structural similarity, and given the finding that Stx3—like GGA proteins—is mono-ubiquitinated and appears to play a role in cargo sorting in the MVB/exosomal pathway, we hypothesized that the Habc domain of Stx3 may be a ubiquitin-binding domain. We report here that Stx3 indeed binds to ubiquitin and K63-linked polyubiquitin chains, but not K48-linked polyubiquitin chains. Structural modeling and mutagenesis experiments suggest that the mode of ubiquitin binding could be similar to that of the GAT domain and may involve conserved hydrophobic interactions and a salt bridge. These results suggest that syntaxin function may be regulated by ubiquitin binding, and that syntaxins may function in protein sorting in addition to their established role in membrane fusion.The structural similarity between the Habc domain (1–146) of Stx3 pulls down with ubiquitin-coated beads much more efficiently than the entire Stx3 cytoplasmic domain (3.78 fold change increase)   of Stx3 with ubiquitin-coated beads to observe any interaction in a pull-down assay  is located on the opposite face of the 3-helix bundle of the GAT domain and encompasses residues in helices B and C24, but no 3D structure is available for this interaction.The GAT domain of GGA3 has been shown to have two distinct binding sites for ubiquitin. Site 1 has been studied in most detail and encompasses residues from the C-terminal half of helix A and the N-terminal half of helix B  in very similar positions as E246 and E250 of GGA3-GAT, and these residues may be predicted to engage in a salt bridge with R42 of ubiquitin. Similarly, L59, L77 and L80 of Stx3 would form a hydrophobic pocket that may interact with I44 of ubiquitin, analogous to the hydrophobic pocket formed by L227, M231 and L247 of GGA3  or double glutamate (E78A/E83A) mutations were generated. Introducing all four mutations simultaneously resulted in an insoluble GST fusion protein that could not be analyzed.3  could bind to polyubiquitin even in the absence of highly conserved primary sequence similarity. In this regard, it is interesting that the L77A/L80A mutations in Stx3 appear to disrupt only the binding to K63-linked Ubi3, which may suggest that longer ubiquitin chains can compensate by interacting with additional residues simultaneously.The ability of the mutants to bind to K63-linked polyubiquitin chains was assessed using the same assay as in Fig. 3 Fig. . Given tabc domains are highly similar, and the fact that hydrophobic and ionic residues known to mediate ubiquitin-binding of the GAT domain may have equivalent residues in the Habc domain of Stx3 . This may add a high level of complexity to the regulation of ubiquitin binding of Stx3, as previously suggested by Sims et al.35 for other UBA domains. While the purpose of the ability of syntaxins to bind to mono-ubiquitin and K63-linked polyubiquitin chains remains to be elucidated, several possibilities can be envisioned. Binding of K63-linked polyubiquitin chains to the Habc domain of a syntaxin may interfere with the ability of that syntaxin to bind to other regulatory proteins that are known to interact with the Habc domain such as members of the munc13, synaptotagmin and munc18 families of SNARE regulators. Thereby, K63-linked polyubiquitin chains, possibly attached to specific regulatory proteins, may regulate SNARE function and therefore membrane fusion in certain vesicle trafficking pathways. Another possibility is that binding of K63-linked polyubiquitin chains to the Habc domain of a syntaxin would interfere with the ability of the Habc domain to engage in an intramolecular interaction with the SNARE domain of that syntaxin. This would result in a “constitutively open” conformation of that syntaxin and, again, may regulate membrane fusion functions. Another possibility emerges from our recent finding that Stx3 can undergo ubiquitination at a cluster of lysine residues located between its SNARE domain and transmembrane domain34. It may be possible that the Habc domain could engage in an intramolecular interaction with this covalently attached ubiquitin thereby locking such modified Stx3 in a “constitutively closed” conformation. Finally, it is possible that binding of K63-linked polyubiquitin chains to the Habc domain of Stx3 may be unrelated to a function in membrane fusion but rather relates to a different function. Such a possibility may be supported by our recent finding that Stx3 that is covalently ubiquitinated at the lysine cluster proximal to its transmembrane domain, enters the endosomal pathway, traffics to intraluminal vesicles of MVBs, and is eventually excreted with exosomes34. We reported that a non-ubiquitinatable mutant of Stx3 (termed Stx3-5R) is not only unable to enter the MVB/exosomal pathway but also interferes with the recruitment of a specific apical exosomal cargo protein, the orphan G-protein coupled receptor GPRC5B, into this pathway. This suggested that Stx3 normally plays a role in cargo recruitment in a fashion that is dependent on its ability to be ubiquitinated. Interestingly, the Stx3-5R mutant was also found to disrupt the secretion of human cytomegalovirus (hCMV) virions, a result that—combined with other findings—suggests that hCMV exploits the MVB/exosomal pathways for virion production and secretion34. In this regard, Stx3 bears