Starch and cell wall polysaccharides (dietary fibre) of cereal grains contribute to the health benefits associated with the consumption of whole grain cereal products, including reduced risk of obesity, type 2 diabetes, cardiovascular disease and colorectal cancer. The physiological bases for these effects are reviewed in relation to the structures and physical properties of the polysaccharides and their behaviour (including digestion and fermentation) in the gastro-intestinal tract. Strategies for modifying the content and composition of grain polysaccharides to increase their health benefits are discussed, including exploiting natural variation and using mutagenesis and transgenesis to generate further variation. These studies will facilitate the development of new types of cereals and cereal products to face the major health challenges of the 21st century. •Resistant starch and cell wall polysaccharides have important impacts on human health.•These effects are reviewed and their mechanisms discussed.•Improved crops can be developed by classical breeding, mutagenesis and transgenesis. They are the major source of calories and protein to the diets of humans and livestock (including poultry).Cereals are the dominant crops in world agriculture, with a total of 2500 million tonnes being harvested globally in 2011, comprising 704 million tonnes of wheat, 723 million tonnes of rice, 883 million tonnes of maize  . SimilarThe present article therefore focuses on the contributions of cereal carbohydrates to a healthy diet, focussing on wheat with comparative data on other cereals being included where appropriate.1.1Carbohydrates can be classified according to their molecular size and degree of polymerisation, with each group being subdivided according to the number and composition of monosaccharide units. This classification includes sugars (monosaccharides and disaccharides), oligosaccharides, starch (amylose and amylopectin) and non-starch polysaccharides.Carbohydrates account for about 65–75% of the mature wheat grain , with sid-glucose but they differ in their level of branching, with amylose being essentially linear and amylopectin being highly branched. These branches can directly affect starch characteristics and functionality as differences in their chain length distribution and clustering, and the ability to form a double helical conformation, may contribute to the crystalline features of the amylopectin . AX comprises a backbone of β-d-xylopyranosyl residues linked through (1 → 4) glycosidic linkages, with some residues being substituted with α-l-arabinopyranosyl residues at either position 3 or positions 2 and 3  grain are arabinoxylan (AX) and -β- 2 and 3 . Substit 2 and 3 . The araBy contrast, β-glucan has a simpler structure, comprising only glucose residues which are linked by (1 → 4) and (1 → 3) bonds. In general, single (1 → 3) linkages are separated by 2 or 3 (1 → 4) linkages but the linkage ratio may vary between tissues and species and longer “cellulose-like” regions of continuous (1 → 4) linkages (up to 14) have been reported in wheat bran β-glucan . The rat22.1All organisms require energy to maintain their life cycles. For humans, carbohydrates are the major energy sources, typically accounting for 45–70% of the total energy intake and expenditure. Being quantitatively the most important dietary energy source for most populations, carbohydrates have a special role to play in energy metabolism and homeostasis. Cereals are the dominant source of carbohydrates in the human diet, providing the major source of energy and contributing significantly to protein intake.In addition to their classification based on degree of polymerisation and sugar composition, carbohydrates can also be classified based on the extent to which they are digested and absorbed in the human small intestine, thus contributing directly or indirectly to the body carbohydrate pool and influencing post-prandial glycaemia (glycaemic carbohydrates). In this classification, carbohydrates which are not digested and absorbed in the human small intestine are distinguished from glycaemic carbohydrates, forming the major part of DF .The carbohydrates that have the greatest impact on post-prandial blood glucose are those that are absorbed relatively quickly from the small intestine. In fact, most metabolic parameters are adversely affected by an abrupt perturbation in the post-prandial period of a metabolic steady state present in the fasting condition. The greater and more rapid the perturbation, the more pronounced are the effects. Therefore this metabolic perturbation could be minimised if carbohydrate digestion and absorption were slowed down. This has resulted in interest in understanding the properties of foods that are able to retard digestion. The type of starch is certainly relevant in this respect, with amylose being slowly digested by α-amylases present in the human duodenum and amylopectin being very rapidly digested because the branched structure provides multiple sites for enzymatic hydrolysis. Moreover, amylose is structurally organised in the form of the double helixes, with the inner part of the helix accommodating the hydrophobic ends of polar lipids which form molecular complexes and reduce the accessibility of the molecule to α-amylase . These pThe contents of fibre and other food constituents that affect carbohydrate digestibility also affect the rise in blood glucose that follows the ingestion of carbohydrates from different foodstuffs, resulting in wide variation between different foods. The glycaemic response to food is also influenced by the method of preparation and other components of the meal.The structure of food has long been recognised as an important parameter governing the health benefits of cereal products, in that the intact structure of a plant food is more important than the composition (including the amount of DF) in determining physiological responses such as those related to glucose metabolism. Many studies have shown the importance of preserving as much as possible of the natural network of cell wall polysaccharides present in plant tissues, particularly in more or less intact cereal  grains .A simple marker for the impact of carbohydrate-rich foods on post-prandial glycaemia is the glycaemic index. This index is based on the increase in blood glucose concentrations  after the ingestion of a portion of a test food containing 50 g of carbohydrate, divided by the incremental blood glucose area achieved with the same amount (50 g) of carbohydrate present in an equivalent portion of reference food (glucose or white bread). Fibre-rich foods often have a low glycaemic index, although some foods with a low fibre content may also have a low glycaemic index.Low glycaemic index/high fibre diets have been shown to have beneficial effects, including lower post-prandial glucose and insulin responses, improved lipid control, and possibly improved insulin sensitivity. Although it is not possible to define precise values for high or low glycaemic index diets, it is clear that the lower the glycaemic index, the greater the metabolic effects .2.2Dietary fibre is one of the major food constituents that influence the rate and extent to which blood glucose increases after ingestion of a carbohydrate food. DF is not digested and absorbed in the small intestine but passes to the colon where it undergoes bacterial degradation. The principal types of DF are the non-starch polysaccharides (NSP), resistant starches (RS), non-α-glucan oligosaccharides (short chain carbohydrates) and some polyols and modified starches. The daily intake of NSP in the diet in European countries varies between 11 and 33 g, all of which will reach the colon .An important health benefit of DF is the capacity to lower the glycaemic response of the foods in which it is present: foods rich in DF release glucose more slowly into the blood which is relevant to the prevention of disorders such as obesity and diabetes .Food viscosity is a potential mechanism by which DF slows down the rates of digestion of starch and the absorption of sugars. Soluble, viscous types of NSP present either in food raw materials or incorporated as ingredients are able to reduce the post-prandial glucose and insulin responses after a meal. The effect is considered to be related to viscosity, which inhibits mixing and diffusion in the intestinal tract and delays gastric emptying. Moreover, high viscosity decreases enzyme diffusion and stimulates the formation of an unstirred water layer which decreases glucose transport to enterocytes. It has been repeatedly demonstrated that reducing the viscosity of DF following acid hydrolysis results in concurrent loss of its clinical efficacy . Severalβ-glucan