Significant reductions in glycaemic responses were achieved without adverse effects on bread texture, appearance or palatability. Starch digestibility analysis and microscopy confirmed the importance of cell integrity in attenuating glycaemic responses. Alternative processing methods that preserve cell integrity are a new, promising way to provide healthier low glycaemic staple foods; we anticipate that this will improve dietary options for diabetes care.The global rise in obesity and type 2 diabetes has generated significant interest in regulating the glycaemic impact of staple foods. Wheat breads  are popular staples, but have a high-glycaemic index, due to the highly digestible wheat starch. Reducing the glycaemic potency of white bread is challenging because the bread-making conditions are mostly conducive to starch gelatinisation. Cellular legume powders are a new source of type 1 resistant starch, where the starch is encapsulated by dietary fibre in the form of intact plant cell walls. The starch in these cell powders is less susceptible to gelatinisation and digestion than starch in conventional legume flours. However, legume cell resilience to baking conditions and the effects of this ingredient on glycaemic responses and product quality are unknown. Here we show that the integrity of cell wall fibre in chickpea powder was preserved on baking and this led to a ~40% reduction in   •Intact cell powder (ICP): a new legume ingredient increased type 1 resistant starch in bread rolls.•ICP lowered starch bioaccessibility and glycaemic response of wheat bread rolls.•Legume cell integrity was retained after baking and digestion.•Wheat breads incorporating ICP had acceptable quality attributes. There iin vivo  protect intracellular starch from digestive enzymes . The impin vivo . In the in vivo . Conventin vivo  which evin vivo . Thus, rA new proprietary process has been developed that enables the preservation of plant cell wall integrity during transformation of whole pulses into leguminous powders . The novWheat bread is a widely consumed starch-staple food and its transformation into a healthier product is likely to positively influence public health . Typicalin vitro studies to provide further insight of the mechanisms of action of dietary fibre, as structurally intact cell walls, in producing type 1 resistant starch and attenuating amylolysis. This investigation has important implications for gastrointestinal and cardiometabolic health especially in relation to glycaemic control. Successful attenuation of glycaemic responses to bread without compromise on quality and cost, represents a significant opportunity for the food industry to create a new generation of food products to support dietary management of type 1 and 2 diabetes mellitus.The aim of the study was to test the resilience of type 1 resistant starch in legume intact cell powders to the bread-making process, and subsequently investigate the glycaemic and product quality effects of bulk incorporation of legume intact cell powder as a wheat flour replacement in white wheat bread. Human studies were performed in combination with 22.1Cicer arietinum L., ~7 mm size, Kabuli-type, Argentine variety, supplied by AGT Poortman Ltd., UK) according to a proprietary process (PCT/GB2019/ 050284) involving hydrothermal processing, wet-sieving and drying , as an ingredient in wheat bread rolls. The ICP was prepared by New Food Innovation Ltd. (UK) from whole chickpeas (d drying . The ICPd drying .The chickpea ruptured cell powder (RCP) was prepared by blending ICP in a Thermomix® TM5  and passing it through a 75 μm aperture analytical sieve. The resulting cell powder (RCP) had a smaller particle size with a higher proportion of ruptured cells and was included for comparison in Study 1 to enable further mechanistic insights into the role of cellular integrity on starch digestibility and glycaemia. The particle size of this ingredient was confirmed through laser diffraction by a method that assumes particles are a spherical shape, as described previously .Other ingredients used to prepare the bread were: wheat flour ; sucrose ; NaCl ; vegetable fat  purchased from Sainsbury's supermarket, Norwich, UK; wheat gluten  and ascorbic acid , purchased from Amazon; and dry Baker's yeast  provided by Lallemand. The wheat flour used in this study contained approximately 14.8% protein, 1.3% fat, 65.9% starch, 1.4% total sugars, 3% dietary fibre, and 13.6% moisture, and 1468 kJ per 100 g and was a major source of digestible starch and wheat protein (mainly as gluten) in the bread roll recipes. The vital wheat gluten contained approximately 76.7% protein, 1.7% fat, 10% starch, 3.3% dietary fibre and 8.3% moisture, and was a further source of wheat protein and starch, particularly in the ICP-enriched rolls.2.2All bread rolls were prepared in a clinical test kitchen . Three bread rolls, each containing different doses of chickpea ICP were produced; these were denoted 0% (control), 30% and 60% bread, as chickpea ICP was added to the recipe at 0, 30 or 60 (w/w) % replacement of wheat flour.All ingredients were measured and combined using the proportions described in One 12-roll batch of each bread type  was produced on each of two consecutive days using a sponge-dough process. Sponge ingredients  were mixed using a pre-weighed 6.9L heavy duty planetary mixer  with a paddle attachment. The sponge was developed for 8 min, then transferred into resealable plastic bags and stored under ambient conditions (22.0 ± 3.0 °C). After 2 h, the remaining dry ingredients and water were combined with sponge mixtures in the planetary mixer (using a 6.9 L bowl and dough hook attachment) and allowed to develop for 8 min. The resulting dough was portioned into bread rolls and immediately proved in a pre-heated high-humidity (100% RH) combination oven  at 38 °C for 30 min. After proving the rolls were lightly spray misted with water and baked by convection at 185 °C for 15 min to achieve an internal core temperature of 95 ± 2 °C. Weight (moisture loss) after baking was recorded for each bread roll. Finished rolls were cooled for 2 h and stored frozen at −20 °C in resealable, opaque polyethylene bags (3 mm thickness). A third batch of rolls were produced under identical conditions at a later date to provide a third replicate for bread quality assessment .2.3Proximate nutritional analysis of ICP and breads baked for the human dietary intervention study were performed by UKAS accredited testing at ALS Laboratories Ltd., Chatteris, Cambridgeshire, UK. Specifically, protein was quantified by Dumas using a Nitrogen conversion factor of 6.25, total fat by NMR, total dietary fibre by AOAC method no. 985.29 (‘Prosky method’), total sugars by ion exchange chromatography, available carbohydrates by ‘difference’ calculation, sodium by ICP-OES and energy was calculated using standard energy conversion factors (EC 2008/100 and 90/496). The dietary fibre measurement includes high molecular weight soluble and insoluble dietary fibres , but does not include some lower molecular weight soluble oligosaccharides , and resistant starch is believed to be only partially measured .Total and resistant starch content of ICP, RCP and bread products were determined in house using the reagents from ‘Total starch kit’ KTSTA-100A and ‘Resistant starch (rapid) kit’ K-RAPRS, Megazyme International, Wicklow, Ireland. Total starch analysis was performed using the smaller-scale DMSO version of the method, as described previously . The sumEnterobacteriaceae, Escherichia coli, Clostridium perfringens, Staphylococci (coagulase positive), Bacillus cereus, Salmonella spp., yeasts and moulds. All microbiological counts were within levels deemed safe for human consumption; no Salmonella spp. were detected. The chickpea ICP was also tested for trypsin inhibitors  of 22-40-01 ) and lec22-40-01  by Campd2.4For each bread type (i.e. dose of ICP), a representative subset of three bread rolls from each of the three separate bake days were subjected to quality analyses. Bread rolls were defrosted in their packaging at room temperature (22 ± 2 °C) for exactly 12 h prior to quality analyses, which included assessment of specific volume, crumb and crust colour, and texture. These tests were done in the same order for each treatment and completed within 2 h to control for quality variation as a result of starch retrogradation and/or drying.3) was determined using AACC method 10-05 (volume based on rapeseed displacement) and the roll mass (g) (Bulk density (g/cmmass (g) . A metalInstrumental measurement of crumb and crust colour was achieved using a bed scanner . For each bread roll type, L*, a* and b* values were obtained using Adobe Photoshop CS6 extended software  from three rolls with eight readings from each roll at different locations on the crumb and crust. Hue angle and chroma were calculated .−1. This was applied to 25 x 25 × 25 mm samples to mimic mastication; crumb ‘hardness’ corresponded to the maximum force required to achieve 40% compression. Five replicate measures were performed on each bread sample. Stable Micro Systems Exponent  software was used to calculate ‘springiness’ and ‘chewiness’ as defined by Szczesniak  equipped with a 5 kg load cell and using a modification of the AACC method 74-09  . Samplesczesniak  and applczesniak .2.52/KI) solution was added to the samples to stain the starch before placing the suspension in a slide with a glass cover and examined.For light microscopy, images were captured using an Olympus BX60 Microscope equipped with a Jenoptik 232 ProgRes camera. Samples were taken before and after digestion and stored in 10% formalin solution until required. A small amount of Lugol's iodine (Ihttp://fiji.sc/). Images on the diffusion of FITC-amylase were obtained in a Zeiss LSM880 confocal laser scanning microscope and processed using ZEN Blue software. FITC-labelled amylase was prepared by mixing pancreatic α-amylase dissolved in 0.1M sodium carbonate buffer pH 9 with FITC in the same buffer at a molar ratio 1:100. After stirring in the dark for 3 h at room temperature, the labelled enzyme was purified in a PD-10 column equilibrated with PBS at pH 7.4. Samples of cooked chickpea cells were stained with calcofluor white and fast green FCF as described before and incubated with FITC-amylase at 37 °C for 2 h.For confocal imaging of the chickpea powders, RCP and ICP were suspended in PBS containing 10 μg/mL calcofluor-white, fluorescein isothiocyanate (FITC) and fast green FCF  to stain cell walls, starch and protein, respectively. Following incubation for 30 min at room temperature on a rotating mixer, samples were centrifuged and washed twice in PBS, mounted on slides and viewed under a Leica SP2 confocal microscope . Samples were imaged using laser excitations of 405 nm, 488 nm and 633 nm and emissions of 410–480 nm, 500–580 nm and 640–750 nm for calcofluor, FITC and fast green, respectively. Image stacks of chickpea cells at 10x