striking similarities to GGA proteins. Both contain a similarly structured ubiquitin-binding domain, both undergo ubiquitination themselves, and both are involved in recruitment of membrane proteins into the MVB pathway. In the case of GGA proteins, this sorting function requires cargo proteins to be tagged with K63-linked polyubiquitin chains32.Our data support the conclusion that the HAltogether, these results suggest that syntaxins contain a ubiquitin binding domain similar to the GAT domain. The implications of this finding are yet to be elucidated but may relate to the regulation of membrane fusion functions and/or point towards a novel function of syntaxins in the sorting of membrane proteins.The cytoplasmic region of rat Stx3 (1–265) and the N-terminal region of Stx3 (1–146), respectively, were cloned into pGEX-4T-3 . pGEX-4T-2-GGA3-GAT plasmid was a kind gift of Kazuhisa Nakayama . Site-directed mutagenesis  was employed to generate the open-conformation mutant (LE165/166AA) in the Stx3 (1–265) plasmid.E. coli Rosetta 2 (Millipore-Sigma) competent cells. When the cultures reached an OD of 0.6, IPTG was added to induce expression of GST-protein. Cells were pelleted and lysed in a buffer containing 10 mM Tris-HCl, pH 8.0; 150 mM NaCl; 500 µg/mL of lysozyme; 50 µg/mL of RNase; 100 µg/mL of DNase; 0.5% Triton X-100; 5 mM DTT; 5 mM EDTA; 1 mM PMSF and a protease inhibitor cocktail (Millipore-Sigma) containing: 100 µM AEBSF, 0.085 µM Aprotinin, 4 µM Bestatin, 1.4 µM E-64, 2 µM Leupeptin, 1.5 µM Pepstain A was subsequently added. CL2B Sepharose beads  were used to pre-clear the lysates, followed by incubation with glutathione-coated agarose overnight at 4 °C with rotating. Beads were washed four times and eluted with glutathione. Eluate content was analyzed by SDS-PAGE. Eluates were then dialyzed into 50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1 mM EDTA and the protein concentration determined using a Nanodrop spectrophotometer (ThermoScientific).GST-fusion protein plasmids were transformed into 55 µl (dry volume) of ubiquitin-coated agarose (Millipore-Sigma) or CL4B Sepharose  beads were washed three times with 1 ml of Buffer A  + 1% FBS. Buffer was removed from pelleted beads using a Hamilton syringe and 55 µL of Buffer A + 1% FBS were added to beads to create a 50/50 slurry. 30 µL of the bead slurry were added to 1 ml of Buffer A + 1% FBS containing 1 µM of purified GST-fusion protein in an Eppendorf tube. Tubes were tumbled end-over-end for 2 h at room temperature. Beads were pelleted and washed four times with 1 ml of Buffer A + 0.005% Tween-20 before pelleting and removing residual buffer with a Hamilton syringe. Beads were re-suspended in 45 µl of SDS-PAGE sample buffer containing 100 mM DTT, boiled, separated on SDS-PAGE, and transferred to nitrocellulose membrane. Membranes were probed with a polyclonal goat anti-GST antibody  and a Stx3-antibody generated by us (available as MAB2258 from Millipore-Sigma) and a donkey anti-goat IgG HRP-conjugated secondary antibody (Jackson Immunoresearch).2O prior to blocking in 5% dry milk in TBST before being probed with mouse anti-ubiquitin antibody P4D1 (Santa Cruz Biotechnology).GST-fusion proteins were incubated in Buffer A containing FBS  while rotating at room temperature with 8 μg of a K48-linked or K63-linked mixture of polyubiquitin chains of 2–7 ubiquitins in length (Boston Biochem). After 1 h, glutathione-coated agarose beads were added to the sample and incubated for one hour. Beads were washed three times with Buffer A containing 0.005% Tween-20, re-suspended in SDS-PAGE sample buffer and treated as above. Membranes were boiled for 10 min in HSPR measurements were performed on a Biacore 2000 instrument and were performed at room temperature in HBS-EP buffer . GST-fusion proteins were captured to a CM5 Sensor Chip  by using a GST capture kit  according to the manufacturer’s instructions. Purified bovine ubiquitin was from Millipore-Sigma (U6253).abc domain of human Stx3 sequence (Q13277) was constructed using the structure of Syntaxin-1A (PDB: 1EZ3) as template and the Swiss-model website for building the 3D model. The structure of GG3-GAT domain: Ubiquitin (PDB:(1YD8)) and Habc of Stx3 were aligned over 88 residues using PyMOL. Figures were generated using PyMOL (The PyMOL Molecular Graphics System).Homology model of the HSupplementary Informations.


Basing welfare assessment standards on protocols developed for industrialised beef exporting countries could enhance the beef production and export standards in Namibia. This paper compares imposed thresholds of welfare measures of categorisation and derived thresholds to see—which was the most appropriate to the range of observations and welfare implication in three cow-calf production systems in Namibia. Using the same thresholds as the New Zealand protocol, regardless of the farm system, commercial herds achieved most welfare measures thresholds, but semi-commercial and communal herds attained acceptable welfare thresholds only in a few measures. For measures with significant welfare implications, the stricter threshold was retained, while derived thresholds appeared more appropriate for commonly occurring traits (but of less welfare importance), and some measures threshold were temporarily adjusted to reflect drought conditions. The