K) and flow behavior indexes (n) after data was fitted to the Power Law and Herschel-Bulkley models. The gradual addition of the ackee aril flour fraction produced an increase in ash, fat, protein, and fiber content; while water and carbohydrate content showed the opposite behavior in the obtained composite flour. Consequently, the partial or full replacement of wheat flour changed the rheological properties of the produced doughs, as well as the quality of the final product. These changes were mostly related to the protein and carbohydrate content of the ackee aril flour fraction. In general, doughs showed a pseudoplastic behavior with thixotropy whose viscosity decreased as the addition of ackee aril flour was increased. Pasting properties of blends involving 25 %-75 % ackee aril flour demonstrate the feasibility of including these flours in products subjected to high processing temperatures such as canned products or even to produce chips and pasta.Wheat is one of the most widely used cereals in the world. However, studies consider wheat flour doughs to be of low nutritional quality, as there is now greater public awareness of celiac disease and gluten intolerance. Therefore, consumers are demanding healthier and more varied food products. Consequently, wheat flour is being replaced fully or partially by flours from other sources with higher quality. Hence, the main objective of this work was to report the effect of blending wheat flour with ackee aril flour, until the total replacement of wheat flour, on pasting and dough rheological properties. Five different levels of blending were analyzed: wheat to ackee aril flour mass ratios of 100 : 0, 75 : 25, 50 : 50, 25 : 75, and 0 : 100. Pasting properties  were analyzed; and steady-state shear measurements were used to obtain consistency coefficients ( Wheat is one of the most widely used cereals for flour production . Wheat fOn the other hand, wheat is not a major crop in Caribbean countries such as Colombia; so, wheat flour importation becomes mandatory to meet the local demand . TherefoThere is an interest in replacing common gluten-free formulations made from gluten-free refined flour, starch , and hydBlighia sapida is a tree native to West Africa that produces the ackee fruit [Sapindaceae family; and its fruit consists of a pod, pulp, and aril covering the seed [Concerning the above, ee fruit . The trethe seed . Arils othe seed . These ethe seed , thus, bthe seed .Ackee is mainly consumed in Jamaica for use in the national dish: ackee and salted fish. For this, the fruit is usually boiled gently for up to half an hour. However, even though the ackee fruit is cheap, has high dietary protein content (over 20 %) and has some claimed disease preventive properties, it is underutilized, and the quantity consumed worldwide is currently relatively low . One of Various studies have been carried out to study the effect of varying flour proportions in wheat flour mixtures on dough rheological properties , 20, 21.The substitution of wheat flour by other types of flour is important from the economic point of view in Colombia and Latin America, since wheat is mainly an imported product in these areas . MoreoveB. sapida) trees of a single genotype are growing in a private farm located within the urban perimeter of the municipality of Fonseca, La Guajira (Colombia). According to Benkeblia [Ackee  for 24 h. Then, the defatted flour was repeatedly washed with clean water to get rid of all excess hexane and dried again at 60 °C for 3 h. Afterwards, the dried flour was milled again and sieved through a metal sieve of 2 mm pore size. Finally, ackee aril flour was sealed in polythene bags in airtight containers and kept in a refrigerator at 4 °C until ready for use.Ackee arils were washed with water and blanched at 55 °C for 10 min. Afterwards, the vegetable material was dried at 60 °C for 72 h in an oven . During the procedure, it was found that dried aril samples were fat-rich, which made it slightly difficult to obtain a fine flour. Consequently, the dried sample was milled with a hammer mill into flour and defatted with hexane using a ratio flour/solvent of 1/10 . Flour mixtures with wheat to ackee aril flour mass ratios of 100 : 0 (T1); 75 : 25 (T2); 50 : 50 (T3), 25 : 75 (T4), and 0 : 100 (T5) were prepared. The negative control sample was 100 % wheat flour (T1). Each sample was blended in a high-speed blender and then sieved through a metal sieve of 2 mm pore size.Physicochemical analyses of wheat flour, ackee aril flour, and wheat-ackee aril flour mixtures were carried out based on the official methods of analysis approved by the AOAC . Ackee a−1, and a shear rate of 2.67  s−1, then held at 95  °C during 150 s, and cooled this time to 50  °C at 12  °C min−1 and the same frequency sweep. Parameters recorded were pasting temperature, peak viscosity, ease of cooking, swelling power, final viscosity at 50 °C, and thixotropy.Pasting properties of the samples were determined using a rheometer TA AR 1500®  with a standard profile as described earlier by Singh et al. , with mo−1–100 s−1 for 300 s in the controlled shear rate mode at 25 °C. The rheological behavior of doughs described by the flow curves was fitted to the Power Law and Herschel-Bulkley models represented by Equation (τ is the shear stress (Pa), τ0 is the yield stress (Pa), −1), K is the consistency coefficient (Pa·sn), and n is the flow behavior index (dimensionless) that signifies the extent of deviation from Newtonian behavior [Steady-state shear measurements were carried out with a rheometer TA AR 1500 using a plate of 40 mm controlled by the TA Universal Analysis Version 5.2® software with triplicate measurements. Dough samples prepared as described previously were equilibrated at 25 °C for about 15 min. Then steady-state shear experiments were carried out in the shear rate (behavior .Experiments were conducted following a single-factor completely randomized design (CRD). This factor was set as wheat/ackee aril flour mass ratio, following the gradual replacement of wheat flour by ackee aril flour. Five different levels of blending were analyzed by using the wheat to ackee aril flour mass ratios of 100 : 0 ; 75 : 25 (T2); 50 : 50 (T3), 25 : 75 (T4), and 0 : 100 . The experiments were repeated three times, for a total of 18 experimental units. The response variables were pasting and rheological parameters.Means and standard deviations were calculated for all data. Analysis of variance (ANOVA) and Tukey tests at a significance level of 5 % were used to determine the influence of blending ackee aril flour with wheat flour on pasting and dough rheological properties. Data processing was carried out by using STATGRAPHICS® Centurion XVI , while curve fitting was done through Origin® 7.5 .K) and flow behavior index (n) parameters obtained after the data were fitted to the Power Law and Herschel-Bulkley models. Yield stress parameters (τ0) were also obtained after data fitting by using the Herschel-Bulkley model.This research focused on the evaluation of pasting and rheological properties of wheat flour (T1), ackee aril flour (T5), and wheat-ackee aril flour blends , with the gradual wheat flour replacement by ackee aril flour. The first part of this section was devoted to the analysis of the physicochemical composition of pure flours and their mixtures. Afterwards, an evaluation of pasting and dough rheological properties was conducted and related to the previous physicochemical results. Pasting temperature, peak viscosity, ease of cooking, swelling power, final viscosity at 50 °C, and thixotropy were analyzed when the pasting properties were studied. Then, steady-shear measurements were used to obtain and analyze the consistency coefficients (p < 0.05). As can be seen in p < 0.05) were found among all formulations regarding moisture and ash content. In terms of fat and carbohydrate content, only T4 and T5 had no significant differences (p > 0.05). This lack of significant differences (p > 0.05) was also observed in pairs T1-T2 and T3-T4 in terms of protein and fiber content.Additionally, statistically significant differences  pasting temperature. Significant differences (p < 0.05) were only observed when pure ackee aril flour (T5) was analyzed. Pasting temperature values found in this work were similar to those reported for cassava dough [Pasting properties of composite flours made by blending wheat flour with ackee aril flour in different mass ratios  are shown in va dough . Pastingva dough , or evenva dough .T1 (pure wheat flour) and T2 (25 % by mass of ackee aril flour) had the highest peak and final viscosities at 50 °C. These parameters were directly affected by the higher carbohydrate content in wheat flour-rich doughs and the resultant swelling of the starch granules present . The frip < 0.05) the swelling power. However, at ackee aril flour mass fractions above 75 % (T4 and T5), there were no significant differences in swelling power (p > 0.05). The ackee aril flour content may have interfered with the adhesive interactions between the starch granules, resulting in lower adhesive forces that made water penetration difficult and thus lowering swelling power [Regarding the aforementioned, the blend level significantly influenced p < 0.0 the swelng power . FinallyK), flow behavior index (n), and yield stress (τ0) parameters were obtained after the data were fitted to the Power Law and Herschel-Bulkley models. These results are shown in R2) were