magnification and a representative chickpea cell at 63x magnification, and average z-projections and image analysis were conducted using Fiji image analysis software (2.6p-hydroxybenzoic acid hydrazide) assay for reducing sugar analysis of samples withdrawn after 0, 10, 20, 30, 40, 50, 60, 75, 90 and 120 min of incubation with porcine pancreatic α-amylase, as described previously  were obtained in R Core Team  and used to illustrate the joint probability of distinct digestion behaviours.Cell powders and representative bread rolls from each treatment and batch were subjected to starch digestibility analysis (amylolysis) using published methods . Bread reviously . Amylolylsewhere . As the 2.72.7.1clinicaltrials.gov as NCT03994276 Study 1 tested the postprandial glycaemic response to drinks containing 50 g of carbohydrate (mainly starch) in the form of ICP and RCP, and a reference drink containing 50 g carbohydrate as glucose. Study 2 tested the postprandial glycaemic response to bread rolls incorporating different quantities of ICP into the recipe  and formulated to contain the same amount of potentially available carbohydrate per serving.Two double-blind randomised controlled studies, with a three-arm cross over design, were undertaken at the metabolic research unit (MRU) of King's College London between June 2019 and January 2020. The trials investigated the effects of chickpea ICP (consisting predominantly of intact cells) on glycaemia. These studies were registered at The studies were conducted in accordance with the Declaration of Helsinki and approved by the relevant research ethics committee in the UK . All participants' data were stored in accordance with the General Data Protection Regulation 2018 and biological samples were handled, stored, transported and disposed of in accordance with the Human Tissue Act (2004). Participants were reimbursed for their time and travel expenses upon completion of each study.2.7.22, blood pressure ≥ 160/100 mmHg, fasted glucose > 6.0 mmol/L, plasma cholesterol ≥ 7.8 mmol/L, plasma triacylglycerol ≥ 5.0 mmol/L, medications that may interfere with the study (e.g. antidiabetic or lipid-lowering drugs), allergy or sensitivity to wheat, alcohol intake >28 units/week and active or recent cessation of smoking (<6 months). Participants were healthy with no history of cardiovascular disease, diabetes or gastrointestinal disorders, as determined by a pre-screening health questionnaire. Body mass index (BMI), blood pressure, liver function, blood cell count, fasting plasma glucose and lipid concentrations were confirmed to be within normal limits during a clinical screening visit that took place before confirming enrolment.Healthy participants aged 18–45 years were recruited using advertisements around King's College London, including circular e-mails and posters. Exclusion criteria included: body mass index (BMI) < 18 or >35 kg/m. In study 1, all 20 enrolled participants were initially fitted with devices for monitoring of glucose concentrations in interstitial fluid, in addition to capillary blood collection during the postprandial period. For study 2, 21 participants were enrolled onto the study, of which 16 were fitted with continuous glucose monitors. In both studies, issues with these monitors  and drop-outs meant that the final data analyses were performed with data from fewer than the enrolled number of participants. The CONSORT diagrams in .The characteristics of participants that were enrolled onto each study are reported in Table S.12.7.3Study 1 assessed the acute 2 h postprandial glycaemic response to three test drinks  all providing 58 g potentially available carbohydrate . Potentially available carbohydrate was provided as 58 g glucose in the form of dextrose powder from Thornton & Ross, Linthwaite, UK); or as starch (50 g) from chickpea powder (100 g ICP or RCP), plus 11 g Nesquick chocolate powder with no added sugar  for flavouring. The moisture contents of all powders were determined by oven-drying method and weights of powder per serving adjusted accordingly to achieve 58 g potentially available carbohydrate per serving. Complete dispersion of the powders was ensured by the use of a protein shaker bottle containing a blender ball. Additionally, participants were asked to shake the bottle periodically during consumption. All drinks were made up to 330 mL with bottled water . Time to drink was recorded and participants were encouraged to drink at the same rate for all 3 test drinks.Study 2 assessed the 4 h postprandial glycaemic response to three bread roll types in which 0, 30 or 60%, w/w, of the wheat flour in the recipe was replaced with chickpea ICP. Replacement of wheat flour with ICP meant that ~12 and 30 g of the total starch in the 0% bread roll was replaced by starch from ICP to make the 30% and 60% bread rolls, respectively. Bread rolls were prepared as described in Section In both studies, participants were fitted with a continuous glucose monitor  capable of recording subcutaneous glucose concentrations every 15 min continuously for up to 2 weeks. The CGM was covered by a protective film and applied to the back of the upper arm at least 12 h before the first visit and remained in place throughout the duration of study 1. Two monitors per participant were used for study 2 as the total study duration exceeded the 14-day recommended period of use of the sensors. On the morning of each intervention, the participants arrived at the MRU after a 12 h fast and having consumed a standard meal that was provided to them  the previous evening. Participants marked responses to palatability questions  using 100 mm Visual Analogue Scales (VAS) at baseline (t = −10), immediately after the test or control meal, and at 30, 60, 90, 120, 180 and 240 min. The glucose data from the CGM was downloaded with an associated reader using Freestyle Libre Pro software  at the end of each visit. Time stamped glucose concentrations were recorded for the 120 min postprandial period, including a baseline measurement 15 min prior to consumption of the allocated test meal (t = 0). In study 1, additional capillary blood glucose measurements were obtained by finger-prick method using an Accu-Chek Performa-nano monitor with test strips  and lancets .2.7.4Time points from the CGM recordings were anchored at t = 0, defined as the closest reading to the time of the participants first bite of the breakfast test meal, and selected every 15 min thereafter. Incremental postprandial glucose responses were obtained by subtracting the baseline glucose measurement(s) from subsequent glucose measurements within the same individual on each occasion. The incremental area under the interstitial glucose response curve (iAUC) up to a specified time point was calculated using only the area under the curve above the baseline (ignoring the area under baseline) according to the method referred to as ‘Method 3 – incremental AUC method’ in 2.8In vitro data was analysed by repeated measures ANOVA, and in vivo data analysed by mixed model ANOVA, with time and treatment  as fixed effects and individual differences as random effects. The number of participants (n) whose data was included in each analysis is indicated in figure legends and throughout the text. Outliers were not excluded. Postprandial glucose response data are provided as Supplementary data, with mean, SD and n shown for each time point. A Greenhouse-Geisser correction was applied to correct for violations of sphericity. Post hoc analyses were performed when significant treatment x time effects were detected, and Tukey's correction for multiple comparisons applied . Statistically significant effects were accepted at the 95% confidence level. Mean values are reported with SD unless otherwise specified and violin plots indicate the distribution of the data.Statistical analyses and graphical representations were done using GraphPad Prism 8.4.1 software for Windows . 33.1Conventional dry-milled legume flours consist mostly of ruptured cells and exposed starch which becomes highly susceptible to digestion upon hydrothermal processing. The novel pre-processing treatment used to obtain ICP differs from conventional dry-milling in that it facilitates cell separation so that most of the starch in ICP remains enclosed by leguminous cell walls .in vitro starch digestibility and in vivo glycaemia was determined by comparing the intact cell powder ‘ICP’ with a compositionally equivalent comminuted powder referred to as ‘ruptured’ cell powder, RCP. The RCP was obtained by grinding the ICP to pass a 75 μm sieve screen, and therefore contained a higher proportion of ruptured cells. The analysis of RCP was also of interest with regard to the commercial viability of the material; for instance, a smaller particle size may be desirable for some applications, and some cell rupture is likely to occur during large-scale industrial production of the powder.The role of cell integrity in Imaging confirmed that the ICP consisted almost entirely of intact isolated cells , where tin vitro amylolysis assay. The RCP had significantly higher starch susceptibility to amylolysis than ICP . After 120 min of amylolysis, 63.0 ± 1.7% of the carbohydrate (starch) had been digested in RCP, compared with 21.7 ± 0.6% in the ICP. Thus, the grinding operation used to obtain RCP led to a three-fold increase in the amount of bioaccessible starch. Overall, the progress of starch amylolysis in both ICP and RCP  and drink type  on glycaemic responses, and a significant drink × time interaction  over 120 min  are shown in  120 min D.45, which was 40% higher after RCP  than ICP , F1.797, 28.74 = 27.38, adj. p = 0.044). Peak glucose concentrations  were significantly higher (adj. p = 0.028) after RCP  compared with the ICP , F1.743, 27.89 = 26.63 (60 and iAUC120 were not significantly different between RCP and ICP drinks (p > 0.05). These data indicate that differences in cell integrity influence primarily the early postprandial glucose response, and are consistent with the slower rate of release of starch amylolysis products from ICP compared with RCP, observed in vitro.Cell integrity had a significant effect on the iAUC = 26.63 D insert.120) for ICP and RCP were significantly smaller , compared with the glucose reference . The attenuated glycaemic response to ICP and RCP are consistent with the in vitro data showing slower release of starch amylolysis products (glucose oligomers), and reduced glucose availability compared with the glucose reference drink.The acute incremental areas under the postprandial curves  than the glucose reference . Peak capillary glucose concentration after RCP  and ICP  were significantly lower than after the glucose drink . Although differences in peak glucose time were observed (30 min for capillary blood and 45 min for CGM), the correlations between capillary and CGM values at both 30 and 45 min were strong and highly significant . The main limitation of the CGM technology was the 14% failure