Their effects and interactions were studied through the response surface methodology to optimise the bread formulation from a holistic viewpoint, which included the nutritional, technological and sensory characteristics. The optimum formulation with the highest quality was the blend made with 10, 4, and 20% of chia, quinoa, and amaranth, respectively. The results showed a significant increase in protein amount, ash, lipids, and crumb firmness compared to wheat bread. The calorie value of the control sample and the optimised formula were significantly similar, bearing in mind the high lipid amounts present in raw materials. Loaf-specific volume slightly decreased in comparison to control bread, as expected in formulations with gluten-free raw materials and a large amount of fibre. The optimised formula presented nutritionally/functionally higher indexes and similar overall acceptability to the control bread (p < 0.05).With regard to constant technological innovations in the bakery sector in order to increase bread nutritional value without affecting its technological and sensory characteristics, we applied pseudocereals/oilseeds to obtain an optimal formulation. A factorial design 3 P6), forming insoluble compounds that inhibit their bioavailability. Some strategies can increase the bioavailability of minerals when using sourdough [In the last few years, scientific studies have demonstrated that the regular intake of wholemeal or whole grain products prevents certain chronic diseases from developing, such as cardiovascular diseases, type 2 diabetes, and certain cancer types. Hence, consumer interest in such products has grown, although consumer acceptability is conditioned by their sensorial aspects despite being nutritional food with biological functionality in our organism ,2. Thankourdough ,18, or eourdough ,15, or courdough , among oConsequently, the industry in this sector has closely examined the strategy of substituting refined wheat flour for wholemeal ingredients with high added value, such as pseudocereals, legumes, and/or oilseed so that more wholemeal foods offering better technological properties are eaten  with better nutritional properties  . AccordiReplacing wheat flour with chia did not lead to loss of end product quality, and consumer acceptability was higher . However3 and by following the response surface methodology (RSM). Another aim was to evaluate the nutritional value of the optimised formulation by considering its contribution to the daily recommended intake of fatty acids (omega), minerals , and dietary fibre (soluble and insoluble) and its protein quality, and to estimate its glycaemic index (GI) values in vitro. Hence, the main objective of this study was to develop top quality bread by substituting wheat flour for an optimum mixture of wholemeal quinoa, amaranth, and chia flours to maximise its physico-chemical, technological, nutritional, and sensorial properties by a factorial design 3Chenopodium quinoa Willd), black chia , and amaranth (Amaranth caudatus) flour  were milled in a hammer type cyclone mill and at standard sieve (0.8 mm)  and stored at 14 °C. Dehydrated yeast  was used as a starter. Commercial wheat flour and whole wheat flour obtained from HARINERA LA META S.A.  was employed for the bread-making process.Quinoa , kneaded, and then left again (15 min). Dough was manually rolled, proven , and baked at 180 °C/29 min. Temperature and volume increase of dough was monitored at regular intervals during fermentation. After fermentation, dough was baked in an electric oven and cooled at room temperature for 60 min for subsequent analyses.The control bread dough formula consisted of wheat flour (300 g), compressed yeast (3% flour basis), sodium salt (1.6% flour basis), and distilled water . The 27 bread formulations with amaranth, quinoa, and/or chia, obtained by factorial design 3Proximate analyses of raw materials and breads were performed in terms of moisture, total dietary fibre (TDF), and starch according to the approved Association of Official Agricultural Chemistry 925.09, 991.43, and 996.11, respectively . Protein3/g) by measuring volume (cm3) by seed displacement  and weight (g), the width/height ratio of the central slice (cm/cm), and colour tristimulus parameters . From the colour parameters, we calculated the total colour difference (ΔE*) by the Equation (1): Samples were analysed at least in triplicate [The analysed technological parameters were as follows: loaf-specific volume . A 2 cm-thick slice of bread was compressed twice by a stainless steel 0.5 cm diameter plunger, moving 1.0 min/s to a penetration distance of 50%, with an interval of 50 s between compressions. The following parameters were evaluated: firmness, springiness, cohesiveness, and chewiness.2/cm2; wall area/total area, cm2/cm2; number of cells per cm2; and mean cell area, mm2.The digital image analysis was used to measure bread crumb structure. Digital images were taken by an EVOCAM-II Macroscope . Images were processed and analysed by the Nis Elements BR 3.2 software  and also Fiji . A single 10 × 10 mm square field of view of two central slices (10 mm thick) of both loaves was used to yield three digital images per treatment. Data were processed using the Statgraphics Plus 16.1.03 software . The chosen crumb grain features were cell area/total area, cmSamples were transesterified to convert triglycerides into fatty acid methyl esters (FAMEs), following the methodology previously described by the American Oil Chemists’ Society . The fat3  and H2O2  attack. Samples were irradiated at 800 W (15 min at 180 °C) by a Microwave Accelerated Reaction System . At the end of the digestion programme, the digest was placed in a polypropylene tube and made up to final volume with distilled water. Measurements were taken in triplicate [The total Ca, Fe, and Zn concentrations were determined in a flame absorption spectrometer at the Analysis of Soils, Plants and Water Service of the Institute of Agricultural Sciences, Madrid (Spain). Each sample (0.5 g) was placed in a Teflon perfluoroalkoxy vessel and digested with HNOiplicate .myo-inositol hexakisphosphate or phytic acid (InsP6) present in raw materials and the residual in the bread formulations after the bread-making process was measured as phosphorus released by phytase and alkaline phosphatase by a simple quantity K-PHYT method [myo-inositol ring. The total released phosphate was measured by a colorimetric technique according to the AOAC method 986.11 [The T method . This med 986.11 . SamplesFor the amino acid analysis, 10 mL of hydrolysed sample was prepared with 4 mL of 6 N HCl. Solutions were capped in a nitrogen atmosphere for 24 h. Amino acids were determined by acid hydrolysis after derivatisation with diethyl ethoxymethylenemalonate in a high-performance liquid chromatography (HPLC) Model 600E multisystem with a 484 UV–VIS detector (300 mm × 3.9 mm) and a reversed-phase column , acetonitrile in the binary gradient, detection at 280 nm, with D,L-α-aminobutyric acid as the internal standard. Solvents were injected into the column at a flow rate of 0.9 mL/min. Temperature remained at 18 °C .The in vitro gastric digestion of bread samples was carried out according to the methodology described by Sanz-Penella et al. . The drya1, a2, ..., na are the amino acid contents in the sample, and as1, as2, …, nsa are the essential amino acid requirements in the protein standard [The essential amino acid index (EAAI) was calculated according to Motta et al.  by applystandard .The nutritional index ) normalises the qualitative and quantitative variations of the test protein compared to its nutritional status. The NI was calculated by the equation of Crisan and Sands , which cTo evaluate the in vitro rate of starch hydrolysis, we followed the method described by Goni et al.  with sliThe parameters measured in the control and optimised bread formulae were appearance, texture, taste, and overall acceptability, evaluated by a panel of 50 untrained tasters who usually purchase wheat bread using a 9-point hedonic scale of global acceptance: (9) “Especially like”; (8) “Very much like”; (7) “Moderately like”; (6) “Somewhat like”; (5) “Neither like nor dislike”; (4) “Slightly dislike”; (3) “Moderately dislike”; (2) “Very much dislike”; (1) “Especially dislike” .3. The 3 studied factors were the percentage of wheat flour replacement with whole chia flour at 3 levels , whole quinoa flour at 3 levels , and amaranth flour at 3 levels . The run conditions of the factorial design in terms of the experimental conditions and coded values are shown in In order to study the effect of replacing wheat flour with nutritious ingredients on the physico-chemical, nutritional, technological, and sensory properties, we used a factorial design 3The design enabled us to approximate the experimental data The response variables were content of lipids, ash, proteins, and calorie value of bread ; piece-specific volume, crumb and crust colour, crumb firmness, and shape ratio ; and sensory evaluation, such as the product’s appearance, texture, taste, and overall consumer acceptability. Twenty-seven formulations were studied with different proportions of amaranth, quinoa, and/or chia, as shown in p < 0.05). Homogeneity of variances was tested using Levene’s tests and normally distributed on the basis of the Shapiro–Wilk test. All statistical analyses and optimisation of multiple responses were carried out with the Statgraphics Plus 16.1.03 software .One-way ANOVA and Fisher’s least significant differences (LSD) were applied to establish significant differences between samples . For the numerical optimisation, all