welfare assessment identified the strengths and weaknesses in thresholds in measures across the farm types, which is envisioned to draw attention for remedial intervention to improve welfare standards of the beef industry.This paper aims to develop standards for a welfare assessment protocol by validating potential categorisation thresholds for assessing beef farms in various beef cow-calf production systems in Namibia. Forty measures, combined from a New Zealand-based protocol plus Namibia-specific measures, are applied to 55 beef farms  during pregnancy testing, and a questionnaire guided interview. The categorised measures on a 3-point welfare score  are subsequently compared with the derivation of thresholds based upon the poorest 15% and best 50% of herds for each measure. The overall combined thresholds of continuous measures across the three farm types show 10/22 measures that posed welfare compromise across Namibia, whereas commercial farms have 4/22 measures, and semi-commercial and communal village farms have 12/22 and 11/22, respectively, with high thresholds. Most measures-imposed thresholds are retained because of significant importance to the welfare of animals and preventiveness of the traits, while leniency was given to adjust good feeding and mortality measures to signify periods of drought. Handling measures  and abrasions thresholds are adjusted to reflect the temporary stress caused by infrequent cattle handling, and faulty yard designs/design and possible cattle breed influence on handling. Hence, Namibia needs prioritised investigation of underlying contributing factors and remediation to reduce the high thresholds. Animal welfare in Namibia has been increasingly acknowledged as important in the production and trade of farm animals and their products. International trading partners for Namibia’s beef are seeking assurances that acceptable animal welfare standards are maintained. This requires concrete animal welfare legislation, including a standardised, validated protocol for assessing beef cattle welfare. Currently, the Farm Assured Namibian Meat (FAN Meat) scheme provides assurance that Namibian beef is produced under a natural, safe environment and is traceable and of good quality, but the scheme is not comprehensive as a welfare assessment protocol . For exaCurrently, welfare standards in Namibian cattle are only routinely assessed at export abattoirs  with no On-farm welfare data from both categorical measures  and continuous measures (recorded as the percentage of cattle affected on each farm) need meaningful interpretation to reflect the welfare status of farms . The resUsing Namibian data we collected in Part 1 of the study , the pren = 17 farms/herds)  were large privately-owned farms  that were individually fenced off in several grazing paddocks. Semi-commercial village farms (n = 20 herds) in Okakarara area (Otjozondjupa region) were government-owned settlement villages predominantly inhabited by farmers who were dependent on livestock production for their livelihood. Multiple families’ (~10–30 families) cattle grazed and shared water on the same permanent communal land with limited or no internal and external border fencing. These farms were eligible to produce beef for export markets. Communal village farms (n = 18 herds) in Opuwo/Kaokoland area,  were exclusive government-owned communal settlements with inhabitants farming with small multipurpose  cattle holdings mainly for subsistence. As on semi-commercial village farms, multiple families’ (~10–30 families) cattle grazed and shared water on the same permanent communal land with limited or no internal and external border fencing. Communal village farms were in an area restricted for animal disease control to produce beef for overseas export markets. The study areas represent their farm types within Namibia, as described by Kaurivi . In summThe protocol used is described in full in Kaurivi . The welp ≤ 0.05), and log10(n+1) was used to transform those variables that were not normally distributed.All data were analysed using IBM SPSS Statistics for Windows Version 24 . Descriptive statistics for continuous measures were used to capture central tendency (median), and range (minimum and maximum). Qualitative methods were used to analyse the frequency of ordinal measures. The Shapiro-Wilk test was used to test for normality ; 1: Marginal welfare ; and 2: Poor/unacceptable welfare (immediate intervention required) . The iniThe New Zealand thresholds were compared with the pre-determined thresholds of each continuous measure in the current data from the three farming systems in Namibia. For example, for good health measures , painful conditions  and absence of injuries or physical impairment the welfare thresholds were kept the same at 2%. For the additional Namibia-specific measures, the thresholds for extraneous (multiple) brands/cuts 5%), long/sharp horns (10%) and external parasites (10%) were based on authors’ opinion of acceptable welfare standards (no concrete thresholds could be found in literature). The threshold (10%) for tail twisting was aligned with that of hitting of cows. The distance to water and grazing categorisation was based on Holechek  and the %, long/sCategorised observational data are illustrated in Feeding measures: Cattle on commercial