rate of the devices.Capillary blood glucose responses E followein vivo glycaemic effects of RCP and ICP were evident only in the first 45 min following test drink consumption, and were not as clear as seen in vitro. While the in vitro assay is highly sensitive to differences in starch susceptibility , is likely the result of higher starch bioaccessibility , 30 or 60% (w/w) of the white wheat flour in a white wheat bread recipe, and determined the effects on microstructure and physical characteristics of the final baked product. These results are presented together with the palatability scores that were collected from study participants (Bread rolls of each type  were bakThe Resistant Starch ‘rapid assay’ method (Megazyme) showed that 15.5% of the starch in the ICP was classified as ‘resistant’, and the proportion of ‘resistant starch’  detected in control (0%), 30% and 60% breads, respectively, corresponded to the proportion of ICP used in each recipe. These data suggest that some forms of resistant starch in the ICP were preserved through bread-making, and the starch amylolysis experiments in the following section provide a more complete indication of bread starch digestion profiles.A similar amount of wheat protein per serving (11.2–13.5 g) was achieved across all bread rolls by addition of vital gluten to replace gluten from the wheat flour. Because ICP has a lower starch content, but higher protein, fat, and dietary fibre content than wheat flour , matchin– ‘Results of colour analysis’). Imaging of breads incorporating ICP . Springiness, cohesiveness and resilience . Loaf volume showed a decrease with respect to ICP replacement level in the bread rolls, but the differences between breads were not statistically significant .All bread rolls had a similar appearance and size A, regardting ICP B demonstting ICP C, with hting ICP  all incrsilience  decreasenificant . Howevernificant  increaseF2,57 = 7.45, p = 0.001), and scores for ‘difficulty in consuming the given volume’ were significantly higher for the 60% ICP bread compared with the control . These scores could reflect the larger serving (mass) of 60% bread, compared to the 0% and 30% breads. These preliminary participant scores of bread quality attributes are encouraging, and further work will be needed to ascertain acceptable limits of wheat flour substitution with representative consumer groups.Participant scores (n = 20) of bread attributes D provide3.3Recent studies of common beans have shown that structural integrity, cell wall permeability and susceptibility to amylase depends on the processing conditions , and it In vitro starch amylolysis of bread rolls revealed for the first time, that starch encapsulated within the ICP retained its resistance following bread baking. ICP replacement of wheat flour significantly attenuated starch amylolysis profiles of the bread  and lowered iAUC120 values in a dose-dependent manner (C∞) and rate constant (k). The mean values and standard errors of these parameters obtained for the 0%, 30% and 60% bread types were C∞ = 68.3 ± 3.3, 51.4 ± 1.7 and 44.9 ± 1.2% and k = 0.040 ± 0.003, 0.045 ± 0.003 and 0.032 ± 0.002 min−1, respectively. The covariance of these parameters is shown in the parameter plots (C∞), and that 60% substitution caused a reduction in rate of digestion as well as the extent. Furthermore, imaging  diffused into damaged cells, but not into intact cells. Together, these findings indicate that wheat, which provided ~75% and ~41% of the total starch in the 30 and 60% bread rolls, respectively, is the main source of starch amylolysis products in the in vitro assay, whereas the ICP starch enclosed within plant cells remains inaccessible to amylolysis despite further processing during bread making.t manner A. Baselier plots A insert, imaging B of breain vivo in healthy participants following a randomised cross over design in Study 2 (in vitro digestion assays and the glycaemic study in vivo.Glycaemic responses (measured by CGM) to 0, 30 or 60% (w/w) ICP bread rolls  were determined F1.47,20.6 = 5.03, p = 0.024), and a highly significant effect of time  and a bread × time interaction . The iAUC120 , with 30% and 60% bread iAUC120 values ~40% lower than the control. These significant differences in relative glycaemic responses to ICP enriched bread rolls are explained by the lower bioaccessibility of starch from ICP seen in vitro.The breads containing ICP elicited considerably lower glycaemic responses compared with the control bread. There was a significant overall effect of ICP bread on glycaemia C , with lower peaks after ingestion of ICP enriched breads  compared to control . Mean glucose concentrations peaked at ~45 min irrespective of bread type . Additionally, a second glucose peak was observed between 90 and 120 min after consumption of the 30 and 60% breads, and based on the in vitro starch amylolysis kinetics, we can speculate that the first glucose peak may reflect the highly digestible wheat starch, whereas the second peak could reflect the gradual and sustained availability of glucose from digestion of starch in the ICP. The shape of the postprandial curve is more relevant for interpretation of postprandial pathophysiology and appetite than the iAUC  that underpins the low glycaemic effects of whole pulses into bread and other highly processed food products. The results of the acute metabolic study provide strong justification for undertaking further chronic intervention trials of bread and other products enriched with cellular legume powders on glycaemic control in pre-diabetic and diabetic cohorts.This study utilised a combination of Balazs H. Bajka: Conceptualization, Formal analysis, Data curation, Writing - original draft, Writing - review & editing, Supervision, Project administration. Ana M. Pinto: Formal analysis, Investigation, Data curation, Writing - original draft, Writing - review & editing. Jennifer Ahn-Jarvis: Formal analysis, Investigation, Resources, Data curation, Writing - original draft, Writing - review & editing. Peter Ryden: Formal analysis, Investigation, Resources, Data curation, Writing - original draft, Writing - review & editing. Natalia Perez-Moral: Investigation, Data curation, Writing - original draft, Writing - review & editing. Alice van der Schoot: Investigation, Data curation. Costanza Stocchi: Investigation, Data curation. Catherine Bland: Investigation, Data curation. Sarah E. Berry: Conceptualization, Writing - review & editing, Supervision, Funding acquisition. Peter R. Ellis: Conceptualization, Writing - review & editing, Project administration, Supervision, Funding acquisition. Cathrina H. Edwards: Conceptualization, Writing - original draft, Writing - review & editing, Formal analysis, Visualization, Project administration, Supervision, Funding acquisition.There are no immediate conflicts of interest. The methods of preparation of chickpea cell powder (as PulseON®) is covered by a published international patent application but is not commercialised. The patent is authored by CE and PE. SB receives consultancy and shares from ZOE Global Ltd. The other authors  have no conflicts of interest to declare.


Leptalpheus forceps (family Alpheidae) has a unique natural history as an infaunal symbiont of larger burrowing crustaceans. The mitogenome of L. forceps was sequenced, the first for a symbiotic representative of the family and the first for a species outside of the genus Alpheus. The complete mitogenome was 15,463 bp in length and included 13 protein-coding genes, 12 tRNAs, and 2 rRNAs. The gene order matched all known alpheid mitogenomes. Similar to other caridean mitogenomes, the nucleotide composition was A + T biased (62%). A maximum-likelihood phylogenetic analysis of caridean mitogenomes strongly supported monophyly of the family Alpheidae.The snapping shrimp  Leptalpheus forceps is an obligate associate of burrowing ghost shrimps in the families Callianassidae and Upogebiidae, inhabiting the burrow of these hosts  include species that have evolved obligate and facultative symbioses with members of at least eight animal phyla Bruce . The snaWilliams . While mL. forceps was collected with a hand-operated extraction corer  following the manufacturer’s instructions. We prepared genomic DNA library using the Nextera XT Library Preparation Kit  following the manufacturer’s instructions. The library was sequenced  on the Illumina MiSeq Nano v2 platform at the Clinical Genomics Center at the Oklahoma Medical Research Foundation. We obtained 767,201 paired-end reads with a total of 192 million bases. A partial mitogenome assembly was generated by mapping our reads against the Alpheus japonicus mitogenome in Geneious Prime ver. 2019.1.1 using blastn. We used a 672 bp putative cox2 sequence from this partial assembly as a seed sequence to assemble the entire mitogenome using NovoPlasty ver. 2.7.2  for an Alpheidae + Palaemonidae clade as well as for the family Alpheidae, with L. forceps diverging from the four Alpheus mitogenomes (Leptalpheus than Alpheus, as these genera are considered only distantly related within Alpheidae.The mitogenome of ogenomes . The seq


Trypanosoma brucei that are required for PFR formation, PFR assembly factors 1 and 2 . Deletion of either PFR-AF1 or PFR-AF2 dramatically disrupted PFR formation and caused a reduction in the amount of major PFR proteins. The existence of cytoplasmic factors required for PFR formation aligns with the concept that processes facilitating axoneme assembly occur across multiple compartments, and this is likely a common theme for extra-axonemal structure assembly.Eukaryotic flagella are complex microtubule-based organelles that, in many organisms, contain extra-axonemal structures, such as the outer dense fibres of mammalian sperm and the paraflagellar rod (PFR) of trypanosomes. Flagellum assembly is a complex process occurring across three main compartments, the cytoplasm, the transition zone and the flagellum itself. The process begins with the translation of protein components followed by their sorting and trafficking into the flagellum, transport to the assembly site and incorporation. Flagella are formed from over 500 proteins and the principles governing assembly of the axonemal components are relatively clear. However, the coordination and location of assembly of extra-axonemal structures are less clear. We have discovered two cytoplasmic proteins in  Summary: Two cytoplasmic proteins are shown to be required for paraflagellar rod formation, which are extra-axonemal structures found in the flagella of trypanosomes. Trypanosoma brucei, have shown that the flagellum is a complex organelle containing over 1000 proteins  cytoplasmic assembly and maturation of the outer dynein arm complex, (2) transport of the complex into the flagellum and (3) docking of the complex to the microtubule doublet  the cytoplasm, (2) the transition fibre-transition zone and (3) the flagellum. Protein components are synthesised in the