the independent variables were left within their predetermined range, while the dependent variables were optimised by taking into account the nutritional, technological, and sensory aspects.The chemical composition of the flours herein employed is found in Lipid content in the quinoa, amaranth, and chia flours was significantly higher than the lipid content in refined wheat flours because the germ is removed during the refining process, as are outer bran layers, which is where the biggest quantity of fat and fibre is found . NonetheVariation in lipid content is generally due to inter-species differences, environmental factors, and crop-growing practices ,43,49. TTotal fibre content was significantly higher in the chia flour compared to the wholemeal quinoa, amaranth, and wheat flours . Of the Generally speaking, the wholemeal flours made with ancient crops presented a significantly higher ash concentration, which was directly related to a higher mineral content, where chia stood out, followed by quinoa and amaranth, and finally by wheat . Regardia1: 0.214, p < 0.01), followed by the quinoa  and amaranth  flours , which was not detected with quinoa  contents with beneficial health effects, as previously reported ,23. The ) flours . A signih quinoa . This efa1: 0.906; p < 0.01), followed by amaranth flour , and finally by quinoa flour . Protein and ash contents varied between 14.25–20.26% d.m. and 0.91–3.27% d.m., respectively. The nutritional value of formulations rose as the amount of chia, quinoa, and/or amaranth wholemeal flour did in formulations. This increase mostly responded to the effects of the linear coefficients in each studied ingredient a1 > a2 > a3, respectively , to 5.5 ± 0.1 mL/g (CB). This parameter was negatively affected when gluten-free ingredients were included and contained a higher proportion of fibre, which led to a poor retention of the carbon dioxide produced during fermentation [p < 0.01), quinoa , and chia  flours (10: 5.0 ± 0.1 mL/g and Ch20: 4.9 ± 0.1 mL/g). Increasing chia substitution in wheat flour lowered gluten content, and the specific volume was also reduced, but not as significantly as in the formulations with amaranth or quinoa at the same substitution level . Moreover, the effect of gluten dilution and the difference in the specific volume among the formulations with chia, amaranth, and/or quinoa flours could have been due to the high proportion of mucilage that chia seeds possess, forming hydrophilic complexes among ion groups and gluten proteins [The specific volume of all the studied formulations varied from 0.90 ± 0.06 mL/g, which corresponded to the formulation with a higher substitution percentage  flours . The higproteins . This teproteins .20Q40A40). This parameter positively correlated with chia flour , but with no interactions; that is, the more this ingredient was added, the higher crumb firmness values became (Ch10: 1.12 ± 0.06 N and Ch20: 9.0 ± 0.3 N). This result indicates that the inclusion of chia/mucilage does not always reduce/maintain crumb firmness, and it depends on the substitution level in formulations [40: 2.29 ± 0.32 N) and amaranth (A40: 1.22 ± 0.41 N) wheatmeal flours, but to a lesser extent in the latter. Given the interaction between the inclusion of quinoa and amaranth flours, a synergy was generated with increased crumb firmness , as shown in a33: 0.008; p < 0.01), which would imply that the formulations substituted only for amaranth flour would have closer values to the control bread’s firmness values (0.96 ± 0.03 N). The crumb firmness parameter varied from 0.96 ± 0.03N (CB) to 29.59 ± 0.71 N  with a reduction from 2.13 ± 0.02 cm/cm (CB) to 1.66 ± 0.03 cm/cm (Am20Ch20). Conversely, the interaction coefficient between the amaranth and quinoa flours (a23) left this parameter at 2.14 ± 0.28 cm/cm (Q20Am40); that is, loaves with the same shape, but a smaller volume. This would mean that formulations with amaranth and chia flours could be used to obtain more circular-shaped loaves. A similar tendency has been previously reported when substituting wheat for wholemeal amaranth flour [p < 0.01) [The shape ratio parameter  showed an interaction coefficient between the amaranth and chia flours (th flour . An incr < 0.01)   varied within the 3.4 ± 0.8 to 25.0 ± 0.3 and 5.6 ± 0.3 to 20.2 ± 0.3 ranges, respectively, compared to the control sample. A loaf’s crust colour is given mostly by non-enzymatic browning, by the Maillard reaction, and by caramelisation to a lesser extent. Differences in crust colour among formulations were due mainly to raw materials’ colour, followed by different chemical reactions while baking that depend on the employed flour to a greater or lesser extent (ΔE* factor for crust and crumb were significantly relevant (p > 0.05) was chia flour, which can be easily explained by the raw materials’ colour parameter values (a*) and darker (lower L*) colouring than the other flours  . However, this tendency was the opposite to that observed in flours, and the highest colour change value was found for amaranth flour (ΔE* = 14.7 ± 0.3) instead of for quinoa flour (ΔE* = 9.66 ± 0.06) when both were compared to wheat flour. The biggest colour difference for amaranth flour can be explained by the red-violet pigment of the betacyanins present in Amarantus caudatus, which confers its grain a slightly redder colour, unlike white quinoa in which this pigment was not detected [40Ch20 formulation (ΔE* = 22.4 ± 0.4), as the quadratic coefficient indicated.The difference in crust colour and crumb colour  . The effdetected ,59. Therdetected . Finally20 , which remained some way behind those obtained in control bred (CB)  (12) or quinoa and amaranth (a23).The scores in all the studied formulations for all the investigated sensorial attributes were similar to or lower than those obtained by the control bread, but were never superior. The linear coefficients of the sensorial attributes studied for the quinoa factor indicated that the more this ingredient was added, the worse the loaf, texture, flavour, and the product’s overall acceptability became . Howevery like”) . No signa1), as the proportion of chia flour increased in the formulation, bread appearance slightly improved, with a maximum in the experimental domain (Ch10A20: 7.67 ± 0.08) in accordance with the quadratic coefficient (a13), but it had no effect on texture, flavour, and acceptability. Conversely, for higher quinoa substitutions, sensorial parameters were negatively influenced , that is, a loaf made with wheat flour substituted for 33% wholemeal chia, quinoa, and amaranth flours at the 10%, 4%, and 19% flour basis proportions, respectively. To obtain the optimum formulation, we assigned conditions per response: (1) for the nutritional value, the lipid, protein, and ash contents were maximised, and the product’s calorie value was minimised; (2) for technological quality, the loaf-specific volume was maximised, while the central slice aspect/crumb firmness ratio, as well as differences in the crust colour and crumb colour parameters compared to the control sample, were minimised; (3) consumer scores given to the sensorial attributes appearance, texture, flavour, and the baked product’s overall acceptability were maximised. The formulation with all these attributes was formulation Ch10Q4A19. Its nutritional, technological, and sensorial characteristics were compared to the values that the model predicted. Hence, the obtained experimental results differed from those predicted by a percentage error between 0.52% and 6.7%, except for the two sensorial attributes , and optimised bread can be found in Regarding crumb texture, the optimised formula generally had a similar profile to that of the wholemeal wheat bread. Although elasticity and chewiness presented no significant differences among formulations, a lowering trend was observed in both parameters compared to the control bread. The crumb firmness parameter differed significantly among formulations, where the optimised bread formulation obtained an intermediate value between the control and wholemeal wheat breads . 2 retention capacity during bread-making fermentation and oven impulse, which leads to more compact crumbs and a smaller specific volume in the optimised product versus the control  of the optimised and wholemeal wheat breads, in comparison with the control bread, exceeded a value of 5. This indicates that consumers would notice colour differences at a glance , but significantly differed from those of the control bread. The optimised bread crust was a darker colour, conferred mostly by the chia flour included in the formulation  to 8.7 ± 0.8 (optimised formulation) between “Very much like” and “Especially like”.P6), affects the bioavailability of the minerals in the human intestine, which is indicated by the inhibition threshold values given for the InsP6/mineral molar ratio [The nutritional quality of wholemeal flours with these ancient seeds is generally higher than those of cereals, not only for their high mineral content but also for their content of dietary fibre, unsaturated fatty acids, and proteins of high biological value . Nonethear ratio . Phytatear ratio . Nonethear ratio . Thus, iP6/Ca ratio below 0.24  and linoleic acid (LA), respectively . For adup < 0.05) higher for the optimised bread than the control bread. This same tendency has been previously reported when adding quinoa flour to wheat bread [The IVPD of the optimised bread was lower than in the control bread , which cat bread .Generally, pseudocereals such as quinoa and amaranth, and oilseeds such as chia, are an excellent source of dietary fibre ,42,76,77The physiological action that occurs after eating food containing dietary fibre is more effective when the soluble/insoluble fibre ratio remains at 1:2 . This raThe optimised bread’s starch content was significantly lower than in the control bread . This co10Q4A19: chia 10%, quinoa 4%, amaranth 19%) as far as its nutritional characteristics were concerned . It is a product with acceptable and better quality parameters than wholemeal wheat products , and with excellent sensorial characteristics that are similar to products made with refined wheat flour. This optimised product can contribute to providing products with a beneficial impact on consumer health with high acceptability.The response surface model herein employed was suitable for optimising bread formulations from the nutritional, technological, and sensorial points of view. Combining healthy ingredients such as chia, quinoa, and amaranth helped to develop a product with optimum characteristics (Ch