found.After the steady-shear measurements carried out in this work, consistency coefficients  n values in T3, T4, and T5  when evaluated by the Power Law model. The above suggested a decrease in the pseudoplastic behavior (decrease in shear thinning). However, these treatments did not present significant differences among them (p > 0.05). For the data evaluated by the Herschel-Bulkley model and regarding n values, the flow behavior was similar and there were no significant differences among treatments with wheat flour content higher than 25 %. A decrease of shear thinning with the addition of ackee flour was observed earlier during the pasting experiments.It is also observed in K) of doughs evaluated by the Power Law model decreased with the higher presence of ackee aril flour, with statistically significant differences (p < 0.05) up to treatments with 75 % of ackee aril flour. This suggested the dependence of pseudoplasticity on the content of ackee aril flour. The highest consistency index was obtained for T1 (dough with 100 % wheat flour), while the lowest consistency index was obtained for T4 and T5 . The previous treatments decreased their consistency index by almost 30 % compared to T1. In general, these results suggested that starch granule interactions depended on the amylase/amylopectin ratio, which determines the strength and behavior of starch gels [p < 0.05) was only found when T5 was analyzed. In addition, an increase in the yield stress (τ0) was observed with the content of ackee aril flour in doughs, which reflected the presence of some cross-linked structures during gelatinization and cooling [On the other hand, the consistency index  canned goods, pasta, baked goods that require high temperatures, soups, sauces, sausages, jellies, children's food, and other products of the bakery lines.


Each test meal provided 50 g available carbohydrate, and test foods were matched for energy and macronutrients. There was no significant difference in blood glucose iAUC  between the coarse stoneground flour bread and the fine stoneground flour bread  mmol·min/L) and between the coarse stoneground flour bread and the fine roller-milled flour bread  mmol·min/L). The mean difference in insulin iAUC for fine stoneground flour bread compared with the fine roller-milled flour bread was −6.9%  and compared with the coarse stoneground flour bread was 9.9% . There was no evidence of an effect of flour particle size on postprandial glycaemia and insulinaemia among older people with risk factors for type 2 diabetes, most of whom were normoglycaemic.Wholegrain flour produced by roller-milling is predominantly comprised of fine particles, while stoneground flour tends to have a comparatively smaller proportion of fine particles. Differences in flour particle size distribution can affect postprandial glycaemia in people with type 2 diabetes and postprandial insulinaemia in people with and without type 2 diabetes. No prior studies have investigated the effect of wholegrain flour particle size distribution on glycaemic or insulinaemic response among people with impaired glucose tolerance or risk factors for type 2 diabetes. In a randomised crossover study, we tested the 180-min acute glycaemic and insulinaemic responses to three wholegrain breads differing in flour particle size and milling method: (1) fine roller-milled flour, (2) fine stoneground flour, and (3) coarse stoneground flour. Participants ( Since carbohydrate sources like bread and grain-based dishes are staple foods in the cuisines of people worldwide, it is important to examine how the benefits traditionally associated with wholegrain consumption are influenced by modern food processing methods. Wholegrain consumption can improve acute postprandial glycaemia compared with refined grains  and is aAnother group for whom we have limited knowledge about the metabolic effects of grain particle size is people with prediabetes or impaired glucose tolerance (IGT). This group comprises a large (7.7%) and growing proportion of the world’s population ,13, apprTo address this gap in knowledge about the benefits of processed wholegrains, the aim of this study was to measure the acute metabolic response to breads made from roller-milled wheat flour and two stoneground wheat flours, each differing in their grain particle size distribution, among people who are overweight and obese, inactive, and older in age, as these factors are associated with having prediabetes, insulin resistance, and increased risk of type 2 diabetes ,17,18,19This randomised, acute, controlled crossover trial was designed to test the 180-min blood glucose and insulin responses to three wholegrain breads differing in flour particle size among participants with known risk factors for type 2 diabetes. Each participant underwent a meal test on three separate mornings in randomised order to test the acute glycaemic and insulin responses to the three wholegrain wheat breads made with (1) fine roller-milled flour, (2) fine stoneground flour, and (3) coarse stoneground flour.Using a standard deviation of the difference for iAUC  of 152 mmol·min/L , the samParticipants were randomised to the order that they would receive the test foods using a Williams square design , which e2, and not meeting New Zealand’s physical activity guidelines of >150 min moderate to vigorous intensity activity per week [Participants were males and females with known risk factors for type 2 diabetes aged 55–75 years old, BMI > 28 kg/mper week ,17,18,19People who expressed interest in participation and confirmed that they met the study eligibility criteria were invited to attend a screening visit held at the University of Otago Human Nutrition Clinic. Participants provided information on demographics, medical conditions, medications taken, and allergies and intolerances. Duplicate measures were taken for body weight to the nearest 0.1 kg, height to the nearest 0.1 cm using a calibrated stadiometer, and waist circumference to the nearest 0.1 cm (measuring the narrowest point between the iliac crest and lower ribs). Systolic and diastolic blood pressure was taken near the end of the screening visit after participants had been seated for 10 or more minutes and was measured by a digital automatic blood pressure monitor , using the average of three consecutive tests taken in direct succession. Glycated haemoglobin (HbA1c) was measured in capillary blood from a finger prick with the Tina-quant Haemoglobin A1c Gen.2 kit, using the hemolysate application. These were analysed using a cobas c 311 .Test meals consisted of bread providing 50 g available carbohydrate (approximately 3–4 slices) served with 250 mL water. Breads were isoenergetic and were matched for macronutrient composition, water, and sodium.Test breads, varying only by the type of wholegrain flour used, were prepared in the food-grade laboratories at the Department of Food Science (University of Otago) by baking six loaves per batch. Ingredients are listed in All flour was defined as wholegrain in that the bran, germ, and endosperm were all present in the same relative proportions as they exist in the original, intact grain . Flour wNutrient composition of the three test breads is described in Participants arrived at the clinic  between 07:00 and 07:30 a.m., having fasted at least for ten hours. Participants chose which mornings to attend for testing, with the restriction that they were not able to choose to attend on consecutive days, giving a minimum washout period of at least one full day. They participated in a total of three tests on three different mornings testing the three different breads. Participants were blinded to which of the test breads they consumed at each visit. On the day prior to each test morning, participants were instructed to avoid alcohol, to choose the same evening meal prior to each test morning, and to keep consistent the duration and intensity of any physical activity undertaken. Upon arrival at each testing session, adherence to these conditions was confirmed, and if the conditions were not met, the participant was rescheduled.