cytoplasm, then sorted and directed to the flagellum via the transition fibre and transition zone, with many subsequently transported to the flagellum tip by the intraflagellar transport system before being incorporated into the flagellum structure . Not alln humans . In addi doublet . Thus, aChlamydomonas, vane structures found in protists such as the fornicate Aduncisulcus paluster and the paraflagellar rod (PFR) in T. brucei and other Euglenozoa  and is attached to the axoneme via microtubule doublets 4 and 7 . Howeverfied PFR . The extassembly . The twotructure  and a prtructure .The incorporation of axonemal proteins into the flagellum requires processes within three different compartments . We know that extra-axonemal structure assembly occurs within the flagellum of cells such as trypanosomes and sperm ,b. In trHere, we identified two cytoplasmic proteins, which together form a complex that is required for PFR formation. This suggests that assembly of extra-axonemal structures, as with the axoneme, requires processes in all three compartments. Presumably, such cytoplasmically localised phenomena reflect a need for the construction of major flagellar structures to be regulated and coordinated to ensure delivery of correct stoichiometric amounts of components during precise temporal windows and via particular transport systems.PF09728). Results from TrypTag, a global survey of trypanosome protein localisation, showed that the mNeonGreen (mNG)-tagged Tb927.10.8870 protein localises to the cytoplasm and is excluded from the flagellum and the nucleus . However, we noticed that these null mutants took a longer time than normal to grow after the transfection, so we therefore measured the growth rate of the null mutant . The Ty-tagged protein ran at the expected size on the western blot and its localisation was restricted to the cytoplasm, as observed with the mNG-tagged protein. The growth rate of the add-back cell line was similar to that of the parental cell line, and cells with abnormal numbers of nuclei and kinetoplasts were much less frequent than compared to the null mutant  of cells with one flagellum had a bulge at the distal tip of the flagellum in comparison to only ∼3% of comparable parental (n=92) and add-back (n=165) cells . This suggests that flagellum assembly in these cells was disrupted. To determine whether there were changes in the trypanosome flagellum upon deletion of Tb927.10.8870, we stained the parental, null mutant and add-back cells with monoclonal antibodies that detect an axonemal component, TbSAXO (mAb25), and the PFR component PFR2 (L8C4) (Careful examination of the light micrographs of null mutant cells showed that 52% (5) cells B. When n2 (L8C4) A. In then=100), whereas the null mutant had a slightly longer mean flagellum length, 19.9 µm (n=100) and the add-back cells had a shorter mean flagellum length, 16.4 µm (n=100). This suggests that there is a potential connection between PFR assembly and flagellum length.We further analysed the effect of PFR-AF1 deletion by generating detergent-resistant cytoskeletons of parental, null mutant and add-back cells, which were subsequently stained with L8C4 and ROD1, a monoclonal antibody against Tb5.20 , a compoFig. S2B). This again showed that the loss of PFR-AF1 disrupted PFR formation, with PFR2 not integrated into the PFR structure. The PFR2 detected in the detergent-soluble fraction ran at a consistently lower molecular weight than the PFR2 in the whole-cell lysate or detergent-insoluble fraction  A, a–f. Il mutant . Howeverl mutant A, c–d. Tl mutant A, d. Howl mutant B. For thThe cytoplasmic localisation of PFR-AF1 suggested that this protein is unlikely to be involved in the transport of PFR protein components into and along the flagellum or their incorporation into the flagellum. The assembly of certain axonemal components, such as the outer dynein arms, occurs in the cytoplasm before the assembled complex is transported into the flagellum . To inveFig. S2C,D). This suggests that PFR2 was being degraded in the PFR-AF1 null mutant in part by the proteasome, and that PFR-AF1 was important for PFR protein stability.An alternative explanation for the effect of PFR-AF1 deletion on PFR assembly is that PFR-AF1 is a cytoplasmic factor required for the specific translation of PFR proteins and its loss results in reduced PFR protein expression. To test this hypothesis we treated cells with MG132, an inhibitor of the proteasome, in order to stabilise proteins that would normally be degraded without having an effect on translation. If PFR-AF1 is important for PFR protein translation, then the addition of MG132 to the null mutant would not alter the amount of PFR protein in the cell. However, after addition of MG132 to the null mutant for 8 h we saw an increase in the amount of PFR2 relative to mAb25 signal, although the amount of PFR2 did not reach parental levels . The growth rate of the Tb927.7.1360 null mutant was compared to that of the parental cell line and we found that it was consistently slower growing (n=240) of Tb927.7.1360 null mutant cells with one flagellum, as also previously seen in cells without PFR-AF1. Taken together, this suggested that Tb927.7.1360 was likely affecting PFR formation.To understand the function of Tb927.7.1360, we generated a cell line in which both alleles of  growing A. Imagin growing B,C. In aFig. S3B, We investigated the effect of Tb927.7.1360 deletion on the structure of the axoneme and PFR by staining the cells with the axoneme-binding antibody mAb25 and the PFR-binding antibody L8C4 D. There Chlamydomonas. Initial research on these structures described their structure and assembly kinetics, and was followed by later work cataloguing and functionally analysing their constituent parts , the axoneme and basal body (KIF9B), or the transition fibres (FOPL) . The delThe residual PFR structure observed by TEM when PFR-AF1 was deleted was similar to that observed with PFR2 RNAi knockdown. However, PFR2 was still present in the PFR of the flagellum in the null mutants, so it is likely that composition of this residual structure is different to that observed in the PFR2 RNAi mutant . InteresIn sperm, the ubiquitin conjugating enzyme UBE2B was shown to be important for the positioning and assembly of the longitudinal columns of the fibrous sheath . AdditioThe PFR has a regular, ordered structure and it is unlikely that this structure would arise spontaneously, suggesting that additional proteins are required to assist its assembly. Based on the assembly model outlined above, one can conjecture a number of different functions for such additional proteins including cytoplasmic assembly, post-translational modification, transport, and flagellar-based chaperones. As the PFR contains nearly 200 proteins , it is uThe role of the PFR-AF1–PFR-AF2 complex in PFR formation still needs to be defined. The cytoplasmic localisation of these proteins and the possible roles for PFR-AFs discussed above may indicate that PFR-AF1 and PFR-AF2 are involved in the assembly of multi-subunit complexes. For example, do PFR-AF1 and PFR-AF2 act as a scaffold onto which PFR proteins can assemble before disengaging to allow the PFR unit to be transported to the flagellum? An enrichment of PFR proteins was not seen in the PFR-AF1 immunoprecipitation suggesting that any interaction between the PFR-AF1–PFR-AF2 complex and PFR proteins is likely to be transient and that the methods used here were not sensitive enough to detect such an interaction. Moreover, we do not know which of the nearly 200 PFR proteins are present in these putative PFR protein complexes. Although there is evidence for interaction between PFR proteins, none of these interactions involve PFR1 and PFR2 . There iEuglenozoa but it can vary in appearance greatly between different species, with A.deanei and S.culicis having a much-reduced PFR structure that does not contain PFR2  was added without disturbing the pellet. After 1 h at room temperature, samples were washed three times in 0.1 M sodium phosphate buffer, pH 7, for 5 min and post-fixed in 1% osmium tetroxide in 0.1 M sodium phosphate buffer, pH 7, for 90 min at room temperature under agitation. Samples were washed in distilled water three times for 5 min and stained in 2% uranyl acetate for 12 h at 4°C. Samples were washed three times in water and dehydrated in increasing concentrations of acetone  and embedded in Agar-100 resin (Agar Scientific). Thin sections were post-stained in 2% uranyl acetate and 3% lead citrate. Images were obtained using a Hitachi H-7650, FEI Tecnai G2 Spirit or Jeol 1400Flash, operated at 120 kV.Cells were fixed by addition of glutaraldehyde into the cultures to a final concentration of 2.5%. Cells were harvested by centrifugation at 800 g for 5 min and primary fixative was added . After 2 h, cells were washed twice in PBS and settled onto round glass coverslips for 5 min. Coverslips were washed two times in PBS and sample dehydration was performed using increasing concentrations of ethanol  for 5 min in each step. Samples were dried using a critical point dryer. Coverslips were mounted onto SEM stubs using silver DAG (Agar Scientific) and coated with gold using a sputter coater. Images were acquired on a Hitachi S-3400N scanning electron microscope.Fixation was performed by adding glutaraldehyde into the cell culture to final concentration of 2.5%. Cells were harvested by centrifugation at 800 The primary antibodies used in this work and their dilutions were as follows: monoclonal mNeonGreen antibody  diluted 1:100 for western blotting, monoclonal RFP antibody  diluted 1:1000 for western blotting, monoclonal BB2  diluted 6 cells were harvested by centrifugation at 800 g for 5 min, washed in PBS and incubated with 50 µl of 1% NP-40 in PEME  containing protease inhibitors (Roche) for 5 min on ice. To obtain whole-cell samples after the treatment, 50 µl of 2× Laemmli buffer was added to the cell lysate. Soluble and insoluble fractions were separated by centrifugation at 20,000 g for 10 min at 4°C. To the soluble fraction, 50 µl of 2× Laemmli buffer was added. The insoluble fraction was resuspended in 50 µl of 1% NP-40 in PEME and 50 µl of 2× Laemmli buffer. All samples were incubated at 100°C for 5 min. For proteaseome inhibition experiments, MG132 (Sigma-Aldrich) was added to cells at a concentration of 20 µM for 8 h before preparation of whole-cell samples.2×106 or 2×107 cell equivalents were loaded onto a 12% SDS–PAGE gel. After transfer, membranes were blocked with blocking solution (3% milk and 0.05% Tween 20 in PBS) for 1 h, incubated with primary antibody diluted in blocking solution for 1 h, washed three times in blocking solution and incubated with secondary antibody diluted in blocking solution for 1 h. Proteins were detected by ECL. Protein band signal intensity was measured in ImageJ , the background was subtracted and then the signal normalised to the mAb25 loading control.1×10g for 5 min, washed in