Potato pulp is a nuisance waste from the production of potato starch, containing large amounts of dietary fiber; therefore, addition of the pulp to food may have a positive effect on the nutritional value of food products. To increase the amount of dietary fiber, shortcrust pastry cookies were baked by replacing some of the flour (20–100%) with an equivalent amount of dried potato pulp. In all the tested variants, correct confectionery products were obtained. The color of finished product (measured using Konica-Minolta CM-5 spectrophotometer) and mechanical properties of raw dough and baked cookies  were determined. Furthermore, the samples were subjected to sensory consumer assessment . Colorimetric tests of the cookies showed a slight shift in their color from yellow toward green but at the same time lowering its saturation. Strength tests show that only the replacement of more than 40% of flour by the dried pulp resulted in a significant increase in the hardness of baked products. Regarding the tests with consumers, cookies containing up to 40% potato pulp were acceptable. Such a large addition of potato pulp to shortcrust pastry cookies creates new opportunities for this waste management.  The usual proportions of the major ingredients are: three parts of flour, two parts of fat, and one part of sugar, but they can deviate slightly to achieve the preferred final effect. For the production of shortcrust pastry, usually refined flour is used, although it is devoid of many important nutrients . Thanks According to the definition proposed by the European Food Safety Authority, dietary fiber consists of carbohydrate polymers with three or more monomeric units, which are neither digested nor absorbed in the human small intestine . In manyThis study aimed to assess the quality of shortcrust pastry cookies prepared using a basic recipe in which several percentages of wheat flour was replaced with dried potato pulp . The co22.1Shortcrust pastry cookies were prepared according to a basic recipe (without the addition of dried potato pulp), and next, the recipe was modified so that a part or all of the wheat flour was replaced by an equivalent amount of dried potato pulp .In the experiment, commercially available products were used: refined coarse-ground wheat flour type 500 , margarine Kasia (Unilever de Argentina S.A.), granulated white sugar (Krajowa Spółka Cukrowa S.A.), fresh eggs , 18% cream , as well as baking powder and vanillin sugar (Winiary S.A.). Dewatered potato pulp, obtained from Przedsiębiorstwo Przemysłu Ziemniaczanego S.A. in Niechlów, was dried to 91.03% dry matter content. The dried pulp had a starch content of 34.2%  and total dietary fiber content of 52.7%, soluble dietary fiber 11.3% and insoluble dietary fiber 41.4% .The cookies were baked in three replicates in the laboratory in a combi steamer  under controlled conditions: at 210°C in dry air (relative humidity set to 0%). The cookies were gradually cooled down to room temperature, and then, they were placed in plastic bags to prevent drying.2.2L*a*b* color space defined by the International Commission on Illumination in 1976. It expresses color as three values: L* for the lightness from black to white, a* from green to red, and b* from blue to yellow. Furthermore, the hue angle (H) and chroma (C) of samples, which reflect color perception by the human eye, were calculated. The calculations were made using the formulas given in Konica-Minolta’s user manual [Color was measured using Konica-Minolta CM-5 spectrophotometer. Results were expressed in the CIE r manual . The stu2.3Fmax (N) and energy E (mJ) needed to cut the cookie [The raw dough cut into cookies (circles 50 mm in diameter and 5 mm thick) as well as the baked cookies after cooling down were subjected to the tensile test using INSTRON 5544 Tensile Tester with software version 5.11. The unbaked and baked cookies were cut with a single shear blade (head movement rate 100 mm/min). A head with a measurement range of up to 100 N was used for the measurements. Based on the measurements, the maximum force e cookie ,20 were 2.4The baked cookies were also subjected to organoleptic consumer assessment, taking into account their shape, cohesion, and structure, as well as taste and smell of the cookies. All the attributes were evaluated on a 7-point hedonic scale, from 1 (“definitely dislike”) to 7 (“definitely like”), by a group of 65 consumers . The evaluations were conducted in a sensory laboratory.2.5α = 0.05.The results of instrumental tests were subjected to the statistical analysis using STATISTICA 13 software: one-way analysis of variance and distinguishing of homogeneous groups by the Duncan test at the significance level of 33.1H) ranged from about 73° in the control cookies to about 77° in the cookies with the maximum amount of dried potato pulp  of the cookies depending on the content of potato pulp. Increasing the content of dried potato pulp caused a significant decrease in color saturation, from 36 for control cookies to 23 in those where flour was completely replaced by the dried potato pulp. Gómez et al. [C and a decrease in hue angle in cookies with increasing amounts of sweet potato flour.Much greater changes were observed in values of color saturation . The presented results indicate that the increased proportion of potato pulp caused the formation of a harder surface layer on the dough, and the cohesion of the inner part of the dough was most similar to the control in the case of dough with 40% and 100% of flour replaced by the dried potato pulp.The maximum force energy E  needed tResults of strength tests of baked cookies were slightly different . MaximumA similar relationship was observed for measurements of the energy needed to cut the baked cookies: it was the lowest for control cookies (about 12 mJ), but cookies with 20–50% of flour replaced by potato pulp formed a statistically homogeneous group, with energy values varying from 24 to 41 mJ. However, complete replacement of flour with the pulp resulted in an 8-fold increase in the energy needed to cut the cookie. The strength and the energy needed to break or bite cookies are some of the major parameters affecting the acceptance of a food product by consumers .3.3To assess how the consumers perceive the texture of the tested cookies, the products were subjected to sensory consumer assessment on a 7-point hedonic scale .In the sensory tests, control cookies were rated the highest. Increasing proportions of potato pulp resulted in lower scores for cookie structure, taste and smell, and cohesion (consistency). The structure of cookies with potato pulp was rated lower because it was more compact and not typical of shortcrust pastry according to consumers. This problem might be partly solved by longer baking of the cookies with higher potato pulp content. The sensory properties can be affected by the addition of dietary fiber that is present in potato pulp. Many authors have noticed that cookies with a high fiber content are not well accepted ,25,26,27Coefficients of correlation of consumers rating with instrumental measurements of cookie structure  were theOur findings are consistent with earlier studies. Baumgartner et al.  studied It can be concluded that the production of shortcrust pastry cookies with up to 40% of flour replaced by dried potato pulp can be recommended. Mechanical properties of such cookies and their acceptance by consumers are similar to those of control cookies. The potato pulp used in this study contains 52.7% total dietary fiber. It means that the use of the dried potato pulp can markedly increase the dietary fiber content of such products, from about 6% when 20% of flour replaced by dried potato pulp, up to 30% when wheat flour is completely replaced by the potato pulp. Menis-Henrique et al. and Schmid et al. stated that heat treatment does not significantly reduce the amount of fiber fraction ,34, but 4The results of this study proved that potato pulp may be an excellent source of dietary fiber that can be used as a substitute of flour in cookies. Moreover, noteworthy is the fact that in this way, potato pulp is used as a source of fiber, and also this burdensome by-product of the starch industry can be utilized. The addition of potato pulp in an amount of up to 40% does not cause any remarkable change in their crispness and hardness, but shifts the color of the cookies from yellow towards green and causes a significant decline in their color saturation. It is a success that consumers rated the cookies with the addition of pulp in an amount up to 40% as acceptable, which means that a large amount of this waste can be utilized in the confectionery industry.