® Insyte Autoguard shielded I.V. Catheter 20G) placed into a forearm vein by a trained health professional for sampling of blood insulin and a one-off sample to measure glycated haemoglobin. After cannulation, participants remained seated for ten or more minutes, and then blood samples were taken for baseline blood glucose and insulin measurements: two capillary blood samples were taken within 5 min via fingerprick to assess fasting blood glucose concentration, and a 2 mL venous blood sample was taken via the cannula to measure fasting insulin concentration. Immediately after baseline tests, participants were given their test meal and 250 mL water to consume within 10 min or as close to this time as they could reasonably manage. The time taken to finish eating the test meal was recorded. Blood glucose and insulin concentration were measured via a single fingerprick and a 2 mL venous blood draw, respectively, at 15, 30, 45, 60, 90, 120, 150, and 180 min from the time of meal commencement. Alcohol swabs were used to clean the finger prior to each fingerprick. Participants remained seated unless they needed to visit the bathroom in an adjacent room.Participants had a cannula , and outcomes were within the specified ranges.Capillary blood glucose concentrations were immediately analysed using a HemoCue Glucose 201+ analyser. Each HemoCue Glucose 201+ analyser was tested against the Glucotrol® Vacutainer K3EDTA). Immediately prior to the venous blood collection, 1 mL of blood was drawn from the cannula and discarded to ensure that the sample did not include blood trapped in the cannula. After each 2-mL sample was collected, the cannula was cleaned with a 3 mL saline flush. Venous blood samples were held during the test morning for up to 3.5 h in an insulated container containing an ice pack. Blood samples were then centrifuged, and plasma samples were stored in duplicate at −80 °C for up to 4 months. Plasma insulin was measured with a Elecsys Insulin kit (Cat. No. 12017547) using a Cobas e 41 analyser . The coefficients of variation for the Elecsys Insulin Kit were 2.31% and 2.04% for the low and high controls.To measure plasma insulin, each 2 mL venous sample was drawn directly into an EDTA-coated vacutainer .Incremental area under the blood glucose curve (iAUC) was calculated over 180 min using the trapezoidal method, ignoring the area below baseline. The same method was used to determine insulin iAUC except that it was calculated over 150 min because venous blood samples at 180 min were not available for several participants due to blood clotting and blocking the cannula.a priori, since time taken to eat is known to affect postprandial glycaemia [p-values were calculated. p < 0.05 was considered statistically significant with no adjustment for multiple tests.Mean differences in blood glucose and insulin iAUC between test foods were estimated using mixed effects regression models with blood glucose or insulin iAUC as the dependent variable, test food as the independent variable, and participant as a random effect. Insulin iAUC values were log-transformed for analysis to improve homoskedasticity of the model residuals. Model estimates were back-transformed and presented as percentage differences in the dependent variable between test meals. For both blood glucose and insulin iAUC, robust variance structure was used. Variables for randomisation order and time taken to eat the test food were also included in the models. Adjustment for these variables was decided lycaemia , and ordlycaemia . Mean diWe also conducted an exploratory sensitivity analysis to determine whether having features of glucose intolerance moderated the effect of flour particle size on postprandial blood glucose and insulin iAUC because of the high level of heterogeneity in glucose tolerance status amongst the participants. Participants categorised as having features of impaired glucose tolerance had either or both of fasting blood glucose ≥ 6.1 mmol/L or HbA1c ≥ 40 mmol/mol. One participant with normal glycated haemoglobin (34 mmol/mol) was categorised as having features of glucose intolerance because they were clearly hyperinsulinaemic , and their fasting blood glucose was considered to be impaired (5.6 mmol/L) according to the American Diabetes Association . The mixThirty-three participants attended a screening visit, but eight of these were excluded from participation . Hyperte2), and four had a BMI > 28 and <30 kg/m2. Further details of participant characteristics are described in Eleven participants (48%) took medications for cardiovascular disease. Participants self-reported that they engaged in a median of 20 min of moderate to vigorous intensity exercise per week . Fasting blood glucose ranged from 4.2 to 7.4 mmol/L. Nineteen participants had obesity  had a marginally larger mean difference in glycaemic response between bread made from coarse stoneground flour and fine roller-milled flour (mean difference (95% CI) = 37.5  mmol·min/L) and between bread made from coarse stoneground flour and fine stoneground flour (mean difference (95% CI) = 39.5  mmol·min/L)   comparedl·min/L) . Amongstl·min/L) .n = 8). These differences were not statistically significant, and nor were the subgroup sample sizes sufficient to detect significance. Since the exploratory sensitivity analysis relies on a small and poorly defined sample, these findings are speculative and cannot inform a definitive conclusion. In contrast, insulin iAUC outcomes did not follow the trends observed for blood glucose iAUC for either the primary analysis or the sensitivity analysis. Consumption of both the roller-milled flour bread and the coarse stoneground flour bread resulted in a higher insulinaemic response compared to fine stoneground flour bread in the primary analysis (n = 23). These differences were larger among those with features of glucose intolerance (n = 8).Among people with known risk factors for type 2 diabetes, there were no significant differences in glycaemic or insulinaemic response to breads made with coarse stoneground flour, fine stoneground flour, and fine roller-milled flour, although the glycaemic response to the bread made from coarse flour was marginally lower than the two breads made from fine flour. When results were stratified by degree of glucose intolerance status, the differences in glycaemic response between the coarse stoneground flour bread and the two breads made from fine flour were amplified among people with features of glucose intolerance  in wholegrain flour affects postprandial glycaemia or insulinaemia. The proportion of ultra-fine particles differed between all three flour types used to make our test breads, whereas the proportion of fine flour (<180 μm) was similar between the fine stoneground flour and the fine roller-milled flour . Our finOur unexpected findings for postprandial insulinaemia did not align with findings from prior studies ,8,9. In Stoneground flour was chosen for the coarser flour options because it typically is comprised of a larger proportion of coarser particles than fine particles ,29. HoweA limitation of this study is that we did not obtain information on the cultivars of our wheat and the temperature that the stone mills attained during milling since we used commercially available flours. Heat generated during stone milling can vary from 35 °C to 90 °C ,32,33, aIt is possible that factors beyond that of particle size alone may have contributed to the lack of significant differences in glycaemic and insulinaemic responses among the breads. For practical reasons, our breads were frozen and subsequently thawed overnight in a refrigerator, so formation of resistant starch would be expected . It is nThere is lack of evidence for the importance of grain particle size for people with prediabetes. Our study partially contributes towards building this evidence base, but it would have been stronger had we been able to recruit people with prediabetes. Given that people are frequently unaware of having prediabetes, we considered that the cost and time involved in screening for participants who met the diagnostic of prediabetes or impaired glucose tolerance would have been prohibitive. The post hoc analysis suggests that a future study should confirm whether people with prediabetes respond differently to the effects of grain particle size compared to normoglycaemic people. It should also be noted that this sensitivity analysis was underpowered and was only carried out for exploratory purposes. Therefore, the findings may be useful to hypothesise future research directions but not to draw conclusions from.Many consumers consider stone-milled flour to be more desirable, as it is perceived as a natural product . Our resFrom our research, it does not appear that those with normoglycaemia have additional benefits relating to postprandial glycaemia or insulinaemia from choosing wholegrain bread products made with coarse stoneground flour rather than finer roller-milled flour. However, the findings from our exploratory sensitivity analysis contribute to the basis for a future study to confirm whether people with prediabetes will benefit from coarser flour, in line with what has been shown in other studies amongst people with type 2 diabetes ,8. Given


Plasmodium falciparum, the highly polymorphic var genes encode the major virulence protein, PfEMP1, which bind a range of human receptors through varying combinations of DBL and CIDR domains. Here we present a tool, Varia, to predict near full-length gene sequences and domain compositions of query genes from database genes sharing short sequence tags. Varia generates output through two complementary pipelines. Varia_VIP returns all putative gene sequences and domain compositions of the query gene from any partial sequence provided, thereby enabling experimental validation of specific genes of interest and detailed assessment of their putative domain structure. Varia_GEM accommodates rapid profiling of var gene expression in complex patient samples from DBLα expression sequence tags (EST), by computing a sample overall transcript profile stratified by PfEMP1 domain types.Parasites use polymorphic gene families to evade the immune system or interact with the host. Assessing the diversity and expression of such gene families in pathogens can inform on the repertoire or host interaction phenotypes of clinical relevance. However, obtaining the sequences and quantifying their expression is a challenge. In Varia_VIP was tested querying sequence tags from all DBL domain types using different search criteria. On average 92% of query tags had one or more 99% identical database hits, resulting in the full-length query gene sequence being identified  among the five most prominent database hits, for ~ 33% of the query genes. Optimized Varia_GEM settings allowed correct prediction of > 90% of