PBS and settled onto glass slides for 5 min. For whole-cell preparations, cells were fixed in −20°C methanol for 20 min and rehydrated in PBS for 10 min. For cytoskeleton preparations, cells were first treated with 1% NP-40 in PEME for 5 min, washed in PBS and fixed in −20°C methanol for 20 min. After fixation, the slides were blocked for 1 h at room temperature with blocking solution (1% BSA in PBS) and incubated in primary antibody diluted in blocking solution for 1 h. After washing three times in PBS, slides were incubated in secondary antibody diluted in blocking solution for 1 h. After three washes in PBS, slides were incubated in 20 μg/ml Hoechst 33342 (Sigma-Aldrich) in PBS, washed in PBS and mounted before imaging. For live-cell microscopy, cells were washed three times in PBS supplemented with 10 mM glucose and 46 mM sucrose (vPBS). In the second wash, DNA was stained using 10 µg/ml Hoechst 33342. After the third wash, cells were re-suspended in vPBS, settled onto glass slides, mounted with a coverslip and imaged immediately. Images were taken using a Zeiss Axio Imager.Z1 microscope equipped with an ORCA-Flash 4.0 CMOS camera using a Plan-Apochromat 100×/1.4 NA oil objective. Images were acquired and analysed with ZEN 2 PRO software and assembled for publication in Adobe Photoshop CS6.Cells were harvested by centrifugation at 800 8 cells were harvested by centrifugation at 800 g for 5 min, washed in PBS and resuspended into 1 ml of lysis buffer  for 5 min on ice. Soluble proteins were separated by centrifugation at 16,000 g for 10 min at 4°C. 25 µl of mNeonGreen-trap magnetic agarose previously washed in washing buffer (10 mM Tris–HCl pH 7.5 and 150 mM NaCl) were incubated with 500 µl soluble protein lysate  for 30 min at 4°C tumbling end-over-end. Beads were magnetically separated and washed twice with washing buffer. Samples were eluted into 100 µl 1× Laemmli buffer for western blotting analysis or into 50 µl of 4 M urea for mass spectrometry analysis.2×10T. brucei database and a list of common contaminants. Protein and PSM false discovery rates were set at 0.01. The protein group output file was them imported to Perseus 1.5.2.4, to perform a Student’s t-test.Eluates from the immunoprecipitation were reduced, alkylated and then digested overnight with trypsin. The peptides were separated on an Ultimate 3000 UHPLC system and then directly electrosprayed into the coupled QExactive mass spectrometer. The raw data was acquired on the mass spectrometer in a data-dependent mode and full-scan MS spectra were acquired in the Orbitrap. The raw data was analysed using MaxQuant (version 1.5.3.8) with the integrated Andromeda search engine. For protein identification, peak lists were searched against a


Will increased exposure to osteopathic medicine improve allopathic resident and attending physicians’ support of implementing an osteopathic curriculum in graduate medical education? The overall goal of this quality improvement project was to examine the familiarity and interest of allopathically-trained residents and attendings with osteopathic medicine before, and after, a brief educational workshop.Setting: Mercy Health Saint Mary’s Hospital in Grand Rapids, MI. Participants included a sample of 27 Family Medicine (FM) residents and attendings who had been trained in allopathic medical schools. First, a one-hour lecture on “Foundations of Osteopathic Medicine” was given. The lecture included a PowerPoint presentation followed by a 15-minute hands-on demonstration of osteopathic diagnosis and treatment. A pre and post-workshop survey modified from another larger study was administered to all attendees. Primary selected outcomes included percentage of allopathic resident participation in attendance and pre-post-workshop response differences.Of the 31 allopathic residents in this community-based FM program, 23 (74.2%) were present for the lecture and completed both a pre and post-workshop survey. A total of 24 (78%) of participants had never attended a previous lecture on osteopathic medicine. Respondents’ overall attitude regarding the implementation of an osteopathic curriculum was generally positive after the workshop. Of the 27 participants (23 residents and four attendings) surveyed before the lecture, 23 (85.2%) were initially interested in learning how to perform osteopathic manual treatment (OMT), this increasing to 25 (92.6%) after the workshop. The Mercy Health FM resident respondents were initially first supportive of osteopathic medicine at 59.3%, improving to 77.8% after completing the workshop.These results suggest that many allopathically trained physicians may lack exposure to osteopathic medicine principles. In this allopathic-oriented residency sample setting, a relatively brief workshop increased attendees’ interest in osteopathic medicine. Results suggest that initial and refresher exposure to osteopathic medicine content and OMT practices during residency curricula can be used to elicit support from allopathically-trained resident physicians. By 2020, the merger of the Accreditation Council for Graduate Medical Education (ACGME) and the American Osteopathic Association (AOA) will fully integrate the accreditation of both allopathic and osteopathic residency programs. An all-time high of 3,590 doctors of osteopathy (DO) applied in the 2017 allopathic (MD) residency matching program.During that same year, 17.85% of applicants who matched to PGY-1 Family Medicine (FM) positions were DO.Osteopathic recognition is offered to programs to increase the osteopathic curriculum for DO residents in MD-oriented programs.Since its inception in 1892 by Andrew Taylor Still in Kirksville, Missouri, osteopathic education has retained a distinct identity from allopathic medicine.As the number of DO medical schools increased for a limited set of DO-oriented residency program slots, the AOA allowed DO graduates the option of completing MD-oriented residency programs.In 1998, Dr. Johnson and colleagues suggested that osteopathic medicine educational didactics be offered for MD physicians during residency, and stressed the importance of appointing more DO faculty.In 2008, another MD-oriented program that was unable to become dually accredited developed an osteopathic curriculum and OMT clinic to develop residents’ skills.A 2016 survey indicated that Osteopathic Recognition and OMT refresher courses during residency programs remains an important factor for DO medical students.Still, there has apparently been no recently published survey projects gauging the interest of MD-trained residents or attendings in learning osteopathic medicine and OMT principles. It was therefore the overall goal of this quality improvement project to survey the familiarity and interest in osteopathic medicine from a sample of MD residents and attendings before and after participating in a brief GME workshop.The primary objectives were to: 1. Determine the familiarity and exposure of MD physicians with osteopathic medicine in a community-based MD-oriented FM residency program, 2. Evaluate MD residents’ attitudes towards osteopathic medicine before and after participating in an OMT educational workshop, and 3. To increase knowledge and awareness of the possible indications of OMT in FM for physician participants.Since this project was conducted as a quality improvement Initiative at Mercy Health, it was ruled to be exempt from formal review by the Mercy Health Regional Institutional Review Board.An unvalidated pre and post-workshop survey similar to Allee et al.Each pair of participant surveys was also initially assigned a unique number, so that potential differences between the pre- and post-workshop responses could be compared. There were no other identifiers on the survey, so there was no way to link a specific individual with a specific set of survey responses.The OMT workshop for this project included a one-hour PowerPoint lecture entitled “Foundations of Osteopathic Medicine” which was delivered as a grand rounds lecture. The content of the workshop primarily introduced attendees to the four tenets of osteopathic medicine:The human being as a dynamic unit of functionThe body possesses self-regulatory mechanisms that are self-healing in natureStructure and function are interrelated on all levelsRational treatment is based on these principles The lecture was followed by 15-minute hands-on demonstration of OMT techniques which included counterstain, myofascial release, muscle energy and high velocity low amplitude OMT treatments. All residents and attending participants were members of the ACGME-accredited Mercy Health Grand Rapids FM Residency program located in Grand Rapids, Michigan.Prior to the OMT workshop, resident and attending physician attendees completed the 20-item survey questionnaire asking about their ratings concerning dimensions of OMT training for allopathically-trained physicians. To examine for pre-post-workshop differences, attendees were asked after the workshop to complete a largely-identical survey questionnaire. Survey items were on a three or four-category Likert-type scale that was later conservatively collapsed into equivalent-sized numerical categories. .Preliminary descriptive statistical analyses confirmed that the distribution of survey item opinion responses were non-normal  in nature as might be easily expected from a smaller respondent sample in a single FM residency program. In response, a series of non-parametric Wilcoxon Matched Pair Signed Rank t test analytic proceduresOf the 31 allopathic residents in the FM program, 27 (74.2%) were present for the lecture and completed both a pre and post-workshop survey. The training levels of the sample was diverse with seven (26%) being PGY-1 residents, six (22%) PGY-2 residents, 10 (37%) PGY-3 residents and four (15%) attending physicians completing surveys. The majority,  had completed medical school in the United States. (Table 1)Data concerning respondents’ level of familiarity with OMT procedures, as well as varied OMT exposures were also collected. Only one (3.7%) respondent indicated that they were “very familiar” with OMT before the workshop, leaving 13 (48.1%) “somewhat familiar” and 13 (48.1%) “unfamiliar.” Reported types of previous OMT exposure also varied, as 16 (59.3%) had experience through demonstrations, three (11%) through reading, seven (26%) had personal experience, five (18.5%) had exposure through a fellow resident. Only six (22.22%) respondents reported having ever received a past OMT lecture and seven (26%) participants reported having never had any previous experience with OMT. (Table 2)Respondents’ overall attitudes regarding the implementation of an OMT curriculum were also positive and increased significantly after the “Foundations of Osteopathic Medicine” workshop was completed. (p = 0.001) Of the 27 residents and attendings who were surveyed before the workshop, 23 (85.2%) were interested in learning how to perform OMT, increasing slightly to 25 (92.6%) after completing the workshop. (p = 0.560) (Tables 3 and 4)Using the selected analytic software, each of seven key paired pre-post-workshop survey ratings levels were shown to have numerically increased, with four items reaching statistical significance . (Table 4)The results from this study demonstrate that most respondents in this MD-oriented FM residency program had not been very familiar with osteopathic medicine principles or techniques.Table 2 demonstrated that after receiving the “Foundations of Osteopathic Medicine” lecture the overall attitudes toward osteopathic medicine improved with 25 (93%) respondents being interested in learning more about OMT in the future.Following the workshop, no respondents believed that OMT was “not effective” in the treatment of somatic conditions. Both the questions pertaining to somatic and systemic effectiveness of OMT had statistically significant pre-post-workshop increases with p values of 0.006 and <0.001 respectively. (Table 4) This may be due to the fact that workshop lecturers had repeatedly emphasized that “form” and “function” were related, and that systemic disease could can benefit from somatic OMT treatments.Before the workshop, our respondents were generally supportive of implementing an osteopathic curriculum content into MD-oriented programs, with 16 (59.3%) choosing either “strongly support” or “support”, 10 (37%) “neutral” and only one (3.7%) opposed. (Table 3) When asked if the AOA should provide certification for MDs who have tested proficient in the use of OMT, 14 (51.8%) MDs in our program chose “strongly support” despite having very little prior exposure to OMT. (Table 3)With more MDs presumably increasingly interested in learning OMT, hands-on training and refresher experiences similar to Lieber, et al. (2005)It is notable that changes in three survey items  did not increase at statistically significant levels. These survey items could be interpreted as requiring a more personal action/performance from the respondents or systemic developments from the osteopathic profession than the other values/attitude-type survey items. The delivered content of the OMT workshop, sampling error and our small sample size may have also limited our ability to detect significant pre-post workshop differences from these items.Question 2 (see Table 3) asked “To what extent are you interested in learning how to perform OMT.” Since our PowerPoint presentation comprised the majority of the OMT workshop, only fifteen minutes was allotted to demonstrating OMT techniques. This short duration of OMT exposure may have not been enough to significantly influence our post-survey item responses. Both Questions 6 and 7 differences were non-significant and may have also been less immediately relevant to the residents who were surveyed.Our ability to detect some pre-post-workshop differences may have been limited by our small sample size of FM respondents. Due to the size of our sample subgroups, resident and attending responses could not be realistically compared during analysis of final survey results.Although the majority of FM residents  in this study setting were surveyed, only a minority of eligible attendings participated. Two attendings in this residency program were not surveyed as they had been osteopathically trained and the aim of this project was to measure the opinions of allopathically trained physicians.Next steps may involve implementing a lengthier osteopathic curriculum lecture series on “Osteopathic Medicine” for incoming residents and attending faculty. Similar to some earlier cited works,These results suggest that a relatively brief one-hour workshop may serve to improve some MD physicians’ interest levels toward osteopathic medicine and OMT techniques. However, the upcoming ACGME and AOA merger provides both challenges and opportunities for the future of community-based and other GME settings.It may be beneficial for the AOA and ACGME to continue to collaborate and clarify how MDs might be able to learn and implement osteopathic medicine principles and techniques in the future. The conduction of larger-scale studies with multiple residency programs, medical students and other clinical specialties may clarify how to best implement such GME training workshops across the nation.The authors declare no conflict of interest.


NnHXK. 26 lotus cultivars that the 320-bp fragment presented in NnHXK had a lower content of amylose and a higher content of amylopectin. FMGBSS-I8 was developed based on polymorphisms of two haplotypes of NnGBSS. The group containing 32 lotus cultivars with the 210-bp fragment had less amylose content and more amylopectin content. FMAGPL-I1 was developed based on polymorphisms of two haplotypes of NnAGPL (ADP-glucose pyrophosphorylase large subunit gene). The group containing 40 lotus cultivars with the 362-bp fragment had less amylopectin, total starch content and more amylose content. According to the study, FMHXK-E1, FMGBSS-I8 and FMAGPL-I1 are closely related to lotus starch content. It could be provided research basis for molecular assisted selection of lotus starch content improve breeding efficiency.It have been significantly demonstrated that Hexokinase (HXK), Granule-bound starch synthase (GBSS) and ADP-glucose pyrophosphorylase (AGPase) are three critical enzymes in the starch biosynthetic pathway and are related to starch  content in lotus. It is important to develop functional markers in marker-assisted selection of lotus breeding. So far there have been few reports about lotus functional markers. In this study, based on insertion-deletions (INDELs) and single-nucleotide polymorphisms (SNPs), we developed three functional markers, FMHXK-E1, FMGBSS-I8 and FMAGPL-I1. FMHXK-E1 was developed based on polymorphisms of two haplotypes of  Nelumbo nucifera Gaertn), a perennial aquatic herb, is one of the oldest dicotyledonous plants1, which originated and has been widely grown in southern China for thousands of years2. Nelumbo Adans, a surviving living fossil that has experienced the Quaternary glacial period, has an evolutionary history of almost 135 million years. In addition to the evolution values, lotus is also a kind of essential traditional Chinese medicine and food and its rhizome has been widely consumed for over 7000 years in Asia3. Starch content is one of the main factors affecting the lotus root processing and cooking quality. Starch can be divided into amylose and amylopectin in plant4. Amylose is a 200 glucose groups polysaccharide linear molecule with glucose residues linked together by α- glycosidic bonds. Amylopectin is a 300 to 400 glucose groups chain molecule linked together by α-  glycosidic bonds and α-  glycosidic bonds6.Lotus 8. FMs that developed from gene polymorphisms affect phenotypic trait variation9. Therefore, it is necessary to understand the function of genes in the development of functional markers. In recent years, the new type of molecular marker (FM), based on the insertion/ deletion (INDEL) and the single-nucleotide polymorphism (SNP), has been successfully developed and play a broader role in plant molecular marker-assisted breeding12. For example, the functional marker of GBSS has been used to select wheat materials, which is linked with flour quality13. Although traditional SSR and ISSR analysis of genetic diversity have been used in lotus cultivars16, functional markers associated with starch content have not been developed and applied in lotus cultivars. So, it is a critical step towards selecting suitable lotus cultivars to develop functional markers on the identification of amylose, amylopectin and total starch content.Identifying genes and molecular markers associated with trait variation is obligatory for comprehending molecular breeding and crop improvement17. Numerous studies showed that granule-bound starch synthase was encoded by the GBSS gene, which catalyzes amylose synthesis20. AGP is a rate-limiting enzyme which catalyzes ATP and Glc-1-P to pyrophosphate and ADP-glucose (ADPG). ADPG acts as the substrate for the synthesis of amylose and amylopectin under the action of others starch synthases23. HXK could provide a carbon stream for plant starch synthesis which catalyzes fructose to Glc-1-P6. Identifying genes that control starch content could contribute to explore molecular markers about starch content.Starch is only produced through biosynthetic pathway, which involves lots of conservative function enzymes, such as hexokinase (HXK), granule-bound starch synthase (GBSS), ADP-glucose phosphorylase (AGP), soluble starch synthases (SSS), starch branching enzymes (SBE), starch debranching enzymes (DBE) and so onHXK), granule-bound starch synthase gene (GBSS), ADP-glucose phosphorylase gene (AGP), soluble starch synthases gene (SSS), starch branching enzymes gene (SBE), sucrose synthase gene (SUSY), fructokinase gene (FRK), UDP glucose pyrophosphrylase gene (UGP), isoamylase gene (ISA) and pullulanase gene (PUL), were employed to develop functional markers. Our group have resequenced to >13× raw data of 69 lotus accessions (accession no. SRP095218). On the basis of these genomic data and the starch biosynthetic pathway, the SNP and Indel were identified in lotus. We validated the Indel and SNP in 46 different cultivars and further analysis revealed that three enzymes, HXK, GBSS and AGPL, significantly associated with starch content are encoded by Indel or SNP of genes. Finally, we respectively developed three functional markers, FMHXK-E1, FMGBSS-I8 and FMAGPL-I1, from HXK, GBSS and AGPL.In this study, 10 genes, hexokinase gene  and the percentage in total starch (22.09%) are both significantly higher than the percentage of amylopectin in dry matter (3.13%) and the percentage in total starch (11.53%). Correlation analysis showed that the significance differences of amylose and amylopectin content in total starch with diversity bands of marker reached a high level. In FMHXK-E1, with diversity bands of marker, correlation analysis showed significant differences in amylose and amylopectin content in total starch that P value reached a high level at 0.010 and 0.008(P ≦ 0.01) respectively. P value reached a very level at 0.007 (P ≦ 0.01) of amylose content in dry matter  and in total starch (23.56%) of 14 lotus cultivars missing the 210-bp fragment are both higher than the amylopectin percentage in dry matter (3.51%) and in total starch (13.09%) of 32 lotus cultivars with the 210 bp fragment. Apparently, there was a significant correlation between the PCR bands diversity and starch (amylose and amylopectin) content. In FMGBSS II−1, with diversity bands of marker, correlation analysis showed significant differences in amylose and amylopectin content in total starch that P value at 0.040 and 0.022(P ≦ 0.05) respectively. The P value at 0.026 (P ≦ 0.05) of amylose content in dry matter , a 37-bp insertion in the second intron exhibited significant association with wheat Psy1 (phytoene synthase 1) activity30. Similar