varies in solubility, with a higher proportion of the total fraction being soluble in oats and barley than in wheat. The basis for this variation is still not completely understood, but it is likely that chain length (shorter chains being more soluble) and the ratio and distribution of 1 → 3 and 1 → 4 linkages both contribute. A significant relationship between the amount of β-glucan in cereals and the plasma glucose peak, or area under the glucose curve, has been observed. Similarly, a linear dose-dependent decrease in glycaemic responses was noted in response to breads containing proportions of barley β-glucan ranging from 0.1% to 6.3%. It has been suggested that viscosity of β-glucan could account for 79–96% of the effects on glucose and insulin responses after consumption of 50 g glucose in a drink model .Reduced insulin responses have consistently been observed following the ingestion of β-glucan and other soluble viscous types of fibre; as in the case of glycaemia, dose is an important factor in determining insulin responses to β-glucan. A consistent dose-dependent decrease in insulin secretions was observed in overweight individuals in response to oat β-glucan, with significant changes reported at a dose of at least 3.8 g of β-glucan . DF presA significant proportion of DF that enters the large bowel will be fermented by the commensal bacteria that live in the colon. Fermentation is an anaerobic process and, therefore, produces the short chain fatty acids (SCFA) acetate, propionate and butyrate as the principal end-products, together with methane, hydrogen, carbon dioxide and lactate. SCFA can influence liver glucose production and, thus, the plasma glucose concentrations .Among SCFA, butyrate has attracted particular interest because it is associated with unique benefits, such as the ability to promote a normal phenotype in colonocytes by repairing damaged DNA  and induButyrate is the major energy source for the colonic epithelial cells and also has differentiating properties in the cells, arresting cell division through its ability to regulate gene expression. This property provides a credible link between the dietary intake of fermented carbohydrates, such as NSP, and protection against colorectal cancer .Propionate is absorbed and passes to the liver where it is taken up and metabolised aerobically. This molecule moderates hepatic lipid metabolism. Acetate is the major SCFA produced in all types of fermentation, the molar ratio of acetate:propionate:butyrate usually being around 60:20:20. Acetate is rapidly absorbed, stimulating sodium absorption, and passes to the liver and then into the blood from where it is available as an energy source. The fasting levels of blood acetate are about 50 μmol/L, rising to 100–300 μmol/L between 8 and 12 h after meals containing fermentable carbohydrate. Acetate is rapidly cleared from the blood with a half-life of only a few minutes and is metabolised principally by the skeletal and cardiac muscle and the brain. Acetate reduces free fatty acid oxidation in humans and its absorption does not stimulate insulin release .Foods containing high levels of fibre are often made from whole grains or whole foods such as fruit and vegetables and are thus bulky with a low energy density. This may promote satiety and therefore may be expected to encourage weight loss, or prevent weight gain. In addition, some types of fibre, present in oats, barley, pulses, vegetables and fruit, are viscous and may act to delay gastric emptying and reduce the rate of absorption of glucose and fat from the small intestine, which may further contribute to feelings of increased fullness and satiety. The colonic metabolism of fibre after ingestion of the meal may delay the onset of hunger before the next meal as a consequence of the production of short-chain fatty acids and other satiety signals .A further mechanism by which whole grain may influence body weight regulation is via a probiotic effect modulating the intestinal flora. The available evidence, primarily from studies in animal models, suggests that the gut microbiota affect nutrient acquisition and energy regulation. Microbiota composition has also been shown to differ in lean vs obese animals and humans. Of particular interest among the possible mechanisms determining this relationship is the hypothesis that the metabolic activities of the gut microbiota facilitate the extraction of calories from ingested dietary substances and help store these calories in the host adipose tissue for later use. In fact, the gut bacterial floras of obese mice and humans include fewer Bacteroidetes and correspondingly more Firmicutes than those of their lean counterparts, suggesting that differences in the extraction of calories from ingested food substances may be due to the composition of the gut microbiota. The focus on Bacteroidetes, however, seems to be controversial. It has been hypothesised that more specific modulations of the gut microbiota community occur in obesity (instead of those at the wide phylum levels); this hypothesis is supported by several studies . It is n2.3Epidemiological observations consistently show an association between consumption of whole grain cereal foods and prevention of several non-communicable diseases; in particular, several studies show that subjects who ingest three or more portions of foods per day based on whole grain cereals have a 20–30% lower risk of CVD than subjects who consume predominantly refined cereals. Similarly, a high intake of whole grain cereals and their products, such as whole wheat bread, is associated with a 20–30% reduction in the risk of type 2 diabetes .Several prospective studies have also shown a more general inverse relationship between the consumption of fibre-rich foods and the risk of type 2 diabetes. It is noteworthy that in all of these studies the intake of cereal fibre in particular was inversely associated with the incidence of diabetes . In factNo intervention studies have so far determined the effects of fibre, glycaemic index and/or whole grains on the prevention of diabetes, although the most important independent studies aimed at lifestyle modification also included increases in fibre consumption and, in particular, consumption of whole grain fibre in the intervention group.Results from human intervention studies with surrogate end points consuming whole grain cereal products are not always consistent since the outcomes may be influenced by a number of factors such as the type of cereals consumed and their fibre contents together with other dietary factors which may be relevant for health such as the polyphenol content and the overall diet composition .The benefits of regular intake of whole grain and cereal fibre may be mediated by the improvement of one or more risk factors for type 2 diabetes and cardiovascular diseases, such as insulin resistance, dyslipidemia, inflammation and oxidative stress . HoweverIt is possible that the benefits of whole grain consumption in reducing the risks of type 2 diabetes and CVD could also be mediated by mechanisms that have not yet been investigated, particularly in relation to the post-prandial metabolism. A large body of evidence indicates that metabolic abnormalities in pre-diabetic insulin-resistant subjects and in diabetic patients are related more tightly to the post-prandial condition than to the fasting state. Indeed, increases in blood glucose, insulin and lipids in the post-prandial period are risk factors for adverse cardiovascular events that can also be detected in the absence of altered fasting parameters . It can 2.4The digestibility of starch is of great importance to human health  with a vRS has beneficial physiological effects  associatAn improvement of blood glucose regulation has been associated with the consumption of cereals rich in RS . RS is sThe metabolism of RS in the large bowel is beneficial because it is associated with the production of higher levels of butyrate compared to other types of dietary fibre . RS can in vitro starch digestibility of pasta enriched with commercial resistant starches at 10%, 20% and 50% for RS2 and 10% and 20% for RS3 with pasta made from 100% durum wheat semolina, The resultant RS content of pasta was increased from 1.9% to 21% in the blend made with 50% RS2 and was not reduced on cooking. Significantly, the results indicated that substitution of semolina with 10% and 20% RS2 and RS3 had no significant effects on pasta loss on cooking, texture and sensory properties, with only a minimal reduction in pasta yellowness. The incorporation of commercial high amylose starch in baked products such as cakes, muffins and biscuits has resulted in significant increases in total DF content and, at the same