domains placed among the four most N-terminal domains, including the DBLα domain, and > 70% of C-terminal domains. With this accuracy, N-terminal domains could be predicted for > 80% of queries, whereas prediction rates of C-terminal domains dropped with the distance from the DBLα from 70 to 40%.var sequence and domain composition is possible from short sequence tags. Varia can be used to guide experimental validation of PfEMP1 sequences of interest and conduct high-throughput analysis of var type expression in patient samples.Prediction of The online version contains supplementary material available at 10.1186/s12859-022-04573-6. PfEMP1 variants are inserted into the erythrocyte membrane to bind specific human endothelial receptors [var genes of 6–12 kb [var exon1) is composed of multiple DBL (Duffy binding like) and CIDR domains capable of binding specific human receptors. DBL and CIDR domains are classified into a few main domain types, DBLα-ζ and CIDRα-γ, which have been further divided into domain subtypes [Pathogens can evade the immune system through polymorphic protein families interacting with host molecules. Examples of such gene families are the PIR and xoplasma . The moseceptors . The PfE 6–12 kb –5. The esubtypes . Due to subtypes .var2csa and var3 genes, which encode atypical domain compositions. The anchoring intracellular part of PfEMP1 is encoded by the var exon2.The domain composition of each PfEMP1 can vary but follows a general pattern  pipeline, the user provides one or more partial var sequences, which are used for searching a var database for near identical sequences. Hit sequences are clustered based on their full-length sequence similarity, and the domain composition of the longest gene sequence representing each cluster is visualized in circular plots and tabular output files , batches of DBLα ESTs, such as those generated by high-throughput sequencing of multiple patient samples , are cluvar genome database containing all assembled var gene contigs and their domain annotations was generated. The database includes 2400 Illumina sequenced parasite isolates  assembled in [P. falciparum clones [var genes of 755 P. falciparum isolates from the Community Project. The var genes of the 755 isolates were assembled using the var gene assembly pipeline as described in [https://github.com/ThomasDOtto/varDB/tree/master/Datasets/Additional755/. Not all genes from clinical isolates are full-length var genes, and in general, the exon 2 is missing from many of these genes. As we are interested in the prediction of sequences and domains, sequences shorter than 3 kb were excluded. We merged the new dataset with the existing, resulting in a total of 205,595 var gene sequences. From translated amino acid sequences, domain subtypes described in [https://github.com/ThomasDOtto/varDB/. As annotation was performed on complete and incomplete var sequences, these criteria may leave some sequence ends with incomplete domains unannotated.For Varia, a new mbled in , 15 Pacim clones  as well ribed in  and the ribed in . We usedvar sequence prediction from this database, we randomly selected 40 k DBLα sequences from the database. Specifically, we extracted the first 150 nucleotide bases starting from the LARSFADIG motif found in all DBLα domains. These sequences were blasted  against four different partitions of the database: (1) the newly generated var sequences for this paper (41 k sequences), (2) the “normalised dataset” defined in [var gene diversity  (3) the full dataset analysed in [To explore the var gene database is biased towards genomes from South East Asian parasite isolates [var genes of African isolates compared to Asian isolates. Separating the 40 k 150 bp query sequences by African or Asian origin showed that Asian sequences were more likely to have a database match (97%) than African sequences (85%). The hit rate drops with the length of query sequence, and for full-length sequences just over 50% of the sequences have complete matching hits. Increasing the database size from 160 to 220 k makes little difference, but larger databases increase the hit frequency  and more frequent correct annotations compared to DBLε and DBLζ domains. This observation can be explained by the differences in the distribution of diversity within these main domain types. DBLα, β, δ and γ domains are highly diverse in sequence and diversity is homogeneous. Conversely, DBLε and DBLζ domain sequences distribute into distinctly different groups of highly similar sequences. Thus, as identical DBLε and DBLζ domains are found in many different PfEMP1 variants, correct predictions are difficult to make from these domain tags.var genes, a correct annotation was identified for 72% of the query genes at 99% sequence similarity threshold. The correct annotation was found among 14 suggested predictions, on average. At this 99% similarity threshold, prediction of the exact query DNA sequence, defined as 99% identity over at least 80% of the sequence, was successful for 29% of the DBLα domain tags. Lowering the similarity threshold to 90% resulted in a limited increase to 78% of the query genes with correct annotation among the suggested domain compositions, and an increase in the number of alternative suggestions. For this reason, we recommend applying the highest similarity threshold resulting in database hits when using Varia_VIP to search for putative domain compositions and sequences of query genes, to allow manageable experimental validation (e.g. by PCR) of the query sequence.As expected, lowering the similarity threshold increased the proportion of queries with correctly predicted annotations but at the cost of a higher number of different predictions. Specifically, for the DBLα domain tags, which are found in the 5′-end of all var gene database is biased towards genomes from South East Asian parasite isolates [As noted above, the current isolates . At 95% var gene expression in patients, a rapid prediction of the single most likely PfEMP1 domain composition of a large number of sequences is required. For this, we built Varia_GEM, which, based on all hit sequences to a DBLα EST, returns the likely consensus domain annotation of the query gene. As the ability to predict correct annotations depends on domain type and distance to the query tag, Varia_GEM was designed to assess domain annotation for each domain position relative to the PfEMP1 N-terminal. For each domain position  the tool determines if a specific PfEMP1 main domain type or domain subtype is dominant (> 66%) among hit gene sequences. If this is the case, the tool returns the consensus annotation in a tabular format, along with quantitative data of frequency of ESTs associated with the domain or domain composition, to allow quantitative analysis of PfEMP1 domain traits with e.g. clinical features pertaining to the origin of the sequences.In some studies, for example of var gene database, as they would be amplified by the commonly used DBLα EST primers [Average main domain accuracy: An annotation was considered incorrect if the predicted main domain type was wrong, or if a non-existing domain was predicted .Average domain subtype accuracy: An annotation was considered incorrect if the predicted domain subtype was wrong, or if a non-existing domain was predicted .To determine parameters for optimal domain type prediction by Varia_GEM, we tested main domain type and domain subtype predictions at different identity threshold values. We extracted 220 randomly selected DBLα0, 1 and 2 tag sequences  from the  primers . The cogvar gene domain compositions (coverage) was calculated for each domain position.Average coverage (main domain type): The proportion of genes with a correctly predicted main domain type at said position. A domain annotation was considered incorrect if a wrong main domain type was predicted, or a domain annotation was missing .Next, the frequency by which the tool could predict the correct Lowering the blast identity threshold and thereby including a higher number of hits resulted in fewer predictions and fewer correct domain annotations Fig. B. HoweveMost frequent and correct domain predictions were generated using the 99% blast identity threshold. This threshold is likely to be too stringent for analysis of sequencing data, which may contain various minor sequence errors. Instead, a 95% threshold was chosen as default for Varia_GEM. This threshold results in a 0–10% drop in main domain type coverage and domain subtype accuracy, as well as a 0–3% decrease in main domain type accuracy, on average. Using the 95% identity threshold, the effect of the number of blast hits on domain prediction was investigated Fig. C. This sP. falciparum var genes from short DNA sequence tags. Previous studies have shown that the extensive diversity of the var genes is ancient and in large depends on recombination [var genes. Moreover, prediction is hampered by an ambiguous clustering of PfEMP1 domains into specific subtypes. Main domain types  are well defined : Linux ,Programming language: Bash and Python,Other requirements: Database can be downloaded from https://github.com/ThomasDOtto/varDB, as indicated on VARIA GitHub page. Varia builds on several basic bioinformatics tools, like NCBI BLAST, mcl, Circos and samtools. License: GNU General Public License v3.0,Any restrictions to use by non-academics: no restrictions.Additional file 1: Information on implementation and usage of Varia. Examples of output.