The endoplasmic reticulum (ER) is the major folding compartment for secreted and membrane proteins and is the site of a specific chaperone system, the calnexin cycle, for folding N‐glycosylated proteins. Recent structures of components of the calnexin cycle have deepened our understanding of quality control mechanisms and protein folding pathways in the ER. In the calnexin cycle, proteins carrying monoglucosylated glycans bind to the lectin chaperones calnexin and calreticulin, which recruit a variety of function‐specific chaperones to mediate protein disulfide formation, proline isomerization, and general protein folding. Upon trimming by glucosidase II, the glycan without an inner glucose residue is no longer able to bind to the lectin chaperones. For proteins that have not yet folded properly, the enzyme UDP‐glucose:glycoprotein glucosyltransferase (UGGT) acts as a checkpoint by adding a glucose back to the N‐glycan. This allows the misfolded proteins to re‐associate with calnexin and calreticulin for additional rounds of chaperone‐mediated refolding and prevents them from exiting the ERs. Here, we review progress in structural studies of the calnexin cycle, which reveal common features of how lectin chaperones recruit function‐specific chaperones and how UGGT recognizes misfolded proteins. The endoplasmic reticulum is the major folding compartment for secreted and membrane proteins and is the site of a specific chaperone system, the calnexin cycle, for folding N‐glycosylated proteins. Here, we review progress in structural studies of the calnexin cycle, which reveal common features of how lectin chaperones recruit function‐specific chaperones and how UGGT recognizes misfolded proteins. CNXcalnexinCRTcalreticulinUGGTUDP‐glucose:glycoprotein glucosyltransferaseCypBcyclophilin BPDIprotein disulfide isomeraseERendoplasmic reticulumThe endoplasmic reticulum (ER) contains two major folding pathways for protein substrates . One is 3Man9GlcNAc2 glycan. Calnexin  is one of four lectin chaperones in the ER. Calnexin and its soluble homolog, calreticulin, combine a lectin‐like glycan‐binding domain with a flexible arm, the P‐domain that recruits other chaperones. The other major components of the pathway are UDP‐glucose:glycoprotein glucosyltransferase (UGGT), the protein disulfide isomerase ERp57, and the ER glucosidases Glu I and Glu II [The pathway dedicated for N‐glycosylated proteins is named after calnexin, the first protein discovered in the pathway . Upon end Glu II , 7, 8  and ERp29, carry out the isomerization of peptide bonds and a general chaperone function, respectively.There are intricate relationships between calnexin cycle and antigen presentation pathways. Calnexin cycle proteins calreticulin and ERp57 do not only chaperone MHC class I heavy chains, but are also a part of the peptide‐loading complex (PLC), which also includes transporter associated with antigen processing (TAP), β2‐microglobulin, and tapasin. PLC is required for loading of antigenic peptides onto MHC class I heavy chains for presentation to the immune system . UGGT plThis review provides an overview of proteins involved in calnexin cycle with an emphasis on recent structural insights Table . These iCalnexin and calreticulin are the most abundant representatives of a small family of lectin chaperones residing in the ER. The other members are the tissue‐specific homologs calmegin and calreticulin 3. Proteins in this family share common structure consisting of a glycan‐binding lectin domain and a very unusual arm‐like structure, termed the P‐domain due to the abundance of proline residues Fig. . The lecStructures of the lectin domains of calnexin and calreticulin Table  show a j1Man3 tetrasaccharide, the Glc(3)‐Man(D1)‐Man(C)‐Man(4) branch of the monoglucosylated Glc1Man9GlcNAc2 glycan [The details of glycan binding were revealed by the high‐resolution structure of calreticulin in complex with Glc2 glycan   and Man(C) mainly use their O4–O6 edges for interactions with the lectin domain. In particular, O4 of Man(D1) engages in three direct hydrogen bonds with Tyr109 and both the side chain and backbone carbonyl of Asp317. Asp317 is required for binding because it also makes direct hydrogen bonds with O4 and O6 of Man(C) Fig. . The affeticulin  suggestiThe glycan‐binding surface is essentially identical in calnexin and calreticulin. The residues that are involved in carbohydrate binding are highly conserved and adopt very similar conformations in both proteins. In the cell, calnexin and calreticulin display overlapping but distinct patterns of interaction with substrate glycoproteins , 35, 36.The lectin domains of both calreticulin and calnexin contain a solvent‐exposed disulfide bridge on the edge of lectin site. Previous studies showed that treatment with reducing agents dithiothreitol and tris(2‐carboxyethyl)phosphine (TCEP) abrogates carbohydrate binding by calreticulin , 39. The2+‐binding sites [2+ storage in the ER [2+ ions [The calreticulin lectin domain structures also defined the location of a high‐affinity calcium‐binding site , 41. Theng sites  and is rn the ER . Likewisn the ER . More ren the ER  and its [2+ ions , 46.Sequence identify among the lectin chaperones is highest in the P‐domains. The domains are hairpin‐like structures composed of multiple type I and type II motif repeats , 47. TheIn the folded P‐domain structure, the type I motifs interact with type II in a head‐to‐tail fashion forming four modules each containing a small hydrophobic core of two tryptophans and a lysine Fig. . The haiCooperative interactions of chaperones are crucial for efficient protein folding in the ER. Calnexin and calreticulin often serve as a scaffold bringing together N‐glycosylated proteins with the ER‐resident chaperones. ERp57, a protein disulfide isomerase, was one of these proteins originally identified and established as a part of calnexin cycle pathway , 49. MorERp57  possesses oxidoreductase activity , 51, 52 a, b, b′, and a′. The N‐ and C‐terminal a and a′ domains contain CGHC catalytic motifs, while the b and b' domains have lost the catalytic cysteines  and lowest in the b and b′ domains (~ 20%).Structurally, ERp57 consists of four thioredoxin‐like (TRXL) domains termed nes Fig. . ERp57 iTRXL = βαβαβαββα (Pfam Thioredoxin_6 family PF13848) is a very stable and common domain consisting of a central five‐stranded β‐sheet covered by two α‐helices on each side. It is a derivative of classical thioredoxin fold TRX = βαβαββα (Pfam Thioredoxin family PF00085). Approximately twenty proteins in the large family of protein disulfide isomerases contain at least one thioredoxin‐like domain with a βαβαβαββα sequence of secondary elements. Some are catalytically (redox) active domains containing CxxC motif at the N terminus of helix 2, while noncatalytic domains do not contain catalytic cysteines and either play structural role or are involved in protein interactions. The bacterial thiol disulfide oxidoreductase, DsbA, that functions analogously to PDIs, displays a modified version of thioredoxin fold, a DsbA‐like thioredoxin fold DSBA = βαβ–αααα–αββα (Pfam DSBA family PF01323) with an extra four‐helical subdomain that caps one side of the domain  precludes us from identifying individual contacts responsible for the interaction. Moreover, the exact placement of the P‐domain relatively to ERp57 needs to be adjusted. This conclusion follows from steric clashes for a number of residues upon restoring their side chains missing in the model, for instance Lys274 of ERp57. Secondly, the structure does not explain the role of critical residues , which are required for the binding. Therefore, a high‐resolution structure of the complex would be very informative in pinpointing structural determinants of the binding.Cyclophilin B (CypB) is a peptidyl‐prolyl cis‐trans isomerase (PPIase) found in the ER , 58 and The crystal structure of CypB in complex with the P‐domain from calmegin provided a mechanism for recruitment of PPIase activity to misfolded N‐glycoproteins and suggested that CypB functions as part of the calnexin cycle . The strm as estimated from NMR studies [Kd of 6 µm) [m) [The single most important residues from each protein are Lys97 of CypB and Asp338 of the P‐domain , as mutations of each these residues abolish the interaction . Lys97 o studies . This isof 6 µm)  and to c µm) [m) .in vivo. CypB and calnexin/calreticulin co‐localize in the ER and are associated with multichaperone ER complexes [H antibody heavy chain. The heavy‐chain CH1 domain possesses three cis‐prolines in its native state, and its folding is markedly accelerated by CypB [It is very likely that calnexin/calreticulin and CypB interact omplexes , 63. Theomplexes . The ass by CypB . Future Calnexin and calreticulin have been both found to interact with ERp29 , 66, 67,m measured using NMR and surface plasmon resonance [The NMR studies identified P‐domains of calnexin/calreticulin and the D‐domain of ERp29 as the domains responsible for the ERp29–calnexin/calreticulin interactions . In factesonance , 16.The ERp29 D‐domain shows an unusual fold where two C‐terminal antiparallel helices are partially solvent‐exposed by extending out from a three‐helix bundle. These solvent‐exposed helices form the binding site for the P‐domain Fig. . In partThe D347K mutation in the calnexin P‐domain results in no binding to ERp29. The same mutation was previously shown to abrogate calnexin binding to ERp57 and CypB , 77. TheThe P‐domains from calnexin, calreticulin, and calmegin are all able to specifically bind the D‐domain of ERp29. The D‐domain of ERp29 is unique in the human genome, but conserved in ERp29 homologs from other species with sequence conservation highest in the P‐domain‐binding residues. Therefore, it is likely that calreticulin/calnexin binding is a conserved ERp29 function across species.Drosophila ortholog of ERp29, functions in embryo development through processing of a Golgi sulfotransferase, Pipe [in vivo, the D‐domain can be swapped, suggesting a functional conservation of that domain [Windbeutel, the se, Pipe , 78. Twose, Pipe , 79. These, Pipe . In partt domain . In anott domain .The dimerization of ERp29 allows for the assembly of larger chaperone complex with two lectin chaperones bound to one ERp29 dimer. While the functional implication