time, softer texture  with economic and social benefits. More recently, the availability of large genomic resources and bioinformatic tools together with new sophisticated technologies to detect mutations in target genes have led to the development of a technology termed TILLING . This approach combines classical chemical mutagenesis and PCR-based screening to identify mutations in a given gene of interest (3) or ethyl methane sulfonate (EMS) which are able to alkylate nucleotides to induce point mutations which are randomly distributed over the entire genome. EMS alkylates guanine bases that will then pair with thymine instead of cytosine, ultimately resulting in a G/C to A/T transition. Different high-throughput technologies are available for mutation detection . This includes 129,000 ha of transgenic insect resistant maize in five EU countries. Furthermore, transgenesis is increasingly being applied to improve grain composition and quality, as reviewed by Transformation systems are now available for the major cereals  and most minor cereals  , with tr44.1Triticeae tribe, wheat, barley and rye, have a bimodal size distribution with larger ellipsoidal A-type granules ranging from 10 to 40 μm in diameter and smaller spherical B-type granules between 2 and 10 μm. Starch granules in maize have a polyhedral or spherical structure, with a size ranging between 2 and 30 μm, whereas rice granules exhibit a polyhedral shape and size ranging between 3 and 8 μm , branching and debranching enzymes (SBE and DBE), which can be found in the soluble fraction of the endosperm, bound to starch granules, or both, are involved in amylose and amylopectin synthesis, through two different pathways which have ADP-glucose as a common substrate . DetaileDetailed accounts of the role of the different enzymes involved in starch biosynthesis have been provided by several authors  and onlyA single starch synthase, the granule bound starch synthase (GBSS), is involved in the synthesis of amylose. This enzyme is also known as waxy protein reflecting the typical phenotypes of seeds lacking amylose. In diploid cereals such as maize, barley and rice, the inactivation of genes encoding GBSS results in starch containing only amylopectin. In polyploid species, such as bread wheat, the waxy phenotype may result from the complete inactivation of genes present at three different loci located on the short arms of chromosomes 7A and 7D and on the long arm of the chromosome 4A. The inactivation of one or two of these homoeologous genes results in the production of partial waxy lines  which caindica rice than in japonica rice and this has been related to the presence of two types of waxy alleles, Wxa and Wxb. Rice varieties with the Wxa allele produce more GBSSI than varieties with the Wxb allele. The fact that many indica varieties possess the Wxa allele, while japonica varieties usually have the Wxb allele, therefore accounts for the higher amylose content in endosperm of indica rice than in japonica rice  have been shown to contribute to amylopectin synthesis through effects on the synthesis of glucan chains of different length, with SSI appearing to play a direct role in the synthesis of short glucan chains (DP 6–12) .SSIIa genes present on the homeologous group 7 chromosomes. Loss of this activity in wheat, rice and barley   and the other in the leaf (SSIIb). d barley  results d barley . Studiesd barley . No clead barley . Branchid barley . Two majd barley . No majod barley . Branchiin wheat , the abseat (ae) , with a It should be stressed that reduction or elimination of starch branching enzyme activity will not result in the production of true amylose; the major effect will be the reduction of branch points in the amylopectin fraction that will result in the formation of an amylopectin polymer with amylose-like properties .Triticeae.In addition to the enzymes described above, two types of debranching enzyme (DBEs) have been identified in cereals: isoamylases and pullulanases (limit dextrinases). Both hydrolyse α-1,6 linkages of amylopectin but they differ in substrate specificity. The absence of isoamylase activity is associated with lower starch content, the formation of a highly branched polymer (phytoglycogen) and changes in the number and size distribution of starch granules . In part4.2In recent years the manipulation of the amylose-amylopectin ratio in cereals has been identified as a major target for the production of starches with novel functional properties and improved health benefits. Together with natural mutants, induced mutations have been extensively explored in order to extend the narrow range of genetic variation for starch characteristics in crops. The studies carried out on maize, rice, wheat and barley have indicated that lack of activity of starch synthase SSII or SBE (SBEIIa or SBEIIb) are more effective in increasing amylose content in the seeds.ae) allele by ae mutants lack SBEIIb activity  was identified by japonica rice varieties  compared to the wild type (28.7%).More recently, As mentioned above, the development of TILLING technology has allowed the generation and detection of novel allelic diversity in genes involved in starch biosynthesis by several research groups. SSIIa and waxy genes and found single null genotypes for each of the three homoeologous loci of the target genes. Another application of TILLING to modify starch composition in wheat has been reported by SBEIIa and SBEIIb in durum and bread wheat. Screening of the bread wheat TILLING population identified 65 mutations in SBEIIa alleles. In durum wheat, 58 and 35 mutants were detected for the SBEIIa and SBEIIb genes, respectively. The same genes have been targeted by TILLING in the bread wheat cultivar Cadenza by SBEIIa. The crossing of knock-out mutants gave a set of single and double null SBEIIa lines. Pyramiding of two null homeologs resulted in an increase in the amylose content by up to 21% compared to the control line Cadenza. Although total starch content decreased slightly, 100 seed weight did not differ significantly among the single and double null genotypes with respect to the control. More recently, SBEIIa and SBEIIb genes. Single gene mutants showed no significant increases in the contents of amylose and resistant starch, but when a double mutant was obtained, by combining a SBEIIa-A knock-out mutation with a SBEIIa-B splice-site mutation, an increase of 22% in amylose content and 115% in resistant starch content was observed.TILLING resulted in the identification of more genetic diversity than had been reported by other research groups analysing wild and cultivated wheats. An EMS-mutagenised population of the bread wheat cultivar Cadenza was screened by SBEIIa genes. Combining these new alleles of SBEIIa resulted in high amylose durum and bread wheat lines containing 47–55% amylose and with elevated levels of resistant starch compared with wild type wheat. The high amylose lines also had reduced expression of SBEIIa, changes in starch granule morphology and altered starch granule protein profiles as evaluated by mass spectrometry.Similarly, SSIIa and waxy genes in 57 inbred rice accessions.4.3SBEIIa genes and increase the amylose content of bread wheat. Suppression of SBEIIa resulted in high amylose contents of the transgenic lines . The transgenic line showed altered starch granule morphology, a marked decrease in the proportion of chain lengths of DP 4–12 and a corresponding increase in the chain lengths greater than DP 12.RNA interference (RNAi), offers a robust approach to manipulate gene expression with the ability to simultaneously suppress members of multigene families, in order to modify crop quality traits  includinAgrobacterium for cv. Ofanto), the silencing of SBEIIa genes produced similar effects. The Agrobacterium- and biolistic-mediated transformants showed various levels of silencing between independent transgenic lines. The abundance of mRNAs of SBEIIa genes varied in the transgenic lines from nearly undetectable to wild type level, confirming that the RNAi construct, designed to reduce the expression of SBEIIa genes, was effective in inducing gene silencing. An increase in amylose content was found in all transgenic lines but the percentage varied between lines, presumably due to differences in copy number and/or positional effects. One of the lines showed a high value of 75% amylose content, while in the other lines, the content varied between 31 and 56%.The same approach was used by ae mutants of maize, which result from reduced expression of the SBEIIb gene. Analysis of amylopectin chain length distribution indicated that the SBEIIa and SBEIIb isoforms play distinct roles in determining the fine structure of amylopectin. A significant