study had certificated SNP A/G polymorphisms significant associated with Dhn1 and Rsp41 activity for the drought resistance10.There was the explanation allelic variation at some loci might indeed be causal for the trait variation for this finding. And INDELs or SNPs polymorphisms were performed to significantly affect characters of agronomic33. Target lotus root with suitable starch content could be selected from different lotus root cultivars and our results illustrated these functional markers were credible as reliable markers for molecular assisted selection of lotus starch content. The markers are closely linked to the target genes. Compared to the traditional way that measuring the starch content of rhizomes in the later stage of lotus growth, it provides the possibility for selection in the early stage and single plant can be selected as the test object. It can greatly reduce the blindness of the breeding process, shorten the breeding life and improve breeding efficiency.At present, conventional breeding methods are inefficient in improving the starch traits of lotus roots. For this case, functional markers developed from genes that control enzymes involved in starch, is more wisely to select plants in the seedling stage. It can accelerate genetic breeding process by improving the lotus root quality and traits. The experiment of this study is an association tests for starch content traits in advanced cultivars and breeding materials of lotus root. Three functional markers, FMHXK-E1, FMGBSS-I8 and FMAGPL-I1, were developed based on sequence polymorphism among genotypes. In FMHXK-E1 and FMGBSS-I8 markers, amylose and the amylopectin content in the dry matter and total starch have significant differences with fragment polymorphism, but they are different in degree. The difference between FMHXK-E1 and starch content is extremely significant (P≤0.01), FMGBSS-I8 and starch content are significantly different (P≤0.05). In FMAGPL, total starch in dry matter had a significant difference (P=0.042) with fragment polymorphism. All of the loci were selected based on co-localization of functional candidate genes for starch content46 different cultivars of lotus roots were provided by the Guangchang Bailian Institute of Jiangxi Province, China Table . Genomicet al.34 based on at least three main roots selected from 46 lotus roots. Then the proportion of each component were calculated according to amylose content and amylopectin content. 36. Full length cDNA sequence of NnAGPS1 (GeneBank accession no. KJ476823) and NnAGPL (GeneBank accession no.KJ476824) were isolated and deposited to the GeneBank. Genomic DNA sequence of NnAGPS1 (GeneBank accession no. KJ476825) and NnAGPL (GeneBank accession no. KJ476826) were also obtained and sequenced21.The whole genome sequence of Chinese Lotus had already been made publicAccording to Genomic DNA and cDNA sequences of genes, primers were designed by using the software Primer Premier 5.0 meeting the following constraints: GC content of 40–60%, 18–22 nucleotides in length, no secondary structure, and no consecutive tracts of a single nucleotide. All primers were synthesized by AuGCT Corporation .Taq DNA polymerase, 1.4 μL genomic DNA, 1.5 μL forward primer (20 μM), 1.5 μL reverse primer (20 μM), 2.5 μL of 10× buffer, 0.6 μL dNTPs (5 mM each), and 15.5 μL ddH2O. The PCR cycling was performed in a LifePro Thermal Cycler and amplifyed under the following conditions: 94 °C for 5 min, 35 cycles of 30 s at 94 °C, 30 s at 51–63 °C, 45 s at 72 °C, and a final extension for 5 min at 72 °C.The total volume of 25 μL mixture containing 1 μL The products of PCR amplified were mixed with loading buffer, denatured for 6 min at 95 °C, and utilized with a size standard marker of pBR322 DNA/Msp 1 (TIANGEN) to each lane. The products were analyzed by polyacrylamide gel (PAGE) electrophoresis on 6% acrylamide, visualized by silver ion staining and photographed.Based on the p/a(present/absent) of DNA bands , all materials could be divided into two groups. The number of samples from each group were analysis by Excel double sample equal variance t-test method. Based on the p/a amplified bands, we divided respectively amylose, amylopectin and total starch content into two groups. Two groups data were performed for correlation analysis.


This study evaluated the use of a free vascularized bone graft with and without cartilage from the medial femoral condyle (MFC) in patients with recalcitrant scaphoid non-union, with a special focus on union rates and the osteochondral graft for proximal pole destruction.Thirty-eight avascular scaphoid non-unions in 37 patients who were treated with a free osteoperiosteal or osteochondral MFC graft were retrospectively evaluated (mean follow-up 16 months). Bone union, the scapholunate and the radiolunate angles were evaluated on X-ray images. The range of motion, grip strength, VAS, DASH and PRWE scores were evaluated clinically.The overall union rate was 95%. Bone union was achieved in 27 out of 29 (93%) scaphoids treated with a free osteoperiosteal MFC grafts and in 9 out of 9 (100%) scaphoids treated with a free osteochondral MFC graft. The range of motion remained almost unchanged, while grip strength increased significantly (34 kg vs. 44 kg) and the VAS (22–5), DASH (59–19) and PRWE (62–30) score decreased significantly. The scapholunate (71°–65°) and radiolunate (28°–18°) angle decreased. No major donor site morbidity was observed. Postoperative complications were observed in eight cases (21%).The vascularized medial femoral bone graft leads to a good functional outcome in the treatment of scaphoid non-unions. The graft provides adequate blood supply and structural stability to the scaphoid. A proximal pole destruction can be replaced using an osteochondral graft with promising short-term results preventing carpal osteoarthritis and collapse. Owing to its frail blood supply, the scaphoid shows an increased susceptibility to the development of a posttraumatic non-union with consecutive progressive carpal instability after fracture. Healing of a scaphoid fracture is especially critical and prolonged in proximal pole fractures  . Langer Treatment of scaphoid non-union typically consists of surgical treatment using a bone graft for better healing potential. Despite increasing controversy about whether to use a vascularized or avascularized bone graft, some studies in the past suggested that to improve bone healing, for some indications, the use of a vascularized bone graft can lead to a more favorable outcome than the use of an avascularized bone graft . TraditiMunk et al. performed a systematic review of the literature regarding scaphoid non-union and reported an average union rate of 78% for proximal pole scaphoid non-union, including those with avascular necrosis (AVN) that were treated with conventional non-vascularized bone grafts . With thSeveral different vascularized bone grafts exist which can be used , 41, 44.Data on the outcome of this graft, especially on the osteochondral graft for proximal pole destruction, is rare.Therefore, this study evaluated the use of a free vascularized bone graft with and without cartilage from the MFC in patients with recalcitrant scaphoid non-union. A special focus was set on the osteochondral graft for proximal pole destruction and union rates.All patients who underwent surgical treatment of scaphoid non-unions using a free vascularized osteoperiosteal or osteochondral MFC graft between April 2008 and March 2016 at our institution were identified and included. A total of 38 wrists in 37 patients  were reviewed. The mean age at the time of surgery was 26.2 years (range 17–42 years). The right wrist was affected in 19 (51%) patients and the left one in 17 (46%) patients. One patient had surgical treatment on both wrists (3%). Mean time of persisting non-union until the surgery was 51 months (range 2–184 months) and mean follow-up time was 16 months (range 6–53 months).The indication for a vascularized MFC bone graft was based on patient’s symptoms and the correlation with preoperative radiological findings. All patients complained of wrist pain, reduced grip strength and/or reduced range of wrist motion. In all cases, preoperative CT scans showed an established non-union with a proximal pole fragment associated with bone resorption and cystic changes at the fracture site. A preoperative gadolinium-enhanced MRI examination was available for 31 (84%) patients. In all of those, either a compromised or completely missing perfusion of the proximal fragment was seen.Due to a fragmented, unstable and avascular proximal pole or previously antegrade screw fixation with destruction of the proximal pole, 9 24%) out of the 38 scaphoids were treated with a free vascularized osteochondral MFC graft and the remaining 29 (76%) scaphoids were treated using a free vascularized osteoperiosteal MFC graft. Two exemplary patients  are shown in Figs. 4% out ofStandard dorsopalmar and lateral radiographs of the wrist were taken.Assessment was performed by a reviewer who was not involved in the treatment and was blinded to the functional outcome but not to the method of treatment. The pre- and postoperative radiographs were assessed for bone healing, the scapholunate and the radiolunate angle. Changes in the scapholunate angle were evaluated to assess palmar rotation of the scaphoid. The presence of a dorsal intercalated segment instability (DISI) was assessed by changes in the radiolunate angle on lateral radiographs. All radiological parameters were compared to the uninjured contralateral side. In cases with any doubt about the signs of bone healing, CT scans were performed.Functional examination of all patients was performed by one single examiner who was not involved in the surgical treatment. Active range of motion (ROM) and grip strength were measured in a standardized manner for both wrist joints with a digital analysis system . Final functional results were compared to the preoperative measurements and to the uninjured, contralateral side. Wrist pain was evaluated using a visual analog scale . Functional subjective outcome was assessed using the Disability of the Arm, Shoulder, and Hand (DASH) Questionnaire. It has a 0–100 point scale with 0 representing the best result. Additionally, at final follow-up the Patient-Rated Wrist Evaluation Score was recorded. This scale ranges between 0 and 150 points, with no points indicating an asymptomatic wrist.The procedure was performed in the same manner as elaborately described earlier by Iorio, Bürger and Higgins and was usually conducted with a team of two to four surgeons , 17, 20.A standardized aftercare protocol was followed with an above elbow cast fixation for 4 weeks (to minimize rotation forces on the wrist and the pedicle in the early postoperative phase during forearm rotation), followed by a below elbow immobilization for another 6 weeks with K-wire removal thereafter. The palmar plate was removed in two