of that is currently unclear, this may lead to a tighter binding of multiglycosylated protein substrates. ERp29 dimerization may also play a role in glycosylation‐independent chaperone function by promoting direct binding of nonglycosylated substrates to calreticulin and calnexin.Comparison of the crystal structures of P‐domains from calnexin luminal domain , calretiA number of residues are highly conserved in the P‐domains. Some of these such as tryptophan and lysine play a structural role, while others are involved in protein binding. Among the latter, a methionine, aspartic acid, and glycine residue at the tip of the P‐domain (the MDG‐binding motif) are absolutely conserved in all family members and are crucial for the ERp57, CypB, and ERp29 binding. The helical turn projects the key binding residues, methionine and following aspartic acid , to their binding partners. The aspartate residue makes key salt bridges with its counterparts, Lys97 of CypB and Arg223 of ERp29. It is tempting to speculate that it interacts with Arg282 of ERp57, but a higher resolution structure is needed to confirm this. Significantly, the calnexin P‐domain D347K mutation abolishes binding to ERp57 , CypB, aRemarkably, the binding sites for the P‐domain are formed from strikingly different structural scaffolds Fig. . The ERpm, suggesting no strong preference to any of the partners. While one lectin chaperone can only bind one other associated chaperone, the dynamic nature of interaction likely prevents folding bottlenecks or dead ends. Thus, calreticulin and calnexin appear to act as plurivalent adaptors that recruit other chaperones to assist in different aspects of protein folding, such as disulfide bond formation, proline isomerization, or general chaperone activity. The affinity of the calnexin/calreticulin for monoglucosylated glycans is roughly an order of magnitude higher (0.7 µm) [The interactions of calnexin/calreticulin with ERp57, CypB, and ERp29 form a highly interconnected cluster of protein–protein interactions within the ER. The binding affinities to all three proteins are in the same range of 5–15 µThe sequence conservation of the P‐domains is in sharp contrast with the diversity of binding sites on ERp57, CypB, and ERp29. This suggests that these chaperones became specialized for glycoprotein folding through convergent evolution of their P‐domain‐binding sites. The remarkable versatility of the tip of the P‐domain to interact with different structural scaffolds hints at the existence of other protein partners yet to be discovered.in vitro [Do calnexin and calreticulin work only as a scaffold by bringing together protein substrates and other chaperones, or do they provide some chaperoning themselves? Because the substrate would be likely positioned between lectin site and another chaperone bound to the tip of P‐domain, it is reasonable to expect some contacts between the protein substrate and the interior side of P‐domain. Indeed, the P‐domain truncation mutants of calreticulin display decreased ability to suppress protein aggregation in vitro , 83.Kd [in vitro [Another interesting aspect is the ability of calreticulin and calnexin to bind directly to nonglycosylated hydrophobic peptides with micromolar Kd , 84, 85 in vitro , 87, 88.in vitro , 83. Conin vitro , 41, butin vitro .in vitro binding of nonglycosylated substrates [in vivo.More recently, a surface distant from lectin site was identified as responsible for bstrates . In partbstrates . The conExperiments using T7 phage display system revealed interactions between calreticulin and protein disulfide isomerase‐related (PDIR) protein . The intPDIR consists of four thioredoxin‐like domains, but has a unique architecture in PDI family, as it contains an N‐terminal noncatalytic domain followed by three catalytic domains. Crystal structure of the noncatalytic domain identified a conserved positively charged surface, a prime candidate for interacting with the negatively charged P‐domain . Indeed,2+ ions [Early studies reported interactions between calreticulin and PDI, though the binding was not observed in the presence of Ca2+ ions . This wo2+ ions .There are intriguing similarities in glycoproteins processing by calnexin cycle and ER‐associated degradation (ERAD) machineries. They both heavily rely on the state of the glycan, which is recognized and captured by ER lectins, CNX/CRT and UGGT in calnexin cycle and the ER degradation‐enhancing α‐mannosidase‐like proteins (EDEMs) in ERAD. Both systems also display functional and specific interactions with a number of PDIs, where the latter often responsible for reduction and/or reshuffling disulfides in glycoprotein clients. Those include CNX/CRT‐ERp57 and UGGT‐Sep15 pairings in calnexin cycle, while EDEM1‐ERdj5 and EDEM2‐TXNDC11 display reminiscent functional cooperativity in ERAD , 97, 98.Does glucosidase II interact with calnexin/calreticulin, or it gets recruited via another member of calnexin cycle? How do they compete for monoglucosylated proteins? This has important implications on the rate with which glycoproteins escape from calnexin cycle. One study showed the preference of glucosidase II for folded versus misfolded monoglucosylated substrates when in the presence of calreticulin but not on its own , but theN‐glycoproteins that are difficult to fold undergo multiple rounds of folding with assistance of ER lectin chaperones. By reglucosylating misfolded proteins, UGGT plays the role of a checkpoint allowing misfolded proteins to rebind to the lectin chaperones and preventing their exit from the ER. UGGT expression is elevated upon ER stress and is a part of unfolded protein response . UGGT alMammalian UGGTs are approximately 1500‐residue proteins, where the N‐terminal ~ 1200 residues are responsible for sensing misfolded substrates and the C‐terminal ~ 300 residues harbor a glucosyltransferase 24 family (GT24) A‐type catalytic domain Fig. . For a lThe structure consists of four N‐terminal αβ‐sandwich domains, followed by a saddle‐shaped pair of β‐sandwich domains that seat the catalytic domain Fig. . Overallβαβ−αααα−αββα), the helical subdomain arises from residues inserted into the middle of the thioredoxin fold, while in the first UGGT domain (αααα−βαβ–αββα), the helical elements precede the thioredoxin fold. Even more striking, the TRXL4 and β‐sandwich domains are folded with discontinuous regions of the primary sequence  itself to other members of PDI family , 108. MoThere is still a lack of clarity in the mode of catalytic domain involvement. Early theories proposed a great deal of mobility between the N‐terminal part and catalytic domain, while the full‐length UGGT structures invariably showed the catalytic domain firmly entrenched in the β‐sandwich surface . At the d of 20 nM) to ER oxidoreductase Sep15 [UGGT1 binds with high affinity . The Sep15 redox domain contains selenocysteine (U), which is separated from cysteine by a single residue in Sep15 catalytic motif (CxU). This is a deviation from typical oxidoreductases, including PDIs, which possess CxxC catalytic motif. The NMR structure of this redox domain revealed significant differences from thioredoxin . In partDrosophila ortholog possesses a cysteine as opposed to selenocysteine. The redox potential of Drosophila Sep15 is −225 mV [Why does Sep15 contain selenocysteine in place of one of the cysteines in its active site? Selenocysteine likely modifies the redox potential affecting the potency of its oxidoreductase activity; however, it does not appear to be a requirement for Sep15 function as the  −225 mV , which l −225 mV  and thio −225 mV . This suSep15 possesses a distinct cysteine‐rich N‐terminal domain, which is responsible for binding to UGGT1 . Six invWhat is the role of Sep15 in the function of UGGT1 and the calnexin/calreticulin cycle in general? Previous studies showed enhancement of UGGT1 and UGGT2 activities upon binding to Sep15 , 103. ReRecent years have seen new exciting developments in structural understanding of folding pathways of glycoproteins in the ER and brought new potential members of the calnexin cycle into the light. Despite this progress, many questions still remain unanswered. The molecular details of UGGT action are still not fully understood. What is the basis of Sep15 involvement in UGGT function? Future studies of UGGT complexes with Sep15 and substrates would clarify many of these aspects.On the calnexin/calreticulin side, recent insights provided an exciting view of structural organization of these proteins and how they recruit their helpers assisting in folding N‐glycosylated substrates. Calnexin and calreticulin have been traditionally viewed as chaperones, but in light of recent studies they rather appear to function as scaffolds. We now have a much better understanding of their scaffolding function, where the P‐domain works as a long flexible arm that recruits a folding assistant and brings it to a glycosylated substrate captured via the lectin domain. It also became apparent that this process is much more complex than originally thought and involves multiple folding assistants besides ERp57. Because glycan‐based interactions are approximately 10‐fold stronger resulting in a longer lifetime of the bound state, calnexin/calreticulin likely shuffles through multiple chaperones assisting with different aspects of protein folding of any single substrate. Based on recent developments, it would not be surprising if additional chaperone partners of calnexin/calreticulin will be discovered in future years. It will also be interesting to see whether there are other ways in which calnexin/calreticulin can bind chaperones (such as PDIR) and whether this could lead to the formation of multichaperone complexes to assist with folding of specific substrates.An important unanswered question in the field is the interplay of calnexin/calreticulin and UGGT with glucosidase II. Are activities of lectin chaperones, glucosidase II, and UGGT coordinated in any way? How does glucosidase II get recruited into calnexin cycle and how it competes with calnexin/calreticulin for monoglucosylated substrates? There is still much to learn about calnexin cycle pathway, and the future years will undoubtedly bring us more exciting discoveries.The authors declare no conflict of interest.GK and KG wrote the review.