reduction in the frequency of branches in amylopectin occurred only when both SBEIIa and SBEIIb were reduced, whereas there was a significant increase in the branching frequency of amylose when SBEIIb alone was reduced. Functional interactions between SBE isoforms and a possible inhibitory role of SBEIIb on other SBE isoforms have been suggested.In order to define the roles of different isoforms of SBEIIa and SBEIIb in determining amylose content, chain length distribution, and branching frequency of starch in barley, ae phenotype was obtained in rice by The indica rice cultivar Te-qing using a double antisense construct. One of the transgenic lines showed a large increase in amylose content, to 64.8% compared to 27.2% in the normal cultivar. Scanning electron microscopy also showed a drastic change in the morphology of the starch granules in the transgenic line. Granules of smaller size, elongated shapes and filamentous structures with large voluminous bodies with spherical or ellipsoidal profiles were present in the transgenic line, compared with polygonal shapes characteristic of the starch granules present in the normal cultivar.SBEI, SBEIIa and SBEIIb) in barley by RNAi, producing transgenic lines with starch granules containing only amylose. This was achieved using a chimaeric RNAi hairpin construct with three elements targeting simultaneously each of the three SBE genes. The starch granules were irregularly shaped and showed modified thermal properties and crystallinity.Recently, 5Increases in the contents of soluble and total DF (AX and β-glucan) are clear targets for improvement, with the latter contribution to viscosity. More subtle differences in the benefits of AX and β-glucan may also exist, but have not yet been explored. The identification, and generation, of variation in the structure and properties of these two polysaccharides will therefore allow the relationships between their structures and properties (such as viscosity and production of SCFA) that affect health benefits to be defined and exploited to produce new types of cereals with enhanced benefits.5.1d-glucan) (about 2%) and glucomannan (about 7%) (d-glucan) is deposited in the early stages of endosperm development -β-bout 7%) . Calloseelopment , and it elopment . The inaWhereas the starchy endosperm of rye is also rich in AX, the starchy endosperm cells of both barley and oats have higher contents of β-glucan than of AX. In barley, the proportions of AX, β-glucan, cellulose and glucomannan have been reported as about 20%, 70%, 3% and 2%, respectively .The bran fraction produced on milling comprises the pericarp and testa , the aleurone layer and, in most milling processes, also the embryo (germ). In wheat the aleurone accounts for about 6.5% of the whole grain, the outer layers for about 7–8% and the germ for about 3% .The aleurone layer is the outer part of the starchy endosperm, which consists of a single layer of cells in wheat, rye and oats but usually three layers of cells in barley (except for the region adjacent to the germ which is a single layer). The aleurone cells have thick walls which account for 35–40% of the dry weight of the tissue in wheat . The aleThe outer layers of the grain have been studied in most detail in wheat, where they comprise about 45–50% cell wall material . The maj5.2p-coumaric acid (p-coumaric acid (0.2–0.3 mg/g tissue)  , with th tissue) . The AX  tissue) .Both AX and β-glucan are frequently fractionated into water-extractable (i.e. soluble) and water-unextractable (insoluble) forms, which are referred to as WE and WU with the total being TOT. Variation in the proportions of these fractions is discussed below.5.3A number of comparative studies have been reported of variation in the contents of AX and β-glucan in wholemeal, flour and bran of wheat  provided ratios of 0.39 and 0.71 for TOT-AX in bran and flour, respectively, and 0.47 and 0.59 for WE-AX in bran and flour . The ratTriticum spelta lines grown in Hungary in 2005) identified seven loci involved in WE-AX viscosity, three of which co-located with the meta-QTL located on chromosomes 1B, 3D and 6B and four additional loci on chromosomes 3A, 5B, 7A and 7B  for AX to be identified in wheat. A and 7B . The mosA and 7B .The heritability of the amount of ferulic acid bound to AX has not been determined in wheat flour, but the heritability of total bound phenolic acids  in whol5.5The existence of genetic variation in AX amount, solubility and composition provides opportunities for plant breeders to produce improved lines using marker assisted selection. However, it is also possible to generate more precise changes in amount and composition using transgenesis and mutagenesis.It is now well-established that the biosynthesis of β-glucan is mediated by the Cellulose synthase-like (Csl) F6 gene in barley  and wheaThe application of mutagenesis to manipulate grain β-glucan was reported for oats by By contrast to β-glucan synthesis, the pathway of AX synthesis involves several enzymes and is still incompletely understood . MitchelSix forms of GT61 were identified as expressed in developing starchy endosperm of wheat, with GT61_1 being the most highly expressed . RNAi waA similar approach was used to functionally characterise the highly expressed GT43_2 and GT47_2 genes, which were putatively identified as encoding xylan synthases based on their relationship to the IRX10 and IRX9 genes, respectively, of Arabidopsis . In bothRNAi suppression of GT43_2 and GT47_2 also resulted in decreased total AX and in reduced viscosity of aqueous extracts. Determination of the concentration and intrinsic viscosity profiles of WE-AX separated by size-exclusion HPLC showed increased proportions of smaller AX chains in the transgenic lines. However, differences were also observed between GT43_2 RNAi and GT47_2 RNAi lines, with the latter having a greater effect . The funFunctional characterisation of the genes responsible for ferulylation of AX has proved more elusive but transcriptomic analysis in wheat  and RNAiIt is clear, therefore, from these studies that the amounts and fine structures of both β-glucan and AX can be manipulated by altering the total and relative activities of biosynthetic enzymes. This could be achieved by transgenesis or mutagenesis (TILLING). However, because in wheat, most  genes encoding enzymes of cell wall biosynthesis occur in three forms, it is necessary to identify and “knock- out” at all three loci to observe effects on cell wall composition. It should also be noted that mutagenesis rarely results in increased gene expression, and is only routinely applicable where the required phenotype can result from gene knock-out(s).6The polysaccharides of cereal grain are not only important sources of calories in the human diet but also have health benefits related to their physical properties, digestion in the small intestine and fermentation in the colon. These contribute to the well-established health benefits associated with the regular consumption of whole grain cereal foods. There is therefore considerable interest in modifying the amounts and compositions of starch and the major non-starch polysaccharides (arabinoxylan and β-glucan) to develop new types of cereals with improved health benefits, notably reduced glycaemic index in the small intestine and improved properties as DF in the colon. This may be achieved by exploiting genetic variation in composition with the ability to generate additional variation using mutagenesis and transgenesis. The development and adoption of such varieties will allow health benefits to be delivered to large populations in low cost staple foods such as bread, noodles and pasta.TILLING technology provides a very powerful non-GM approach to identify useful mutations which can be exploited in conventional plant breeding, as demonstrated by the increasing number of platforms developed in different crops . In addi