farms did not walk long distances to grazing and water (<1.6 km–3.2 km), and there were no emaciated cows observed, and 76% herds were in good body condition, and 88% of cows had good rumen fill. Semi-commercial and communal herds obtained a good welfare score for BCS only in 40% and 17% of herds, respectively, of which most cattle herds in communal villages were classified as more emaciated  than just thin. Cattle walked long distances to water and grazing (>3.2 km), and all village farm herds scored poorly in this. Hazards were mostly marginal across the farm types, with semi-commercial and communal showing 35% and 33% of herds with poor welfare score.Health observations: The worst scores were observed for abrasions and mortality at all farm types. Only 35% of commercial and 25% of semi-commercial herds had a good score for abrasions, and none of the communal herds had a good score for abrasions. The mortality rate was categorically poor in 65%, 95% and 100% of commercial, semi-commercial and communal herds, respectively. Fifty percent of semi-commercial herds had a good score for dystocia, while only 24% and 33% of commercial and communal herds did, respectively. Lameness and extraneous brands/cuts were worst in communal followed by semi-commercial herds. The tick and fly burden were worse in semi-commercial with 20% and 40% herds with a poor score, and 17% and 11% of communal herds had poor welfare for ticks and flies, respectively, while the majority of commercial herds had a good score for both ectoparasites types.Painful management measures: Procedures were performed without the use of anaesthesia or analgesics. All farm types identified cattle with ear tags , while most herds at the village herds had poor scores for ear notching (with knives) and extraneous branding/cuttings. In relation to disbudding, those commercial herds who disbud, the mode for age was 2 months, while it was 6 months at the village herds. Only 29% of herds had cows with long/sharp horns in commercial compared to 95% and 100% for semi-commercial and communal herds, respectively. Communal village farms had the most herds with poor scores for dehorning, castration, and ear tagging/notching.Animal-based stockpersonship measures: Scores were similar across the farm types, except for electrical prodders, which were only used at 5/17 commercial herds, producing a poor score at those herds (>1% cows prodded). Commercial farms had the least herds with poor welfare for fearful/agitated, hitting and tail twisting. All the farm types scored poorly (>2% threshold) for falling/lying.Resource-and management-based stockpersonship measures: Good welfare score was showed for equipment noise and dog noise and health checks, at most herds across the farm types. Commercial farms had less noisy handlers and more herds with >4 times yarding frequency per year (good welfare), and effective yard handling/flow. The communal (95% of herds) cattle yarding frequency/year (2 or fewer times) was the poorest than the other farm types.Derived threshold values for the three farm types are shown in All combined derived thresholds for the 55 beef cow herds are shown in Kaurivi  comparedThe findings indicate that to improve most of the compromised traits, development of interventions should be geared towards what works in village farm herds. Whereas for some other traits, there are herds exceeding the threshold in all herd types, and solutions must be developed for all herd type. Although the long term target is to have improved derived threshold in all measures at all the farms (a target of at least 50% of the herds in good welfare), an initial target to improve the worst 15% of herds might be the logical first step, and then revise the red thresholds, to gradually to pull the recovering herds to better. It is risky to set a target that 90% of the herds currently fail; this may deter farmers from making improvements and instead dismiss the target as unrealistic. See good feeding, with below-average rainfall (since 2013) across Namibia, the 2019 drought (the driest year in 90 years) was treated as a state of emergency by the government [With regards to vernment . The impll since 13 acrossvernment ,22. Thisvernment . In New vernment  and undevernment . For Namll since 13 acrossvernment ,26, espeappropriate environmental issues, dirtiness and diarrhoea were the only measures where the thresholds (both thresholds were 20%) fit for Namibia were reduced. In New Zealand, diarrhoea was faecal soiling, due to the high-water content in the pasture. Thus, a 5% threshold  fit for Namibia where grass grows seasonally and between years, and where diarrhoea cases may also be of pathological nature  was essential. Similarly, a 5% threshold was more fitting for dirtiness, where cattle on drier pasture are less dirty; red thresholds were only up to 3.6% in this study . For measures related to skin alterations  the derived thresholds were more than twice the imposed threshold for the combined herds, indicating high prevalence. The prevalence of swelling was attributed to faulty injections; thus, there is room for improving the skills of injections across all farm types, through awareness and training. Similarly, the high derived thresholds of hair loss at the village herds could have been prevented with preventative vaccination