Niemann-Pick disease type C (NPC) is an ultra-rare, progressive, genetic disease leading to impaired lysosomal function and neurodegeneration causing serious morbidity and shortened life expectancy.The Niemann-Pick type C Clinical Severity Scale (NPCCSS) is a 17 domain, disease-specific, clinician-reported outcome measure of disease severity and progression. An abbreviated 5-domain NPCCSS scale has been developed  and the scale reliability has been established. Additional psychometric properties and meaningful change of the scale need, however, to be assessed.Mixed method studies were conducted to ascertain which NPCCSS domains were most important, as well as to explore meaningful change: 1) surveys in caregivers/patients (n = 49) and 2) interviews with clinicians (n = 5) as well as caregivers/patients (n = 28). Clinical trial data (n = 43) assessed construct validity and meaningful change through an anchor-based approach.Domains identified as most important by clinicians, caregivers, and patients  were Ambulation, Swallow, Cognition, Speech, and Fine Motor Skills, indicating content validity of the 5-domain NPCCSS.2 = 0.97. Convergent validity was demonstrated against the 9 Hole Peg Test, r2 = 0.65 (n = 31 patients), and the Scale for Assessment and Rating of Ataxia (SARA), r2 = 0.86 (n = 49 patients). Any change was seen as meaningful by patients/caregivers across domains. Meaningful change using trial data and interviews with NPC experts (n = 5) and patients/caregivers (n = 28) suggested that a 1-category change on a domain is equivalent to 1-point change or greater in the 5-domain NPCCSS total score.Criterion validity was shown with the 5-domain NPCCSS being highly correlated with the 17-item NPCCSS total score (excluding hearing domains), rQualitative and quantitative data support content and construct validity of the 5-domain NPCCSS score as a valid endpoint in NPC trials. A 1-category change on any domain is equivalent to 1-point change or greater in the 5 domain NPCCSS total score, representing a clinically meaningful transition and reflecting loss of complex function and increased disability.Trial registration NCT02612129. Registered 23 November 2015, https://clinicaltrials.gov/ct2/show/NCT02612129 NPC1 (≈95% of cases) or NPC2 (≈5% of cases) genes [Niemann-Pick disease type C (NPC) is a rare, progressive, neurodegenerative disease arising from autosomal recessive mutations in the s) genes , 4. Mutas) genes . There aThe NPC Clinical Severity Scale (NPCCSS) is a disease-specific, clinician-reported outcome (ClinRO) measure that was developed to characterize and quantify disease progression.  The origThe NPCCSS has shown good inter-rater reliability , 10, conThe NPCCSS allows a comprehensive assessment of the symptom burden experienced by NPC patients. However, in clinical research, a short form version would be beneficial, allowing clinicians to focus on the core symptoms of the condition, evaluate outcome across those specific symptoms and reduce variability, as well as increase feasibility in studies. Additionally, a shortened version would be ideal for use in daily practice research as it is less time consuming for clinicians to complete. Therefore a 5-domain version has been developed to cover Cognition, Swallow, Fine Motor Skills, Speech, and Ambulation Table . The domTo further investigate the 5-domain version of the NPCCSS Table , 2 studiIn this study (OR-SRV-NPC-01), NPC patient advocacy groups in the US and UK advertised the study to their members, with 49 completed surveys (n = 37 US and n = 12 UK) included in the final analyses: n = 43 family caregivers (reporting for n = 22 paediatric patients and n = 21 adult patients) and n = 6 adult patients reporting for themselves. Twenty-eight of these survey respondents also took part in a follow-up telephone interview . Overall, a broad range of ages and levels of NPC disease severity were included. Patient participants represented a pediatric population and an adult population: range 13 months to 65 years; mean (SD) current age 8.1 (5.5) years (median 8.0 years) and 33.3 (14.2) years (median 30.0 years), respectively. The disease severity ranged from ≤ 4 (n = 7); 5–19 (n = 36); and ≥ 20 (n = 6) on the 5-domain NPCCSS. The patients in the survey and interviews were similar in age, age at symptom onset, and disease severity (Table Adult participants experienced their first NPC symptom later in life as compared to the younger age groups Table .Table 3AFor the second study (OR-SRV-NPC-03), 5 NPC clinicians (n = 2 based in US and n = 3 in Europe) participated in an interview. All the clinicians met the eligibility criteria of 5 years plus experience with clinical evaluation and management/treatment of NPC patients and clinical expertise, thus were considered to be experts in NPC.When ranking the top 5 most important domains, Ambulation was most frequently chosen. Other domains frequently chosen  were Swallow, Speech, Memory, Cognition, and Fine Motor Skills. Table The five domains of the 5-domain NPCCSS were included in the 6 most frequently selected domains. When comparing across current age groups ; the impact hand tremors or difficulty coordinating their hands had on their everyday activities like eating, writing, and caring for themselves (Fine Motor Skills); the importance communication has in everyday life, especially in expressing their needs or wants (Speech); the possible risks of choking, aspiration, pneumonia, and even death ; and the impact on patients’ education or work (Cognition and Memory). Although they are captured distinctly in the measure, some participants expressed in interviews that they saw cognition and memory as highly inter-related and, therefore, said that it was difficult to rank one as more important than the other.All 5 NPC expert clinicians were familiar with the original 17 domain NPCCSS scale and agreed that the 5-domain NPCCSS captures the core symptoms of NPC, and probably those that are most important to patients and their families as they affect the patients’ quality of life and functioning the most. Two clinicians highlighted that the symptoms captured in the 5-domain version are present in most patients with NPC, whereas the other NPC symptoms captured in the full 17 domain measure are not.Given the progressive nature of NPC, any level of change in any symptom was often seen by caregivers and patients as meaningful because it was an indication to them that their NPC was further progressing and worsening. In relation to what degree of change  from their current level of severity would be meaningful to them in each domain of the 5-domain NPCCSS, the vast majority 61.9%–88.5%) of participants indicated that a 1-category worsening in each of the domains would be a meaningful deterioration. The feedback per domain is shown in Table 8.5% of pAdditionally, nearly all participants expressed during interviews that slowing the progression of any one of the domains in the 5-domain NPCCSS would be meaningful. The 2 most important domains to slow the progression were considered to be Ambulation and Cognition.There were mixed responses to the question of what specific change in score on the 5-domain NPCCSS is considered meaningful, as clinicians felt that this depended upon patient age, NPC severity, and the domain changing. However, most of the clinicians considered a 1-category change to be meaningful, corresponding to a 1- or 2-point change within a domain of the 5-domain NPCCSS. There were individual differences among clinicians in which domains would be most meaningful to change; however, overall, the clinicians felt that change in any of the domains and delaying progression would be important.In a placebo-controlled clinical trial (CT-ORZY-NPC-002) based on the CGI-I (between baseline and end of treatment), patients who were reported to have “no change” in their condition (n = 18) had a mean (SD) change in the 5-domain NPCCSS total score of 0.83 (2.176) with a median change of 0. The upper 95% CI of those who reported no change was 1.915. The collapsed “worsening” category on the CGI (n = 13) had a mean (SD) change in the 5-domain NPCCSS total score of 2.69 (3.225) with a median change of 2 points (Table 2 = 0.65 (n = 31 patients), and between the 5-domain NPCCSS total score versus Scale for Assessment and Rating of Ataxia (SARA), [2= 0.86 (n = 49 patients).Convergent validity was demonstrated at baseline both between Fine motor skills versus the 9 Hole Peg Test  and betw (SARA),  r2= 0.862  = 0.97 (n = 49 patients), demonstrating a high level of criterion validity.The 5-domain NPCCSS also correlated highly with the total score of the 17-item NPCCSS (excluding hearing domain), rIn the clinical trial  the 15 pThe original 17 domain NPCCSS was developed to characterize and quantify disease progression across all symptoms experienced by NPC patients. However, a smaller number of items in a measure can reduce variability and provide a more focused subset of symptoms. Focusing on the most relevant domains as assessed by patients, caregivers, and clinicians , will help inform treatment effects where there are such effects. An abbreviated tool is also helpful in daily clinical practice. From the mixed methods approach used in the current research, involving both patients and caregivers, and the qualitative clinician interviews, the 5-domain NPCCSS was found to be content valid and suitable for use across ages, age of onset, and severity subgroups. This is supported by prior research from Iturriaga et al.  and CortDifficulties with ambulation, swallowing, and speech were highly salient NPC symptoms to the participants interviewed. After these 3 symptoms, Fine Motor Skills, Cognition, and Memory were those most often discussed by participants as being important because of the impacts on patients and caregivers. Cognition and Memory were seen by many participants as being highly interrelated if not the same thing, thus use of one or other in a grouping of the most important symptoms appears to be acceptable.The 5-domain NPCCSS was shown to have construct validity and be sensitive to change using the data from a recently conducted clinical trial , and hasInterpretation of any outcome measure score is essential to allow understanding of whether observed changes in a condition are meaningful or not. There has been no prior research looking at this specific element of the NPCCSS measure. Data gathered in both the qualitative studies and the clinical trial provided evidence that a 1-category change in any domain, corresponding to a 1–2-point change in total score of the 5-domain NPCCSS scale, is meaningful. This indicates that worsening by 1 category is a reflection of loss of complex function and increased disability.Among caregivers and patients across all levels of severity in each domain, all but one participant reported a 1- or 2-category worsening as being meaningful, with most stating a 1-category worsening is meaningful. Clinicians agreed that the measure captures clinically important and relevant NPC symptoms and domain changes, and that delaying progression was important.The qualitative data provide support and contextualization to the quantitative approaches to determine the minimal clinically important difference for