ZNF277 gene (NM_021994.2), encompassing exon 5, in an individual with severe receptive and expressive language impairment. The microdeletion was not found in the proband's affected sister or her brother who had mild language impairment. However, it was inherited from both parents, each of whom carries a heterozygous microdeletion and has a history of language problems. The microdeletion falls within the AUTS1 locus, a region linked to autistic spectrum disorders (ASDs). Moreover, ZNF277 is adjacent to the DOCK4 and IMMP2L genes, which have been implicated in ASD. We screened for the presence of ZNF277 microdeletions in cohorts of children with SLI or ASD and panels of control subjects. ZNF277 microdeletions were at an increased allelic frequency in SLI probands (1.1%) compared with both ASD family members (0.3%) and independent controls (0.4%). We performed quantitative RT-PCR analyses of the expression of IMMP2L, DOCK4 and ZNF277 in individuals carrying either an IMMP2L_DOCK4 microdeletion or a ZNF277 microdeletion. Although ZNF277 microdeletions reduce the expression of ZNF277, they do not alter the levels of DOCK4 or IMMP2L transcripts. Conversely, IMMP2L_DOCK4 microdeletions do not affect the expression levels of ZNF277. We postulate that ZNF277 microdeletions may contribute to the risk of language impairments in a manner that is independent of the autism risk loci previously described in this region.Specific language impairment (SLI), an unexpected failure to develop appropriate language skills despite adequate non-verbal intelligence, is a heterogeneous multifactorial disorder with a complex genetic basis. We identified a homozygous microdeletion of 21,379 bp in the  SLI affects 3–7% of preschool English-speaking children.5 Similarly, some find that a subset of children with ASD exhibit language profiles resembling SLI.7 However, others report qualitative differences that can distinguish the performance of individuals with SLI or ASD in language tests.10Although a diagnosis of SLI excludes the presence of other clinical conditions that affect language, SLI presents co-morbidity with other neurodevelopmental disorders, such as autism spectrum disorder (ASD). Although ASD is characterized by an impaired communication, language skills can be highly heterogeneous and phenotypic overlaps with SLI in the language domain have been debated. Some studies report that a minority of adolescents with a history of SLI show behavioral traits reminiscent of autism.CNTNAP2: variants in and disruptions of this gene are reported to be associated with language endophenotypes in both SLI and ASD,11 suggesting that CNTNAP2 might harbour susceptibility risk factors that could impair language skills in distinct language-related disorders.Recent studies investigated whether the phenotypic similarities of the language deficits may reflect potentially shared causes. An example of functional link between SLI and ASD, in addition to other neurodevelopmental disorders, is given by 12 such as ASD, schizophrenia and bipolar disorder. Some CNVs are recurrently observed across multiple neuropsychiatric conditions,13 sometimes with variable penetrance of the genes implicated, suggesting a pleiotropic effect. These findings support the hypothesis that these CNVs can represent shared risk variants across different types of neurodevelopmental diseases and might highlight common genes and/or pathways.Submicroscopic copy number variations (CNVs) have been shown to be an important source of susceptibility to psychiatric disorders,ZNF277 gene, found in a child with SLI. This CNV falls in the AUTS1 region of linkage to ASD .18 A fine mapping study of AUTS1 previously reported association of ASD with SNPs in two genes that are proximal to ZNF277, DOCK4  and IMMP2L .19 An additional investigation20 described a rare microdeletion involving the DOCK4 and IMMP2L genes that co-segregated with the presence of dyslexia in an extended family.Here, we describe a novel CNV case involving homozygous loss of material from the ZNF277 may represent a candidate gene for both disorders. Therefore, we went on to investigate the frequency of ZNF277 microdeletions in cohorts of individuals with SLI or ASD. Finally, we used quantitative PCR (qPCR) techniques to further examine the effects of ZNF277 microdeletions on expression levels of genes in the AUTS1 region.On the basis of prior reports of phenotypic and genetic overlaps between SLI and ASD, and given the position of this homozygous microdeletion, we hypothesized that 21 PennCNV22 and QuantiSNP23 algorithms. A manuscript describing this larger data set is in preparation. In the current manuscript, we describe an incidental finding from these data: the identification of a homozygous microdeletion involving exon 5 of ZNF277 (NM_021994.2) . As this microdeletion was in a homozygous form, we predicted that it might be particularly deleterious to the individual and thus selected it for further study, as described below.A genome-wide CNV screen of 512 individuals from families with SLI was performed to investigate CNV burden in individuals with SLI. This study utilized genome-wide SNP data from the Illumina Human OmniExpress (v12.1) beadchip and identified CNVs within the Nexus,The microdeletion was predicted to be present in a single child of a non-consanguineous Caucasian family. The child had a clinical diagnosis of SLI. She did not develop language skills until the age of 4–5 years. She appeared sociable but was dependent on being shown what to do with toys. Her thinking was slightly rigid but no other obvious autistic behaviors were reported or observed. Her non-verbal intelligence was below average (performance IQ=75). She attended a special unit for speech and language impaired children.The proband had two siblings, an older brother and a younger sister. All three children presented with a similar pattern of speech and language impairment, which primarily affected the expressive domain. They all presented with delayed word and phrase speech, unintelligible speech with poor articulation and impaired word retrieval. However, the three children differed in terms of the severity of their impairment and their non-verbal attainment. The younger sister had a slightly higher non-verbal IQ than the proband (PIQ=94) and also had a diagnosis of SLI, although she appeared less severely affected than the proband. The brother had a particularly high non-verbal IQ (PIQ=127) and although he was reported to have had an early speech and language delay, he did not have a diagnosis of SLI. He did attend a special educational unit and at 10 years of age he had a significant verbal performance discrepancy and impaired sentence recall. Both parents reported a family history of speech or language problems.ZNF277 microdeletion breakpoints, as described for the screening of the larger cohort below. For each sample, qPCRs were set up in triplicate and compared against a control gene (ZNF423) and a control subject. The number of copies of each amplified fragment was calculated using the 2ΔΔCt method.24qPCR using iQ SYBR-green Supermix  and four primer pairs across the predicted CNV and the surrounding region was used to validate the presence of the microdeletion and to examine co-segregation in the discovery pedigree. Primer sequences are available on request. All the members of the family were analyzed by qPCR, except for the sister, for whom insufficient DNA was available. The sister was instead tested using a PCR assay with primers spanning the ZNF277 microdeletion were determined by Sanger sequencing using primers flanking the predicted deletion boundaries.The boundaries of the ZNF277 microdeletion breakpoints, which generated a shortened product in the presence of the microdeletion, were used for a PCR-based screening of three separate cohorts: a cohort of families containing individuals with SLI, a cohort of families containing individuals with ASD and a control cohort. A second primer pair that amplifies exon 5 of ZNF277, giving a product only when the allele is not deleted, was subsequently used to test whether the identified microdeletions were in the heterozygous or homozygous form.Primers spanning the 28 In short, these British nuclear families were ascertained on the basis of at least one child who had expressive or receptive language skills ≥1.5 standard deviations (s.d.) below the normative mean for their chronological age and WISC Perceptual Organisation Index  of >77.5 (1.5 s.d. below that expected for their age).The SLI screening cohort consisted of DNA from 1234 individuals from 322 families . This cohort included the 512 individuals who comprised the CNV study but included many additional SLI subjects  and their family members . All families formed a part of the SLI Consortium cohort, which has previously been described in detail.16 The families were collected from different countries  and were predominantly Caucasian (92.5%). In short, this cohort consists of multiplex ASD families in which at least one child meets a clinical diagnosis of ASD under the Autism Diagnostic Interview and/or Autism Diagnostic Observation Schedule (ADOS) or ADOS generic.The ASD cohort consisted of DNA from 1021 individuals from 252 families  and formed part of the International Molecular Genetic Study of Autism Consortium (IMGSAC) cohort, which has previously been described.http://www.hpacultures.org.uk/products/dna/hrcdna/hrcdna.jsp). In addition, we had access to sequence data from 130 unrelated Caucasian samples through an in-house project at the Wellcome Trust Centre for