Covalent Organic Frameworks (COFs) are an exciting new class of microporous polymers with unprecedented properties in organic material chemistry. They are generally built from rigid, geometrically defined organic building blocks resulting in robust, covalently bonded crystalline networks that extend in two or three dimensions. By strategically combining monomers with specific structures and properties, synthesized COF materials can be fine-tuned and controlled at the atomic level, with unparalleled precision on intrapore chemical environment; moreover, the unusually high pore accessibility allows for easy post-synthetic pore wall modification after the COF is synthesized. Overall, COFs combine high, permanent porosity and surface area with high thermal and chemical stability, crystallinity and customizability, making them ideal candidates for a myriad of promising new solutions in a vast number of scientific fields, with widely varying applications such as gas adsorption and storage, pollutant removal, degradation and separation, advanced filtration, heterogeneous catalysis, chemical sensing, biomedical applications, energy storage and production and a vast array of optoelectronic solutions. This review attempts to give a brief insight on COF history, the overall strategies and techniques for rational COF synthesis and post-synthetic functionalization, as well as a glance at the exponentially growing field of COF research, summarizing their main properties and introducing the numerous technological and industrial state of the art applications, with noteworthy examples found in the literature. Reticular chemistry is a recently developed area in organic chemistry and material science, concerning the strategic and rational synthesis of new organic compounds by promoting the controlled growth and formation of regular, porous and crystalline structures ,2. By chThe first major result of the application of reticular chemistry concepts was the development of a new class of materials, so-called Metal Organic Frameworks (MOFs). These new structures, branching out from the family of coordination polymers, consist in the coordination between metal ions (or clusters) and multifunctional organic ligands, capable of forming subunits that extend in two or three dimensions .In a MOF, the metal ions act like coordination points, or vertices, while the organic ligands, whose structures are typically rigid and spatially well-defined, are the backbone of the reticulated 3D structure.4(O)O12C6 clusters in which an oxygen atom is surrounded by four Zn2+ atoms, which in turn are coordinated to six carboxylic groups from the six 1,4-benzenedicarboxylate (DBC) ligands. Since the ligands are symmetrical and bifunctional, each one is further coordinating with two other adjacent zinc ions from nearby clusters, resulting in a regular, permanent cubic-shaped structure -4,4’-diamine, a fluorinated derivative of benzidine. The electron-withdrawing CF3 groups introduced in the structure showed particular affinity towards ibuprofen molecules, with a maximum adsorption capacity of 119 mg g–1 at neutral pH x-TPB-DMTP-COF, is a 2D imine-type framework in which a fraction of one of the monomers possess alkynyl groups, which were further reacted with (S)-pyrrolidine chiral groups post-synthesis dianiline, a C2 symmetric diamine bearing thiazole groups in its backbone , rivalling and surpassing many previously reported water reduction systems [Several water splitting studies have also been reported using COFs as photocatalysts. The reaction consists of obtaining molecular hydrogen and oxygen from water, a process of great interest for numerous technological and industrial applications, since, theoretically, it can be one of the most efficient, practical methods for Htriazine ,207,208,triazine ,211,212,triazine , imine [triazine ,215 or atriazine  have beetriazine . The β-kbackbone . The unu systems .Much like the research developed with respect to catalysis, the potential of COFs for applications related to chemical sensing has recently been broadly investigated. Photoluminescence properties intrinsic to aromatic COFs, due to the conjugated double bonds and ompounds . An examompounds . The imi3 g−1) and a BET surface area of 987 m2 g−1. The highly photoactive COF showed over 21% absolute photoluminescence quantum yields and selective luminescence quenching in the presence of 2,4,6-nitrophenol (TNP)  , proving3+ in aqueous solution [3+, with outstanding selectivity and sensitivity. In a similar approach, Cui and coworkers interestingly prepared COF nanosheets for Al3+ ion detection [3+ in solution, with the fluorescent intensity sharply increasing as Al3+ concentration increases, unlike with several other alkaline or transition metals tested, due to overall weaker coordination interactions with bipyridine groups.Another interesting example are hydrazone-linked COFs for photosensitive detection of Fesolution . The stretection . The as-2 g−1, 0.89 cm3 g−1 pore volume and average pore size of 1.5 nm. The bulk solid was then processed into ultrathin nanosheets via exfoliation by solvent dispersion and ultrasonic techniques, prior to biosensor experiments. Designed DNA probes marked with a fluorescent dye adhere to the pore wall surface which results in the quenching of the COF’s fluorescence. However, when target DNA is present in solution, a chain reaction between the probes and the DNA strand is triggered, and the resulting dsDNA biomolecule, which has very weak bonding interactions with the COF, leaves its surface, thus “switching on” the COF’s fluorescent activity again. This predictable, linear behavior allows for a quantitative detection of DNA and can potentially be used for many other biosensing applications [Peng et al. successfully synthesized a highly crystalline COF for sensitive and selective DNA detection . The 2D ications .p-toluene sulfonic acid monohydrate, which acted as a co-reagent allowing for a slower crystallization process, thereby inducing better porosity and crystallinity properties. The entrapped acid compound also acted as an efficient proton carrier for conductivity testing. The resulting COF membrane, obtained by a slow reaction process at low temperatures for a long period (3 to 4 days) to allow for thermodynamic control and consistent framework error correction, showed high crystallinity and an extremely high proton conductivity of up to 7.8 × 10−2 S cm−1 at 80 °C and 95% relative humidity, higher than the majority of organic polymer materials reported to date [2 g–1 BET surface area and 0.84 nm pore size. In its free form, a proton conductivity of up to 3.96 × 10–2 S cm–1 was reported for NUS-10, at 298 K and 98% relative humidity, one of the highest reported values for similar structures. Furthermore, a mixed-matrix COF membrane consisting of a blend of the resulting COF and nonconductive polyvinylidene fluoride (PVDF) polymer was prepared, affording up to 1.58 × 10−2 S cm−1, in water at 353 K [The promising results obtained for COFs regarding electrochemical energy storage and ionic conductivity also turns these materials into potential candidates in a wide range of new technological applications such as fuel cells or solid-state ionic batteries . In orde to date . In a di to date . One of at 353 K .2 g−1 and pore size and volume of 1.5 nm and 0.97 cm3 g−1 were reported, respectively. The optically-active COF was further tested by using it as a dopant for a reference perovskite solar cell, substantially improving power conversion efficiency by up to 18.0% as compared to the non-doped material [Several COFs have been further tested for implementation as novel lithium  and lithmaterial .Moreover, besides photovoltaic cells ,262, sev2-functionalized boronic acid monomer (BDBA) replaced the non-functionalized counterpart (BDBA) via sequential monomer substitution. The amine functionalization allowed for the conjugation of camptothecin-20-O-hemisuccinate (CPT-Suc) within the COF pore walls. Characterization showed that the CPT ligand increased hydrophobicity of the material, therefore increasing COF stability in water—an important aspect to consider as boronate ester COFs are particularly