the 5-domain NPCCSS. The anchor-based analyses suggested that progressing beyond a 1-point worsening on the 5-domain NPCCSS would be clinically meaningful and, therefore, preventing a 2-point worsening would be a viable treatment goal.A potential limitation to the mixed methods study is the small sample size for the web-based survey (n = 49) and interviews (n = 28), especially when considering the broad age range of patients (1–65 years). While there was a good proportion of adult and paediatric patients represented, there was a limited number of adults reporting for themselves  (which could reflect the nature of the condition), and also a small number of very young patients included . Although the key domains appeared to be generally consistent across age groups in the current survey, there may be some differences in the very young children where Fine Motor Skills were not always ranked as most important. In addition, swallowing impairment tends to begin later and occur when the disease has progressed in severity. While the 5 domains are relevant for all age groups, it is acknowledged that the assessments have some limitations for the very young (0–2 years) age group. For example, ambulation impairments may be difficult to assess in the very young who would not yet be expected to have reached this developmental milestone. An alternative age appropriate measure would need to be utilized for this age group. Furthermore, the interviews focused on those domains highlighted as important or within the 5-domain NPCCSS which means that some of the original 9 domains were not discussed in depth, however the findings suggest that the domains focused on were those that were most important. A further potential limitation is that the patient or caregiver interviews and survey were only conducted in 2 countries (US and UK). Research involving participants from other countries would enable confirmation of cross-cultural validity. Finally, although the qualitative and quantitative data support each other regarding what constitutes a meaningful worsening on the 5-domain NPCSS  the gold-standard, anchor-based approach, was based on a small group of subjects (n = 13) due to the late introduction of CGI into the study. Furthermore because of the late introduction, there was increased risk of recall bias as the ratings for the majority of patients were based upon retrospective evaluations of disease status at baseline. Therefore, it is recommended that future researchers continue to explore this property of the measure.The results of the mixed methods, qualitative studies and clinical trial data are supportive of the 5-domain NPCCSS as a valid measure of NPC. Importantly, through this research, a meaningful change threshold has also been proposed. Nearly all participants expressed in interviews that slowing the progression within any domain on the 5-domain NPCCSS scale would be meaningful to patients and/or caregivers, and this was echoed by clinicians. A 1-category change on a domain equivalent to 1-point or greater change in the 5 domain NPCCSS total score supports a clinically meaningful transition. This supports that each category reflects loss of complex function and increased disability. The anchor-based analyses suggest that progressing beyond a 1-point worsening on the 5-domain NPCCSS would be clinically meaningful and, therefore, preventing a 2-point worsening would be a viable treatment goal.The reliability has also been demonstrated both within clinician (intra-rater) and between clinicians (inter-rater) . This evThis study involved adult patients (aged ≥ 18 years) and caregivers of either paediatric (< 18 years old) or adult patients from the US and UK with a confirmed diagnosis of NPC. There were 2 parts to the study: Part 1 was a web-based survey; and Part 2 was a follow-up telephone interview with a subset of survey participants.In Part 1, participants were asked to rate the severity of their NPC (or the NPC of the person under their care) using a series of questions based upon the 9 major domains of the NPCCSS . The NPCCSS questions and response options had been reworded to be appropriate completion by patients and caregivers while still reflecting the clinician-reported version of the NPCCSS. Definitions for some of the domains were provided. For example, for Cognition, participants were directed to think of cognitive ability as the ability to learn new skills, make decisions, follow instructions, or focus attention; within the measure this is considered distinct from Memory. The 5-domain NPCCSS scores were then used to select a range of severities for the interviews. Participants were also asked to identify and rank the 5 most important domains in NPC from 1 = “the very most important symptom” to 5 = “the least important symptom.” They were asked to identify these from the 9 major domains or given the option to add others.In Part 2, semi-structured interviews (approximately 90 min long) were conducted by trained interviewers following a discussion guide. All were conducted in English. Although the survey asked about the 9 major domains of the NPCCSS, the interviews focused on exploring the 5-domain NPCCSS symptoms and any other symptoms that participants had indicated as being most important in the web survey. Participants were asked why these symptoms were important, how they impacted activities of daily living (ADL) and health related quality of life (HRQoL), what category decline in each domain would be meaningful , and what such a change would mean in terms of impact on ADLs and HRQoL. The response option range of the 5 domains are as follows: Ambulation ; Speech ; Swallow ; Fine Motor Skills ; and Cognition . This information was used to infer what change in total score (range 0–25) on the 5-domain NPCCSS would be meaningful, since it is cognitively challenging to qualitatively discuss the meaning of change at the overall scale level with patients/caregivers.In-depth, semi-structured interviews lasting up to 1 h were conducted with clinician experts over the telephone. The interviews explored the importance and relevance of the 5 domains, the level of change clinicians consider to be meaningful in the 5-domain NPCCSS score, and each of the separate domains.The construct validity and meaningful change threshold for the NPCCSS was derived from a 12-month, prospective, randomized, double-blind, placebo controlled therapeutic study in paediatric patients with a confirmed diagnosis of NPC (NCT02612129) . A totalConvergent validity  was assessed using Spearman’s correlation coefficient due to the categorical nature of the response options. A treatment effect was evaluated using mean change (SD) from baseline across both treatment arms (placebo and arimoclomol). Analysis of meaningful change through the anchor-based approach used the CGI-I as the anchor . The CGIThe CGI-I was collected throughout the trial by the same investigator for each participant. To determine the anchor-based meaningful change threshold, patients were grouped by CGI-I levels. Mean and median change, 95% confidence interval, and standard deviation of the NPCCSS were calculated by CGI-I category.The analysis included all patients, independent of the assigned treatment group, and was performed on blinded data.


Lactobacillus acidophilus LA5 and Bifidobacterium BB12 was produced via fortification with chickpea flour . During refrigerated storage for five weeks, probiotics maintained a viable count above the minimum therapeutic level (106 CFU/g) in all yogurt types. Although there was no significant (p > 0.05) positive effect of chickpea flour on probiotic viability during storage, the addition of chickpea flour has beneficial effects on the viability of both probiotic species in the presence of gastric and intestinal juices, with 0.3% bile. This study also evaluated the physio-chemical properties of probiotic yogurt during storage. Some physicochemical properties of yogurt, such as water holding capacity and susceptibility to syneresis, were enhanced by the addition of chickpea flour. Hence, chickpea flour could be an attractive pulse ingredient in the production of probiotic yogurts for health-conscious consumers.In the present study, probiotic yogurt with  Lactobacillus and Bifidobacterium have been widely used and are considered safe and effective [Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus [Bifidobacterium. Therefore, in probiotic yogurt manufacture, both probiotics and starter cultures are used together as adjunct cultures. Starter culture bacteria are also known to provide benefits and enhance consumers’ immune system [6–107 CFU/mL or g of carrier food product, at the time of consumption. This concentration is known as the minimum therapeutic level [Probiotics are live microorganisms, which confer health benefits on the host, when administered in adequate amounts , such asffective ,4. Fermeffective . Yogurt ffective . Yogurt ffective ,4,7. Morffective . Therefoffective ,9,10. Itmophilus . Generalmophilus . Startere system . Howevere system . To provic level . Effectiic level .The viability of probiotics in yogurt is affected by many factors such as the oxygen content of the product and oxygen permeation through packaging materials . The viaLactobacillus [Cicer arietinum L.) is rich in protein, fiber and other prebiotic substances. As a food ingredient, it may enhance the nutritional and functional qualities of food products. Hence, chickpea flour may be a highly attractive substance to incorporate into the formulation of yogurt. Recent studies have demonstrated successful incorporation of chickpea flour into yogurt [Lactobacillus acidophilus LA5 and Bifidobacterium BB12 and evaluate the potential of the yogurt to confer enhanced in vitro gastrointestinal tolerance of these probiotics. Product quality characteristics, including probiotic viability over five weeks of refrigerated storage, and major physicochemical properties were also evaluated.Pulse flour is becoming increasingly popular in extending or substituting animal-derived products, due to recent interest in sustainable food production and the conferred health benefits ,21. For bacillus . Chickpeo yogurt . AlthougStreptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus). The probiotic Lactobacillus acidophilus LA5 and Bifidobacterium BB12 were also added according to manufacturer’s recommendations . Plain yogurt with no added chickpea flour was produced by placing the inoculated milk in a 250 mL sterile plastic container and incubating at 42 ± 1 °C until pH 4.4–4.5 was reached. Similarly, another three batches of yogurt, each 250 mL, were produced by incorporating 1, 2.5 and 5% (w/v) roasted chickpea flour . The flour was roasted at 60 °C for 10–15 min. After combining flour with the yogurt, it was incubated under the same conditions as the plain yogurt. All types of yogurt were stored at 4 °C for 5 weeks. Yogurt production was repeated three times. According to the manufacturer, the commercial chickpea flour contained nutrients in the following approximate composition: energy, 1510 kJ; protein, 21.8 g; fat, total 6.0 g; -saturated, less than 1 g; carbohydrate, 45.7 g; -sugars, 2.6 g; dietary fiber, 13.4 g; sodium, less than 5 mg; gluten—nil.Skim milk