Human Genetics – the 500 Whole-Genome Sequences Project (WGS500 Consortium29).The control cohort consisted of DNA from 224 non-related UK Caucasian blood donors from the ECACC Human Random Control (HRC) panel  to test whether the allelic frequency of ZNF277, DOCK4 and IMMP2L by qPCR in cDNA derived from lymphoblastoid cell lines from 10 individuals belonging to four ASD families, 5 of whom carried a heterozygous ZNF277 microdeletion, and the expression level of ZNF277 in cDNA derived from blood from two individuals from a single Dutch multiplex ASD family previously described by Pagnamenta et al.20 with an IMMP2L_DOCK4 microdeletion . The housekeeping gene GUSB (NM_000181.3) was used as a normalizer and expression levels were normalized against a control individual. The 2ΔΔCt method was applied to estimate the difference in the expression of the three genes between the samples.24 Statistical significance was calculated with the Student T-test, assuming unequal variance between the two independent sample groups for the expression analysis.RNA samples were not available for the discovery individuals. We therefore chose to examine expression levels of ZNF277. The microdeletion removes exon 5 of the ZNF277 gene, causing a frame-shift mutation and introducing a premature stop codon in exon 7 (NM_021994.2). Given the likelihood of non-sense-mediated mRNA decay, this homozygous microdeletion would thus be predicted to result in a complete lack of functional protein in the affected individual.30During a CNV analysis of SNP array data, a female patient with a diagnosis of SLI was found to carry a novel homozygous microdeletion within the gene qPCR of available family members in this pedigree demonstrated that one copy of the microdeletion was transmitted to the proband from each parent, who were both heterozygotes and reported to have language problems in their childhood: the father had speech impairment and the mother had dyslexia. However, the microdeletion was not transmitted to the proband's brother, who presented with an early expressive speech and language impairment but did not have a diagnosis of SLI . Insuffi32 (January 2012). Subsequently, one larger, overlapping heterozygous loss, was described in a HapMap female control sample (NA12156) . A similar size microdeletion was observed in an in-house sequencing database at the Wellcome Trust Centre for Human Genetics in a heterozygous form (1/130 samples of the 500 Whole-Genome Sequences Project (WGS500 Consortium29)) allowing the accurate detection of the breakpoint boundaries which lie in two LINE elements, L2c and L1M4. Sanger sequencing validated the boundaries in the discovery individual  and confirmed the presence of the microdeletion in the parents and proband but not in either of the siblings  and had a minimum predicted size of 4153 bp. At the time of detection, there were no overlapping deletions described in the Database of Genomic Variants (DGV)siblings .ZNF277 microdeletion in a heterozygous form, 5 of whom were probands , 6 parents  and 5 siblings   affected by SLI . The screening led to the identification of 16 additional individuals with the cy 0.7%) , giving In contrast, the microdeletion was observed in the heterozygous form in 1 of 130 unrelated samples in our in-house sequencing cohort  and 2 of 224 ECACC control individuals  giving a control population allelic frequency of 0.4% (3/708 chromosomes).ZNF277 microdeletions in 4 ASD families. Four mothers carried the microdeletion  and it was inherited by three affected children  giving a frequency of 0.3% (7/2042 chromosomes) across the entire cohort. All of the ASD families were ascertained as multiplex pedigrees and thus included more than one affected child. Unlike the SLI families, in many cases, there was no single designated proband within the family units. All three ASD cases who inherited the microdeletion had affected siblings who did not inherit the microdeletion rendering the derivation of an objective proband frequency problematic.Screening of 252 ASD families  identified heterozygous ZNF277 microdeletion and its relationship to a nearby IMMP2L_DOCK4 microdeletion independently described in an autistic family in a previous publication20  but did not significantly alter the expression of DOCK4 or IMMP2L , although the rarity of the microdeletion meant that this difference did not reach significance when examined with a two-tailed Fisher's exact test (P=0.206).In this study, we identified a novel homozygous microdeletion of exon 5 of the ZNF277 gene within a known ASD risk locus, we postulated that the disruption of this gene may be relevant for both disorders. However, screening of a cohort of ASD multiplex families found that the frequency of ZNF277 microdeletions in individuals with autism was similar to that observed in controls. The ZNF277 microdeletion that we describe was not documented in the DGV, possibly because of the small number of standard array SNPs contained within the deleted segment. However, we specifically searched 33 and occurred at a frequency of 0.3% (6/2248 chromosomes). This figure matches with that observed in our ASD cohort, supporting the current data set and reinforcing our conclusion that the ZNF277 microdeletion does not contribute to ASD susceptibility.Given prior observations of phenotypic and genetic overlaps between SLI and autism, as well as the genomic position of the ZNF277 microdeletions was de novo and that, in many cases, the segregation with language impairment was incomplete. In three families, the microdeletion was not inherited by the proband, whereas in another three families, the microdeletion was inherited by unaffected siblings , a gene which lies head-to-head with the ZNF277 gene, and the first three exons of IMMP2L, decreases the expression level of DOCK4 but not ZNF277. Taken together, these data suggest that ZNF277 microdeletions may have a role in SLI susceptibility that is distinct from the autism risk loci described in the AUTS1 region.Given these data, some researchers propose a ‘dual-hit' model.ZNF277 is an evolutionary conserved zinc finger gene with 12 exons.49 It is expressed in several tissues, including the brain, particularly in the neocortex and hippocampus in early mid-fetal development.50 Although the function of ZNF277 has not been studied in humans, the mouse Zfp277 gene, which shows >80% homology to the human gene at the amino-acid level, has been implicated in the epigenetic regulation of cellular memory.51−/−Zfp277 mice were born healthy and fertile, indicating that the knockout is not lethal,51 consistent with our finding of viability for humans with no functional ZNF277. Interestingly, the Zfp277 protein directly interacts with Bmi-1, a key component of the Polycomb Repressor Complex. This complex has an important role in the maintenance of adult stem cells from numerous tissues, including the central nervous system.5354 In conclusion, we propose that the disruption of ZNF277 may contribute to SLI in a complex genetic model. We further hypothesize that this risk is distinct from the autism risk loci as previously described in this region. Further studies will be required to replicate these findings and characterize the function of the human protein ZNF277, clarifying its potential implication in language development.Sequencing of the breakpoints in the discovery individual demonstrated that the breakpoint boundaries lie in two LINE elements, L2c and L1M4. L2c  belongs to the L2 LINE family and L1M4  to the L1 LINE family, which promote structural variation through non-allelic homologous recombination. BLAST alignment of the entire sequence of these two elements did not reveal extended homology between them. However, sequencing of the breakpoints revealed 2 bp microhomology at the junctions, suggesting that this deletion may to be generated through a microhomology-mediated repair mechanism.During the proof-editing stage of the manuscript, we noted that a deletion of the same size had also been reported in the most recent release of DGV (evs2656841) . These data again supported the expected frequencies found throughout our study ).http://www.ebi.ac.uk/dgva/).The CNV data generated for the discovery family will be archived in the DGVa (