cases after a mean of 8.5 months because of disturbing impingement. The other patients with palmar locking plates (which was intraoperatively sometimes shortened or adapted) showed no complaints which would imply implant removal. The immobilization period was followed by passive and assisted active wrist exercises.t test was used for statistical analysis. Statistical significance was set at p < 0.05.Descriptive data are reported using means, standard deviations and the range. A paired Student’s Differentiated union rates with and without previous surgery are presented in Table All patients completed the follow-up protocol. Bone union was achieved in 36 treated scaphoids . The mean time to union was 16 weeks (range 12–22 weeks) for the patients treated with an osteoperiosteal MFC graft and 18 weeks (range 14–26 weeks) for the patients treated with an osteochondral graft. One patient (3%) achieved partial union . This patient was asymptomatic; therefore, no further treatment was performed. Two patients (5%) had persisting non-unions with the bone graft being continuously resorbed throughout the first postoperative year. One patient remained asymptomatic and denied further treatment. The other patient suffered from ongoing pain and restriction in the range of motion. This patient was treated 7 months after the free vascularized MFC graft with a free vascularized bone graft from the iliac crest that led to bone union after 4 months.The humpback deformity and an associated DISI deformity were improved postoperatively and did not deteriorate during the follow-up. The scapholunate angle decreased by a mean of 6° and the radiolunate angle was partly restored with a mean of 10° improvement. Correction of the radiolunate angle was statistically significant Table .Table 2RThe results for the range of motion and grip strength are shown in Table p = 0.03). The mean preoperative DASH score was 59 points (SD 12) and decreased significantly to 19 points (SD 11) at final follow-up (p = 0.03). The mean PRWE score decreased significantly from 62 points (SD 10) before surgery to 30 points (SD 9) at final follow-up (p = 0.04). No patient had to change his/her occupation.The mean pain level decreased significantly from 22 points (range 8–64) before surgery to five points (range 0–16) at final follow-up (Postoperative complications were observed in eight cases (21%) with seven (18%) being treated with subsequent surgical interventions. Loosened or dislocated implants which led to local irritation represented four out of those complications (one associated with hyperossification). All four implants were removed after an average of 14.5 months. One late surgical site infection was seen 3 months after surgery which was treated with removal of the metal implants (one K-wire), antibiotic therapy and a V.A.C. therapy (vacuum assisted closure) for 3 days. The patient then went to unproblematic bone union. One patient suffered from a venous bleeding that had to be surgically stopped on the second postoperative day. Another patient suffered from a postoperative functional Boutonniere-like malposition in the metacarpophalangeal joint of the thumb and was treated with surgical tenolysis and tendon transfer 7 months postoperatively. Finally, one patient suffered from delayed wound healing of the palmar approach to the scaphoid that eventually healed without surgical treatment.No major donor site morbidity was observed. The only discomforts that patients noticed in the area of the medial femoral condyle were postoperative swelling, pain and hypesthesia in the area around the scar. All of these symptoms disappeared after 1 year or less.This retrospective evaluation showed that the vascularized osteoperiosteal and osteochondral medial femoral condyle graft is a good option for the treatment of scaphoid non-unions showing a high union rate, reducing pain, preventing carpal collapse and improving carpal alignment and wrist function.A special focus on the osteochondral graft even showed a 100% healing rate. Patients whose proximal pole is destroyed have few treatment options. If untreated, this would probably always lead to wrist destruction with consecutive osteoarthritis and collapse with increased pain and limited wrist function. In such cases, only salvage procedures like a radioscapholunate fusion, four-corner fusion or proximal row carpectomy seem imaginable. The findings of the present study showed that this subgroup of patients can have a good outcome if the proximal destroyed scaphoid pole is replaced with an osteochondral vascularized MFC graft. At our institution, the authors are confronted with a rather young and physically active population with a high functional demand. So when in doubt, the decision whether to try a reconstruction or conduct a salvage procedure would tend toward the attempt of reconstruction in most cases.In the previous unsuccessful bone grafting (non-vascularized graft from the iliac crest), the authors also observed a union rate of 100% by using a vascularized MFC graft. The restoration of vascularity at the fracture site seems to have improved the healing potential in those cases.Since pain levels decreased and functional scores increased significantly postoperatively, scaphoid non-unions should not be left untreated. Even the range of motion remained more or less the same despite a rather long postoperative cast fixation  similar to Higgins et Buerger .Previously failed screw fixation with or without bone grafting can be encouragingly treated with a vascularized MFC graft, leading to satisfied patients thereafter.Earlier on, iliac crest bone grafts were used to treat scaphoid non-unions. Bone grafting in the classic Russe technique showed union rates of up to 92% , 14.Other investigators found healing rates of 94% using avascular and 85–97% using vascularized iliac bone grafts , 36, 43.A long-term follow-up study also showed that 81% of the patients were pain free and the progression of osteoarthritis could be stopped in those patients who achieved bone healing . The iliHowever, later surveys showed that despite the appealing union rate, the patients suffered from significant donor site morbidity. Harpf et al. followed a collective of 60 patients after scaphoid reconstruction with a vascularized iliac crest graft and found that 32% of the patients suffered from an impairment of the lateral femoral cutaneous nerve and 63% showed bone deformations at the donor site radiologically . A systeIn 1991, Sakai et al. first described the use of a new osteoperiosteal graft harvested from the medial condylar and supracondylar area of the femur, which was pedicled from the articular branch of the descending genicular artery. It was used on six patients with a fracture non-union of the upper extremity and all of them healed uneventfully after graft implantation . After tThe following studies showed very promising results , 8, 22. Ioro et al. showed in two cadaveric studies that the descending genicular artery gives a very reliable and constant source of perfusion for the harvested graft in terms of dimensions and that it can be harvested with a skin paddle , 20. AddDue to the similar anatomy of the proximal scaphoid pole to the convex surface of the medial femoral trochlea, it was possible to replace the proximal pole entirely using this graft if required with a union rate of 94% . ImproveIn the present study, there was a slight improvement of the average SL-angle which corresponds to the study of Buerger et al. who also observed an average improvement of 6° at final follow-up , 17. As Despite these reported high union rates, it remains debatable today whether a vascularized bone graft is favorable in most cases. On the one hand, an overview by Rancy et al. showed that there is no consensus about which modality is best to predict proximal pole vascularity, and that there is a lack of clear evidence that vascularized bone grafts outmatch non-vascularized bone grafts . The firThe reported union rate of 95% was achieved with different fixation methods of the bone graft. Two non-unions occurred, one with a K-wire fixation and one with a palmar locking plate. Recent studies by Quadlbauer et al. showed a higher union rate in stabilization of scaphoid waist non-unions with two headless compression screws or a plate compared to stabilization with only one compression screw. This study cannot confirm or disprove these results, since the majority of fixations were conducted with K-wires in our population , 33. TheFailed previous screw fixation was associated with a lower union rate (66.7%) in our population. This might originate in the combined volar and dorsal approach that was necessary in this subgroup, rather than in the previous screw fixation itself.The MFC graft was favored by our team throughout the last years due to its good and consistent perfusion in the harvesting area, the good quality of cancellous bone and the possibility to harvest a rather big graft if necessary, as demonstrated by Iorio et al. . In addiFurther, as also hinted previously by Rodriguez-Vegas et al., the authors can confirm a very moderate donor site morbidity, which meets the demands of relatively young patients . This isThe authors only conducted a microsurgical attachment of the pedicled artery and left the veins untouched. This approach differs from the method earlier described by Bürger et al. . The autDespite the good clinical results in this study, complications occurred. However, they were rather not linked to the graft, but the surgery itself. Implant irritations healed uneventfully after removal. One delayed wound healed without surgical treatment, one venous bleeding was successfully stopped surgically and one infection showed further unproblematic bone union after implant removal, antibiotic and V.A.C therapy. The last patient suffered from severe decrease of thumb motion which had to be treated with surgical tenolysis and tendon transfer 7 months postoperatively.There are some limitations of this study. It is a retrospective review and there is no randomization or control group. Therefore, the authors cannot conclude if a vascularized bone graft or an avascular bone graft leads to similar or better results. Also, the question whether one graft is superior to another cannot be answered. Additionally, different surgical approaches and implants were used which might distort the results. Nevertheless, the MFC graft showed good clinical results and gives the option to replace the proximal scaphoid pole preventing carpal osteoarthritis and collapse in the short term.In conclusion, this study showed that the vascularized medial femoral bone graft leads to a good functional outcome in the treatment of scaphoid non-unions. This graft provides adequate blood supply and structural stability to the scaphoid. Additionally, a proximal pole destruction can be replaced using an osteochondral graft with promising short-term results preventing carpal osteoarthritis and collapse.