susceptible to hydrolysis. More importantly, the CPT-COF conjugate was observed to be easily internalized by cell membranes and showed higher cytotoxic activity than the free drug, even at low doses [3@COF-BODIPY-2I@GAG, was obtained by first reacting 2,5-dimethoxyterephthalaldehyde (DMTP) with 1,3,5-tris(4-aminophenyl)benzene (TAPB). The 2D imine-linked COF was then functionalized with the amine-substituted BODIPY-2I. The functionalized material was further modified through the sequential impregnation of CaCO3 and glycosaminoglycan (GAG), yielding the final composite. BODIPY-2I acted as photosensitizer in a photodynamic therapy experiment, whereas GAG served as the target agent for CD44 receptors on the digestive tract tumor cells. The synergistic effect of highly reactive singlet oxygen (1O2), produced by the photosensitive BODIPY-2I, combined with Ca2+ overload promoted by the loaded COF leads to tumor cell destruction. In vitro experiments showed selective tumor cell death induced by both Ca2+ overload and 1O2 oxidative stress by using green light as light source, suggesting the efficiency of COF nanomaterials for the novel approach [Researchers have recently discovered the potential of COFs for use in biomedical applications, such as drug delivery and cancer therapy ,271,272.ow doses , adding ow doses ,276,277.ow doses . The finapproach . Severalapproach ,283,284.It is undeniable that COFs are a very promising new class of porous materials with enormous potential for a wide range of applications, already showing impressive results in several different fields of study and research that can rival and even sometimes surpass already established, well-known, commercially available solutions. However, COF development is still in its very early stages and a few serious challenges still need to be addressed and overcome before COFs can be considered a viable alternative for mass-scale real-use applications. First and foremost, the “crystallization problem” needs to be tackled and properly solved. COF synthesis needs to be standardized, consistent and reproducible. This means researchers need to acquire more knowledge and information regarding the specific mechanisms of COF formation, growth and crystallization, in order to more precisely control the polymerization processes and more accurately take advantage of desired physical properties such as porosity, surface area and crystallinity. Then, for the scale-up challenge, if COF materials are to be employed in the industry on a massive scale, standardized synthetic procedures must first be achieved. This implies discovering new synthetic pathways that allow for a consistent COF material with intact properties in easier, more efficient, faster and cheaper ways. Despite these challenges, however, COFs are set to remain a very encouraging, exciting prospect in materials chemistry.


Campylobacter lari (PglB) catalyzes the glycosylation of asparagine in the consensus sequence N-X-S/T, where X is any residue except proline. Molecular dynamics simulations of PglB bound to two different substrates were used to characterize the differences in the structure and dynamics of the substrate-enzyme complexes that can explain the higher catalytic efficiency observed for substrates containing threonine at the +2 position rather than serine. We observed that a threonine-containing substrate is more tightly bound than a serine-containing substrate. Because serine lacks a methyl group relative to threonine, the serine-containing peptide cannot stably form simultaneous van der Waals interactions with T316 and I572 as the threonine-containing substrate can. As a result, the peptide-PglB interaction is destabilized and the allosteric communication between the periplasmic domain and external loop EL5 is disrupted. These changes ultimately lead to the reorientation of the periplasmic domain relative to the transmembrane domain such that the two domains are further apart compared to PglB bound to the threonine-containing peptide. The crystal structure of PglB bound to the peptide and a lipid-linked oligosaccharide analog shows a pronounced closing of the periplasmic domain over the transmembrane domain in comparison to structures of PglB with peptide only, indicating that a closed conformation of the domains is needed for catalysis. The results of our studies suggest that lower enzymatic activity observed for serine versus threonine results from a combination of less stable binding and structural changes in PglB that influence the ability to form a catalytically competent state. This study illustrates a mechanism for substrate specificity via modulation of dynamic allosteric pathways.The oligosaccharyltransferase of  Asparagine-linked glycosylation (N-glycosylation) is an essential function in eukaryotes . The attCampylobacter jejuni OST and it is thought to have a similar catalytic mechanism  bound to an acceptor substrate peptide, which provided structural insights into the catalytic mechanism and the role of conserved motifs , which attaches a lipid-linked oligosaccharide (LLO) to the consensus sequence N-X-S/T of the acceptor substrate (where X is any amino acid except proline) . OST is echanism . The bacmplicity . These sd motifs . Recent ari PglB . Due to C. lari PglB is composed of a transmembrane domain and a periplasmic domain , a threonine residue at the +2 position (where the acceptor asparagine is defined as position 0) allows for glycosylation 40 times more efficiently in eukaryotes than a serine in this position . In C. lequences . In the equences , red andequences . One obvequences . HoweverIn this paper we focus on the substrate binding phase of the catalytic cycle and assess the differences that could affect the catalytic competency of PglB when bound to acceptor substrates containing threonine or serine at the +2 position using molecular dynamics (MD) simulations. The catalytic activity of PglB requires binding of both the acceptor substrate and LLO, although the order is thought to be unimportant . While LC. lari PglB with the optimal acceptor sequence, GDQNATFG (PglB-NAT), and the peptide variant GDQNASFG (PglB-NAS) were used to discover the detailed differences between threonine and serine at the +2 position in the substrate-bound state of the catalytic cycle. We observed an increased propensity for substrate disassociation in the trajectories of PglB-NAS compared to PglB-NAT. Our results show that threonine at the +2 position forms a network of interactions with the periplasmic domain and EL5 that promotes a more closed orientation of the periplasmic domain. The loss of the methyl group in the serine disrupts the substrate-enzyme interaction and the allosteric communication between the periplasmic domain and external loop EL5, allowing for increased motion of the periplasmic domain and leading to a smaller population of stably bound, catalytically competent states.MD simulations of C. lari with bound optimal peptide (GDQNATFG) were started from the crystal structure (PDB code 3RCE). The missing residues of external loop EL5 (residues 283–306) and missing loop residues in the periplasmic domain (residues 605–607) were built with modeler . In the second phase of equilibration with constant temperature and pressure (NPT ensemble), restraints were removed and a force was applied to any water molecules that entered the lipid bilayer to prevent infiltration and disruption of the bilayer. At the beginning of the second phase, conjugate gradient minimization was performed on all atoms, followed by velocity reinitialization and temperature and pressure equilibration. The final phase of equilibration was performed with no constraints or applied forces using constant temperature and pressure to allow for final density equilibration of the system. Multiple restraint conditions were tried in the final phase of equilibration and the unrestrained equilibration protocol was determined to be the most appropriate to reproduce the coordination of the Mg2+ ion given the uncertainty in the Mg2+ coordination due to resolution of the crystal structure (3.4 Å) (The simulations were performed with NAMD 2.13  using th (3.4 Å) . The coo (3.4 Å) .xyPzT ensemble). Pressure