powder  was added to cow’s milk  at a concentration of 1%, before pasteurization at 80–85 °C for 30 min. The pasteurized milk mixture was cooled to 40–42 °C before adding 0.2 g/L of freeze-dried ABY-10 yogurt culture containing both starter cultures . The titratable acidity of yogurt samples was measured by titrating 9 g of yogurt sample with 0.1 NaOH solution and using phenolphthalein  as an indicator . The totL. acidophilus LA5 in yogurt samples. pH-modified (4.58) MRS agar was used for enumeration/counting of L. delbrueckii supsp. bulgaricus. M17 agar was used for enumeration/counting of S. thermophilus [Bifidobacterium BB12. All plates were incubated for 48–72 h under anaerobic conditions at 37 °C except S. thermophilus, which was incubated under aerobic conditions. In addition to the use of the above selective media, macroscopic observations and Gram-staining have been performed to identify and confirm each microbial species grown on each selective medium in this study.Microbial viability in yogurt was determined using serial dilution and spread plating techniques and expressed as CFU/g . Media wmophilus . Reinforw/v) saline solution to reach a final concentration of 3 g/L. Then, the pH of the solution was adjusted to 2.0 by adding concentrated HCl. Simulated small intestinal juices were prepared by suspending pancreatin  in sterile 0.5% (w/v) saline solution to a final concentration 1 g/L, with or without 0.3% bile salts . NaOH (0.1 M) was used to adjust the pH to 8.0.Simulated gastric juices were prepared by suspending pepsin  in 0.5% , 9 mL of gastric juices or intestinal juices and 1 g of yogurt  were mixed for 10 s using a vortex mixer at maximum setting . The samples were incubated at 37 °C. Aliquots of 1 mL were removed to assess resistance to gastric juices  and to intestinal juices with or without 0.3% bile  [Repeated measures analysis of variance was used for analysis of microbial viability and gastrointestinal survival. Physiochemical properties were analyzed using one-way analysis of variance. The means were separated using the least significant difference at a 95% confidence level.L. delbrueckii subsp. bulgaricus [S. thermophilus, L. acidophilus and Bifidobacterium spp. [Yogurt pH and acidity are important parameters that determine product quality. Yogurt pH values in all samples in the present study were generally within the desirable range (~4.0–4.5) as suggested by previous studies . As predlgaricus , S. therium spp. . Lactoseium spp. , which lp < 0.05). Several studies have reported that the WHC of yogurt is mainly related to the ability of protein and fat globules to retain water [L *, a * and b * values for color between the different yogurt formulations during storage is shown in p > 0.05) in L * value across the four formulations. Noh et al. [Corni fructus did not significantly affect the L * value. However, contrary results have been reported by Costa et al. [L * value may be due to variations in the milk composition and prebiotics selected. Moreover, due to the fact that chickpea flour is visually yellow-green in color, all yogurts fortified with chickpea flour were found to be greener , while a greater yellow coloration as indicated by the b * value only occurred under a high concentration of the flour (≥2.5%).As shown in in water . Chickpein water . Yousseein water  have reph et al.  drew a sa et al.  that thia et al. . Thus, tS. thermophilus in yogurt formulations containing 0%, 1% and 2.5% chickpea flour was relatively stable across the five-weeks storage period. The viable count for yogurt with 5% chickpea flour remained lowest during storage. Although the viability of L. delbrueckii subsp. bulgaricus remained stable over the first two weeks, a significant decline in the viable count was observed in all yogurt formulations at week 3  during storage, the ultimate probiotic count in all yogurt formulations exceeded the minimum therapeutic value of 6 log CFU/g. As shown in L. acidophilus LA5 across all formulations was very similar. However, yogurts with chickpea flour were found to have a clearly lower probiotic viability than plain yogurt from week 2, despite a temporary increase in the 2.5% chickpea flour formulation during week 4. However, L. acidophilus LA5 in all yogurt formulations had similar viability of around 7.40 log CFU/g at the end of storage.Although the viability of both probiotic bacteria in the yogurt decreased significantly  to the end of storage.Previous studies have implicated several factors in the viability of ufacture ,39. For ufacture  observednditions . This isductance . In thisBifidobacterium BB12, a slight decrease within the first four weeks was observed in plain yogurt, while in the last week a significant reduction to 7.70 log CFU/g was recorded. However, the viable count of Bifidobacterium BB12 in all yogurts containing chickpea flour decreased by more than 1 log CFU/g during the first week. The probiotic viability in yogurts with 1% and 5% chickpea flour then remained constant during the later stages of storage. The 2.5% chickpea flour formulation showed a slight increase at week 2 and then remained stable. Although the viability of Bifidobacterium BB12 maintained a high level (>7 log CFU/g) in all yogurt samples to the end of storage, the viable count in yogurt with 5% chickpea flour was significantly (p < 0.05) lower than in the other formulations.Regarding Bifidobacterium [Bifidobacterium ranges from 6 to 7, which is higher than that of the yogurt in the current study [Bifidobacterium. For instance, the proteolytic properties of L. delbrueckii subsp. bulgaricus provide amino acids essential to the growth of Bifidobacterium [S. thermophilus can act as an oxygen scavenger, which further improves the survival of strictly anaerobic Bifidobacterium BB12 [Higher acidity is detrimental to the growth of acterium . It has nt study . As a rent study . Furthernt study . For exant study  indicateacterium . Moreoveium BB12 . Thus, tL. acidophilus LA5 and Bifidobacterium BB12, the addition of chickpea flour has no significant (p > 0.05) positive impact in yogurt formulations.Accordingly, in terms of the viability maintenance of the two probiotic bacteria Bifidobacterium BB12 and L. acidophilus LA5 in the present study. The viability of both L. acidophilus LA5 and Bifidobacterium BB12 in all yogurt samples during simulated gastric digestion showed a significant decline (p < 0.05), though to different degrees . The largest decrease in the viability of L. acidophilus LA5 was noted in yogurt containing 2.5% chickpea flour, which fell from 8.30 to 4.95 log CFU/g at the end of 180 min of digestion. All other samples maintained significantly higher viability at the end of gastric digestion (p < 0.05). Interestingly, the plain yogurt with no added chickpea flour demonstrated satisfactory protection of probiotic species during simulated gastric digestion.As yogurt starter cultures do not possess biological mechanisms to permit satisfactory gastrointestinal survival, the viable number during simulated gastrointestinal transit was assessed only for probiotic  degrees . The viaBifidobacterium BB12 decreased significantly within an hour (p < 0.05). The viable counts in all formulations decreased to less than 1 log CFU/g at the end of 240 min. L. acidophilus LA5 demonstrated significantly higher viable numbers in all yogurts (>4.47 log CFU/g) at the end of digestion as compared to Bifidobacterium BB1 (p < 0.05). As shown in When yogurt samples were exposed to simulated intestinal juices with 0.3% bile salts, the two probiotics showed similar trends in the maintenance of viability. The viable counts of both probiotics in plain yogurts were reduced by more than 8.00 log CFU/g within 60 min. All yogurts containing chickpea flour maintained a relatively high value (>4.50 log CFU/g) until 60 min. At the end of 240 min of exposure, both probiotics in all formulations demonstrated poor viable numbers of less than 1 log CFU/g.Bifidobacterium BB12 as compared to plain yogurt. The protective effect may be due to the relatively higher fat content of chickpea flour, which increased the buffering capacity of the yogurts. With increased fat content, the sensitivity of Bifidobacterium BB12 to the high acid environment was reduced significantly, resulting in higher viability after simulated gastric digestion. However, the formulation containing 5% chickpea flour only had a protective effect for the first 60 min. This could be attributed to the possible microstructural changes in the yogurt due to the addition of a higher concentration of chickpea flour, which may have exposed bifidobacteria to gastric juices directly during prolonged exposure to gastric juices. However, this phenomenon warrants further study. The addition of chickpea flour showed no significant protection of L. acidophilus LA5 in this study. Gastric juice tolerance of probiotics is strain-specific and, as mentioned above, L. acidophilus has a high tolerance to acidic environments, which may explain its high viability during simulated gastric digestion in all yogurt samples.In order to achieve a therapeutic effect for consumers, probiotics must survive in the harsh conditions of the gastrointestinal tract. When probiotics pass through the stomach, the low pH and the antimicrobial property of pepsin are the main detrimental factors affecting the survival of these bacteria . In the L. reuteri, with resistance to intestinal juices and bile salts. Additionally, Patel at al. [Lactobacillus. The protective effects of these ingredients on probiotics are mainly attributed to the physical protection they provide [Generally, the intestinal environment is even more challenging to probiotic survival and colonization, as they are exposed not only to highly alkaline conditions (pH around 8.0), but also to bile . In thisl at al.  reported provide . Moreove provide . In the In addition, further experiments are needed to evaluate the organoleptic acceptance of chickpea flour-enriched probiotic yogurt products.Bifidobacterium BB12 and L. acidophilus LA5 in all yogurt samples was satisfactory (>7 log cuf/g) during shelf-life. The addition of chickpea flour did not confer any additional advantage in maintaining the probiotic viability of yogurts during storage. The addition of 1% and 2.5% chickpea flour showed significant protective effects on Bifidobacterium BB12 during the simulated gastric digestion. Further, the addition of chickpea flour demonstrated a significant positive impact on probiotic survival when exposed to simulated intestinal juices with 0.3% bile salts. Therefore, chickpea flour may be used as a suitable ingredient to improve some physicochemical properties and the functional efficacy of certain probiotics during gastrointestinal transit.In this study, the effect of chickpea flour enrichment on probiotic yogurt was evaluated in terms of the physicochemical properties of the product, microbial viability during five weeks of refrigerated storage and in vitro gastrointestinal transit tolerance of the probiotics. The formulation of yogurts incorporating chickpea flour to a concentration of 5% improved certain physicochemical properties, such as water holding capacity and susceptibility to syneresis. The viability of probiotic