of the endemic lumpy skin disease in the country. Hence, an imposed threshold of 2% was also retained as fitting for these conditions. On the other hand, the much higher prevalence of abrasions on cattle across all the beef farm types is indicative of a welfare concern in the country, so an initial target of 2% would likely be unrealistic. However, abrasions are painful and cause suffering for the animal. Efforts should be directed toward identifying the causes of the abrasions and attempting to remove relevant hazards from the environment, such as faulty yard designs and yard structural materials [For most aterials . Given 5Setting welfare thresholds for any measures should reflect the significance of that trait to the welfare of animals and not necessarily according to the status quo. A good example was the mortality rate that varies in extensive beef systems with the type of production system, the location, and herd management ,29. In tEctoparasites have a significant impact on the welfare of animals  and a high incidence can markedly impair farm productivity ,32. Howepainful management procedures, unlike castration and disbudding that can be performed at an early age (<2 months) and with anaesthesia to mitigate pain [With regards to ate pain , the dowate pain , but theate pain . Farms wate pain ,41,42 coate pain . Alternaate pain ,41,42. HThe threshold (10%) considered as unacceptable welfare for herds with long/sharp horns was motivated by difficulty in handling, injuries, bruises, and condemnation at slaughter  that arestockpersonship and handling measures, it appears that three main issues, namely, (1) yarding frequency, (2) breed variations and (3) yard design/quality contributed to the high derived thresholds of fearful and falling/lying across the farm types. Fearfulness in cattle was associated with adverse handling [For handling ,49 and shandling ,51. Thushandling ), which It was important for animal-based and stockpersonship assessments to be made while cattle were handled for purposes other than the assessment itself. Thus, it might be worth describing and validating yard construction and design quality in a welfare assessment, such as to define what entails a yard, differentiate those with enough holding pens, the race and chute, as well as materials used, e.g., metal, wooden poles, tree poles and thorny bushes see  for the Finally, the study was undertaken at only 55 herds, but all herds represented the prevailing production system in the three regions. The welfare thresholds need to be validated across multiple beef herds in the country, with more assessors and consideration of the prevailing environmental conditions . It is hoped that this categorisation will also be validated by expert opinions to apply the correct decisions when an assessment is made, such as for on-farm benchmarking and certification of cattle or beef from such establishments. The current assessment results and thresholds could also be used as a basis for the welfare status of Namibia farms, such that, especially the village farms prioritise intervention areas of welfare compromise to either attain or maintain beef markets. This study can help to improve animal welfare in other countries with different status of animal welfare with individual thresholds of measures.The study provided thresholds which could be used to categorise continuous animal welfare measures into acceptable, marginal and non-acceptable welfare, hence, providing guidance for when the intervention was needed on Namibian beef farms. Commercial farms attained welfare measures thresholds based on a New Zealand protocol—which suggest opportunities to create a system and a suitable environment that promote animal welfare in Namibia. However, the same thresholds, regardless of the system, were set for the country’s beef herds, and semi-commercial and communal herds showed less acceptable welfare of beef cows. The long-term target is to have improved derived threshold in all measures at all herds, but an initial target could be geared towards improving the worst 15% of herds. Adjusted lenient thresholds to reflect drought condition were suggested for measures related to good feeding and mortality, whilst the originally imposed thresholds were still retained under “normal conditions”. This was a good example of setting the welfare threshold for “what should be and not what is”. Nevertheless, it may be an advantage that the current thresholds were validated in the possible worst-case scenario that can hit the beef industry in Namibia. Some of the imposed thresholds  were retained because of the significant importance to the welfare of animals and preventiveness of the traits. Handling measures  and abrasions thresholds were adjusted to reflect the temporary stress caused by infrequent cattle handling, and faulty yard designs and or constructions and the possible influence of Bahaman cattle behaviour during handling. There is a need to validate this categorisation and scoring of measures on multiple farms, and with more experts to classify Namibian beef farms status correctly. It is to the advantage of Namibia to have a validated, comparable animal welfare assessment protocol based on other beef exporting countries to strive for maintaining good welfare standards across the beef production chains.