and temperature were maintained at 1 atm and 310 K, respectively, using Langevin dynamics and the Nosé-Hoover Langevin piston method. The SHAKE constraint algorithm (Definition of Peptide Binding).The production phase of the data generation used constant temperature, pressure, and constant area in the plane of the bilayer , indicating that the dynamics of PglB is not influenced by the lipid composition of the bilayer. For example, the difference in the peptide-PglB interaction energy for the two lipid types was less than the standard deviation: PglB-NAT POPC = −228.7 ± 24.8 kcal/mol, PglB-NAT POPE = −240.0 ± 26.7 kcal/mol, Δ(NAT POPC-POPE) = 11.3 kcal/mol. Therefore, we combined all the trajectories for better sampling. PglB-NAT spent 5,063 ns in the bound state and PglB-NAS spent 2,780 ns in the bound state and all histograms were normalized to account for the difference in the number of data points. To ensure that we have collected enough of the relevant states to be able to make meaningful comparisons between the two peptides, we assessed the quality of sampling using jack-knife analysis  of the pDefinition of Peptide Binding) excluding the first 50 ns to allow for structural equilibration, figures were prepared with Tableau Software v10.4 , and structures were visualized with VMD using the STRIDE algorithm for secondary structure identification  the largest difference in the Cα-Cα distance between the periplasmic and transmembrane domains (residues 535–537 of the periplasmic domain and residue 279 of the transmembrane domain) and 2) the backbone RMSD (root mean square displacement). From the trajectories of PglB-NAT and PglB-NAS we identified the structures corresponding to the most populated inter-domain distance  and RMSD values. These structures were then validated as representative of the most populated conformation by locating them in the plot of the first two principal components and were found to be located in the regions of highest density .RMSD between average or most populated structures was calculated asbio3d geostas algorithm (http://www.R-project.org) to determine which residues have the least variation in position over the trajectory (the stable core). The stable core residues were determined from the combined trajectories of PglB-NAT and PglB-NAS and consist of transmembrane domain residues 118–124, 126–130, 132, 342–347. Alignments on all atoms of PglB, the transmembrane domain, and the stable core were compared for structural analysis, cross-correlation calculations, and principal component analysis. In all cases, alignment on the stable core identified by the geostas algorithm  was found to perform better than an all-atom alignment. However, previous simulations of PglB have found that the transmembrane domain does not behave as a single rigid domain . To furtlgorithm  in the Rlgorithm  performebio3d package (http://www.R-project.org). The structured C-terminus of EL5 (residues 307–324), which is present in the crystal structure  using the  package  in the Rtructure , is incllgorithm . Initiallgorithm  of the p package .2+ ion was also calculated and the results did not alter the conclusions based on the PglB/peptide interaction energy  using cross-correlations calculated over all bound frames aligned on the stable core. Cross-correlations between PglB, the peptide, and Mg2+ ions were included in the calculations with a threshold of 0.7 (corresponding to an angle between motion vectors of <45°). Community membership was determined using the greedy algorithm  and edge weight between two communities (Eij) are defined asi is the number of residues in each community and max (Cij) is the maximum correlation coefficient between residues in each connected pair of communities. The scaling was chosen to illustrate relative differences between communities without obscuring the underlying structure. Residues of the structure are colored according to community membership with unconnected communities in white in Network graphs overlaid on the structure represent each community by a sphere located at the geometric center of each community. The community radius (RThe number of shortest paths that pass through each node (betweenness), the number of edges of each node (degree), and suboptimal path analysis are calculated from the full network defined by the underlying cross-correlation matrix (where each residue is a node). The nodes contained in the suboptimal paths of PglB-NAT were identified using 500 paths and visualized using the first 100 paths for clarity.Sequence alignments were performed using with the Clustal algorithm  on the rC. lari PglB with the optimal acceptor sequence, GDQNATFG (PglB-NAT), and the peptide variant GDQNASFG (PglB-NAS) were used to discover the detailed differences between threonine and serine at the +2 position in the substrate-bound state of the catalytic cycle. Thirteen replicas with a cumulative simulation time of 6.5 and 5.8 μs were collected for PglB-NAT and PglB-NAS, respectively. We observed a striking difference in the behavior of NAT and NAS peptides: out of the thirteen independent trajectories collected for each system, four trajectories of PglB-NAT exhibit partial peptide unbinding while ten trajectories of PglB-NAS exhibit partial peptide unbinding. Since a stably bound substrate is prerequisite to forming a catalytically competent state, we wanted to understand the critical interactions in the bound state that lead to this behavior. To do so, we needed to quantitatively determine when the peptide was in the bound state versus a partially bound state or fully unbound state in our simulations. We then analyzed the frames where the peptide is bound to identify differences in the bound states of the two peptides.Molecular dynamics simulations of We identified six non-catalytic H-bonds whose absence corresponds to loss of the catalytically relevant H-bonds and disassociation from the binding pocket. These H-bonds were used to define the bound, partially bound, and fully unbound states see . The H-bWe analyzed the behavior of the peptides after becoming partially bound and observed that there is no clear pathway that leads to the full unbinding and dissociation from the binding pocket. After the loss of the H-bonds p+1 O-M318 N  do not involve p+2 at all. To understand the difference in stability of the bound NAT and NAS peptides, we calculated the interaction energy between the peptide residues and PglB. The largest change is observed for p+2, however, the interaction energy while bound is affected beyond this residue and shows a large degree of compensation at peptide residue p-2 .The interaction energy between PglB and the NAS peptide is overall less favorable , consistThe compensation observed in the interactions with p-2 can be explained by the changes observed in the NAS peptide. The aspartic acid at position p-2 shifts relative to its transmembrane domain hydrogen-bonding partners R147 and R331, decreasing the favorable interaction energy with R147  and increasing the favorable interaction energy with R331 , accounting for 80% of the difference in interaction energy at p-2. The remaining difference comes from numerous long-range electrostatic interactions of the charged aspartic acid side chain.For p+2, the key interacting residues of the WWD motif and I572 from the crystal structure  show devComparison of the average structures suggests a difference in the conformation of PglB when bound to NAT versus NAS peptides. The difference between PglB Cα-Cα distances was calculated for PglB-NAT and PglB-NAS to identify regions of PglB that differ in conformation when bound to the two peptides. We found that the distance between the periplasmic domain and the transmembrane domain is increased in PglB-NAS compared to PglB-NAT . The aveAnalysis of Trajectories). The results obtained using either the average or the most populated structure are the same.In contrast, the intradomain Cα-Cα distances of the periplasmic domain and the transmembrane domain differ remarkably little between PglB-NAT and PglB-NAS, with average changes of 0.01 Å and 0.04 Å per residue pair, respectively. The minimal change in Cα-Cα distances within the periplasmic and