She missed her antiretrovirals 10 days before the onset of her symptoms and subsequently doubled her daily dose the following day. She had been on no other medication in the preceding 8 weeks.A 31-year old HIV-infected African woman on nevirapine, tenofovir and lamivudine for more than 4 years presented with an 8-day history of symptoms and signs of Stevens-Johnson syndrome. She was on no other medication. Her viral load was undetectable and she had maintained a CD4 count of between 356 and 387cells/mmHer ARVs were stopped and she fully re-epithelialized with the exception of the lips, over the following 10 days. She was started on a daily single tablet of Odimune® .Nevirapine is the most common offender in cases of antiretroviral-associated SJS in published literature. Lamivudine is very rarely implicated while there are no similar reports with tenofovir.  We concluded that nevirapine was by far the most likely offender in this case. Nevirapine toxicity is associated with high CD4 counts, undetectable viral load and high drug plasma level. We postulate that the sudden increase of the plasma levels of nevirapine in a patient with a high CD4 count and undetectable viral load created a perfect storm for the development of SJS in our patient, who had been on the NVP-containing regimen for many years.Clinicians should be aware that severe adverse drug reactions are dynamic and can occur even when the drug has been in use for a long time. NVP is also used to minimize diarrhea and cardiovascular side effects of protease inhibitors and neuropsychiatric side effects of Efavirenz2. However, NVP is associated with severe adverse reactions, including Stevens-Johnson syndrome (SJS).Nevirapine (NVP)-based antiretroviral regimens have been used widely in developing countries because of its affordability, availability and efficacy3. We report a case of probable nevirapine-associated Stevens-Johnson syndrome occurring more than 4 years after initiation of the drug. This case illustrates that in susceptible persons, a severe drug reaction can occur when there are dose adjustments despite having been exposed to the same drug for a long time.In larger series, the incubation period before developing features of SJS ranges from 10–240 days with a median duration of around 12 days3 and her HIV RNA viral load at the time was unknown. Her last CD4 counts, done 31, 21 and 11 months before developing her current symptoms were 373, 356 and 387 cells/mm3 respectively. The last HIV RNA viral load test, done 11 months prior to the onset of her symptoms showed an undetectable viral load.A 31-year-old HIV-infected black African female presented with an 8-day history of painful swallowing, sore eyes, malaise and a worsening rash. She had been on antiretroviral (ARV) regimen of nevirapine (NVP) 400 mg daily, tenofovir 300 mg daily and lamivudine 300 mg daily for more than 4 years uneventfully. She was on no other medications and had not taken any other medication in the preceding 8 weeks. She had acquired HIV via heterosexual contact – the exact date of HIV infection was unknown. At the time of initiation of ARV therapy, her nadir CD4 cell count was 139 cells/mmShe gave a history of forgetting to take her ARV medication for a day. The following day, 10 days before development of her symptoms she took 2 days equivalent of her ARVs in one day, in her own words “to make up for the missed dose”.9/L. The liver and renal function tests were normal. Her ARVs were stopped and her epidermal necrosis did not extend and eosinophil counts normalized. Over the next few days her skin condition improved and she was discharged 10 days later fully re-epithelialized with the exception of the lips, which were still eroded in areas. On review, 2 weeks after discharge, her skin had normalized except for residual hyperpigmentation. She was started on a single daily tablet of Odimune®, a fixed drug combination of tenofovir 300 mg, emtricitabine 200 mg and efavirenz 600 mg. On follow-up, 4 weeks later she was tolerating the new ARV regimen.On examination, she was normotensive and had a temperature of 38.8°C. She had conjunctivitis and hemorrhagic cheilitis but no involvement of the genital mucosa. She had epidermal necrosis involving predominantly her trunk and face and to a lesser extent palms, soles and extremities, totaling 10% of her body surface area, 3% of which was stripping. Initial laboratory studies showed normal blood count, except for an elevated eosinophil count of 0.70 × 104. Based on these, we concluded that NVP was by far the most likely offender in our case.Clinicians routinely blame NVP as the culprit agent in cases of ARV-associated SJS as it is by far the most common offender in published literature. However, lamivudine has infrequently been reported as a cause of SJS, but there have not been similar reports with tenofovir6. We postulate that the sudden increase of the plasma levels of NVP in a patient with a high CD4 count and undetectable viral load created a perfect storm for the development of SJS in our patient, who had been on the NVP-containing regimen for many years. The impact of NVP drug concentrations on the risk of adverse drug reactions is still controversial. Some studies have shown that above a certain threshold, the risk of severe drug reactions is significantly increased. A recent study has shown that impaired renal function, thus impaired clearance of the drug, is associated with an odds ratio of 8.0 (3.9 to 17) to develop allopurinol-associated SJS in susceptible people7. However other studies have not found this association9. NVP has a long half-life, 25–30 hours, and the effect of doubling the dose possibly increased the plasma drug concentrations considerably resulting in SJS8.Numerous factors have been associated with NVP toxicity, including high current and nadir CD4 counts, undetectable viral load, female sex, abnormally elevated baseline transaminases, history of drug allergy, lower body weight and high drug plasma levels10. There were previous suggestions that the timing of SJS and other NVP-associated reactions coincided with the increase of the drug dose. However, this has been disproved, because as high as a third of cases in larger series occurred during the lead-in period of the dosing regimen before the dose was increased11. Pretreatment with ARVs longer than 1 year has also been reported as an independent predictor of a cutaneous drug reaction. Whether this is truly an independent predictor or a result of low HIV viral load or higher CD4 counts is unclear and further studies are needed to answer this question.The patient had been compliant on treatment for more than 4 years and had undetectable viral load and a high CD4 count the last time they were measured, 11 months earlier. Both of these are independent predictors of NVP-associated SJSIn summary, this case highlights the potentially life-threatening risk of the development of SJS outside the expected period, probably triggered by a spike in serum levels of NVP. The case also illustrates that development of severe drug eruptions, like SJS, is a dynamic processes that may evolve with changing environment in the body. Clinicians should warn their patients against unregulated dosing changes of their NVP-containing regimen.Written informed consent for publication of the clinical details was obtained from the patient. The authors highlight an important report of Stevens-Johnson syndrome (SJS) in a patient on nevirapine-based antiretroviral therapy for more than two years. The median duration to the onset of  nevirapine-associated SJS is two weeks, thus the case report stimulates exploration of the relationship between sudden rise in plasma nevirapine concentration and increased risk of SJS. However, it was not clear whether the authors performed any formal adverse drug reaction causality assessment, even though they stated that the reaction was "probable".I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. This is an interesting case of Stevens-Johnson syndrome probably due to a single double dosing of nevirapine-containing regimen in an HIV-infected woman. There have been a lot of case reports and short series of SJS/TEN due to antiretroviral drugs. However, the singularity of  this case is that the drug had been taken for a long time (4 years), and the triggering was an increase in the dosage. This side effect due to an increase in the dosage has also been reported for allopurinol.Mycoplasma pneumoniae. In my opinion, it is always important to rule out such infections, when a SJS in suspected. This should be included in the article.Most of Lyell's syndrome/TEN are caused by drugs; however, SJS may be triggered by a "new" drug or also an infection such as VHS/VVZ orSome clinical images of the skin and mucosal involvement would improve the quality of the article.I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. The Title, Abstract and all content of the article are clear and relevant.This case could seem to be simple but it actually adds a valuable perspective to our understanding of what might trigger severe drug reactions and adds clinical insights and clarity to what is often an overly complex environment.I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.