transmembrane domains indicates that the overall structure of both the periplasmic and transmembrane domains do not change between PglB-NAS and PglB-NAT. These results suggest that while the structure of the two domains is conserved, their relative positions change and that there should be a “hinge” that allows for rigid domain motion. If true, we would expect the hinge to meet the following criteria: 1) the hinge should have low backbone RMSD between average structures, 2) the hinge should be located in an interfacial region between transmembrane and periplasmic domains, and 3) the magnitude of the displacement observed between the open and closed positions should correlate with the distance from the hinge. In our system, this means we expect that the further a residue is from the hinge, the more different its position will be between the open state and the closed state for the mobile portion of the structure (the periplasmic domain). Two pairs of structures were used to identify the differences between PglB-NAS and PglB-NAT: the average structure and as an additional validation, the most populated structure  versus unlatched (PglB-NAS). The motion should be more restrained in PglB-NAT and the motion should be freer in PglB-NAS. Principal component analysis of PglB (excluding the unstructured EL5 N-terminus) shows that the motion of PglB-NAT and PglB-NAS is similar . HoweverThe motion of PglB-NAT versus PglB-NAS supports the existence of a latch. To verify that our choice of latch and pin residues is correct, we looked at the distance between these residues in the latched (PglB-NAT) and unlatched (PglB-NAS) conditions. For the latch and pin to restrict the motion of the hinge (as we propose occurs in PglB-NAT), the latch and pin complex must be stable (the distances between these residues should not fluctuate significantly). When the pin is unable to close the latch (as we propose occurs in PglB-NAS), we expect to see greater fluctuations in the distance between the pin and latch residues. The histograms of the distances between the PglB residues on opposite sides of the latch (W463 and I572 of the periplasmic domain with T316 of the EL5 loop) show that the latch residues in PglB-NAT predominantly sample a narrow range of distances , gray. TThe behavior of the pin and latch residues explains the interplay between peptide binding and periplasmic domain orientation. The distances between the latch residues in PglB-NAS increase because serine lacks the methyl group and is unable to form the stable simultaneous interactions necessary to pin the latch closed (keeping the two sides of the latch in closer proximity). The NAS peptide therefore shifts its position to interact with the latch residues on opposite sides (periplasmic domain and EL5) because the latch residues are further apart. As the NAS peptide moves towards one side of the latch, it loses the interaction with the other side of the latch (reflected in the increased sampling of near-zero interaction energies . The inc2+ ions). The two largest communities contain most of the periplasmic domain residues . Half of the unconnected residues in PglB-NAS are similarly located in the transmembrane domain. Significantly, the remaining unconnected residues in PglB-NAS are the entire EL5 loop (43 residues) and the peptide (8 residues)  : white. esidues) . The NATesidues) , excludiesidues) . The lacSuboptimal path analysis can show paths of allosteric communication between different parts of the network . The perIn the cryo-EM structure of the human OST complex with acceptor peptide : the loss of the methyl group would eliminate the van der Waals interaction between the substrate residue at position p+2 and I572 of PglB . However, since being bound in the catalytic pocket is a prerequisite to the formation of these H-bonds, the increased partial substrate disassociation observed for PglB-NAS decreased the population of catalytically competent states by roughly half.More remarkably, the identity of the amino acid at the p+2 position of the substrate peptide also affected the periplasmic domain orientation of PglB. The two bound structures are similar, but they differ in ways that suggest either the unbound or the peptide/LLO bound states. Comparison of the average bound structures of PglB-NAT and PglB-NAS with the average unbound structure  shows thRigid body domain motion was found to describe the difference between the conformations of the periplasmic domain in PglB-NAT and PglB-NAS with a hinge near the junction of the two helices containing N448 and Y473, near the interface of the periplasmic and transmembrane domains. Threonine at position +2 acts as a pin in a latch to inhibit the hinge motion by forming interactions with residues in the periplasmic domain and EL5. Loss of the methyl group in serine results in the disruption of these interactions, allowing the latch to open. The pin/latch complex and the H-bonds necessary for stable substrate binding involve many of the same residues. We found that the opening of the latch necessarily disrupts the binding, and thus combined with the more open periplasmic domain orientation produces the fundamental difference observed between the simulations of PglB-NAT and PglB-NAS, namely, the 2.5 times increase in the frequency of partial NAS peptide unbinding. The importance of the newly identified substrate-EL5 interaction is supported by, and may help explain, the conservation of T316 as part of the bacterial TIXE motif . Networkp > 0.8) that we identified as important for substrate binding stability. Substrate residues p+1 and p+3 form backbone H-bonds with the structured portion of EL5; EL5 pins the substrate in the binding pocket and p+1/p+3 play a role in stabilizing the peptide-EL5 interaction. We also found a robust network of interactions between the periplasmic domain and EL5 mediated by the NAT peptide with high connectivity of multiple substrate residues (including p+1 and p+3), facilitating allosteric communication.Neighboring substrate residues at positions p+1 and p+3 play supporting roles in the binding and allosteric communication. Both p+1 and p+3 form strong H-bonds in PglB-NAT (These results offer potential explanations for the observation that the catalytic competency of N-X-S substrates is more sensitive to nearby residues p+1 and p+3 than N-X-T substrates . With thExperimental data have shown that an interplay exists between the catalytic competency and the substrate residues at positions +1, +2, and +3–serine at p+2 increases the sensitivity to p+1 and p+3, and a less optimal residue at p+1 was found to make differences at p+3 more pronounced . This ob-1-N-X+1-S/T, where X-1 and X+1 can be any residue except proline (Certain features that play a role in the behavior observed in our simulations are known to differ between bacterial and eukaryotic OSTs and may contribute to greater sensitivity to serine-containing substrates in eukaryotes . We foun proline ] compens proline  while ba proline .Van der Waals interactions between p+2 and T316 are part of the network of interactions that facilitate allosteric communication between the periplasmic domain and EL5. Although this interaction is directly affected by the lost methyl group, the NAS peptide was able to still form transient interactions with T316. T316 is part of the conserved TIXE motif containing the catalytic E319 in bacteria; in eukaryotes, a different sequence is conserved: SVSE  Supplem. The repFrom our results we conclude that the loss of the methyl group in serine has repercussions far beyond the van der Waals interaction with I572 observed in threonine substrates. The loss of the methyl group destabilizes the entire constellation of interactions present with threonine and disrupts neighboring interactions between substrate and enzyme. This interrupts the allosteric communication between the periplasmic domain and EL5 mediated by acceptor substrates with threonine, allowing for opening of the periplasmic domain. While threonine at the +2 position acts as a pin in a latch to favor more closed periplasmic domain orientations reminiscent of the crystal structure of PglB with acceptor substrate and LLO analog , the mor