Lung cancer is currently the most common cause of cancer-related death in the world and approximately 85% of all lung cancers are non-small cell lung cancer (NSCLC). Previous studies have shown that patients with NSCLC containing high densities of CD8+ memory T cells have a survival advantage. Others have identified an association between relatively high numbers of regulatory T cells and poor prognosis. Our group is interested in better characterizing the immune infiltrates and refining prognostic biomarkers that identify patients at risk of recurrence. Recent advances in multispectral imaging provide an opportunity to evaluate up to 7 markers in a single 4 micron section of formalin-fixed paraffin-embedded (FFPE) tissue. Using this technology, we reported that a high ratio of CD8:FoxP3 at the tumor correlated with the ability to isolate tumor-specific T cells from melanomas . This determination could be further improved by incorporating the ratio of CD8:PD-L1 present in the tumor into the evaluation. Now we are applying these same strategies to the study of NSCLC. Initial analyses have been performed on more than 450 tumor cores from 77 patients with Stage 3A/3B/4 NSCLC and included the following markers: CD3, CD8, FoxP3, CD163 and PD-L1. Additional markers are being evaluated. Ultimately, we expect that this approach could be used to stratify patients for clinical trials. We anticipate that some biomarkers will identify suppressive pathways and be associated with a short progression-free survival. While we expect there will be heterogeneity in escape mechanisms, identification of a specific mechanism could be used to tailor therapy with an agent or agents to overcome the specific immune suppressive pathway operational in that specific tumor. Patients who lack evidence for a specific suppressive pathway would be randomized to treatment with combination immunotherapy that includes a cancer vaccine.Drs. Ballesteros-Merino and Neuberger; and Drs. Reu and Winter contributed equally to this work. Supported by the Harder Family, Lynn and Jack Loacker, Robert W. Franz, Wes and Nancy Lematta, Providence Medical Foundation.


S. pombe, S. cerevisiae and Drosophila suggest DIS3 homologues have a common role in cell-cycle progression and microtubule assembly. DIS3 has also recently been implicated in antibody diversification of mouse B-cells. This article aims to review current knowledge of the structure, mechanisms and functions of DIS3 as well as highlighting the genetic patterns observed within myeloma patients, in order to yield insight into the putative role of DIS3 mutations in oncogenesis.DIS3 is a conserved exoribonuclease and catalytic subunit of the exosome, a protein complex involved in the 3' to 5' degradation and processing of both nuclear and cytoplasmic RNA species. Recently, aberrant expression of DIS3 has been found to be implicated in a range of different cancers. Perhaps most striking is the finding that DIS3 is recurrently mutated in 11% of multiple myeloma patients. Much work has been done to elucidate the structural and biochemical characteristics of DIS3, including the mechanistic details of its role as an effector of RNA decay pathways. Nevertheless, we do not understand how DIS3 mutations can lead to cancer. There are a number of studies that pertain to the function of DIS3 at the organismal level. Mutant phenotypes in  A fine balance must be achieved between the synthesis and degradation of RNAs in the cell. Mutations that cause defects in RNA turnover can have significant consequences on cellular function ,2,3,4. TDIS3 is a highly conserved 3' to 5' exoribonuclease that provides catalytic activity to a multi-subunit complex, the exosome ,7 regulaThis article aims to review the current knowledge of DIS3. We will discuss the structural and catalytic properties of DIS3, as well as its molecular and biological functions, role in disease and common genetic patterns observed in patients, in order to provide a basis on which to investigate the role of DIS3 in cancer progression.S. pombe mutants that were defective in sister chromatid disjoining [DIS3 was first discovered in sjoining . Ortholosjoining . Memberssjoining ,11. Somesjoining  for moresjoining ,32,33. Asjoining . Humans sjoining ,35. DIS3sjoining ,37,38.Structural analyses and RNase protection experiments have revealed a common model for the mechanism of action of DIS3. DIS3 is a highly processive, hydrolytic enzyme with a preference for substrates with phosphorylated 5' termini . It hydrS. cerevisiae exosome complex shows Rrp44 (Dis3) to be anchored at the bottom of the exosome core through interactions with the PIN domain and CR3 motif [In vitro evidence suggests that DIS3 can act independently of the exosome although this has not been shown in vivo [In vitro, when bound to the exosome, the activities of DIS3 and Rrp6 are suppressed through allosteric effects that diminish their RNA binding ability [In mammals, DIS3 functions as one of the three catalytic subunits of the exosome, along with DIS3L and Rrp6, a distributive exoribonuclease which belongs to the RNase D family ,44,45. TR3 motif . Rrp6, c ability . This main vivo [in vivo is also unknown, however they are known to work sequentially in the maturation of 5.8 S rRNA [The central channel of the exosome is only wide enough to accommodate single-stranded RNA, so secondary structures must be unwound from the cap by either the nuclear TRAMP complex or cytoplasmic Ski complex ,49,50. S8 S rRNA .Drosophila [Drosophila S2 cells, Dis3 has shown restricted localisation to the cytoplasm and the localisation pattern differed from cell to cell [Drosophila cells [S. pombe and human DIS3 interact with Ran, which is essential for nucleocytoplasmic transport of proteins and ribonucleoproteins [Subcellular compartmentalisation of exoribonucleases is an important control mechanism in the temporal and spatial regulation of RNA processing and decay. The subcellular localisation of DIS3 homologues and the different exosome subunits has not been investigated in great depth, besides two studies in osophila ,54 and oosophila . It is g to cell . The funproteins ,55.The predominantly observed nuclear localisation of DIS3 is thought to be controlled by two nuclear localisation signals at the C-terminus of the protein . DIS3 isThe continuous synthesis and degradation of RNAs allow the metabolic changes required for proper cellular function. In association with the exosome, DIS3 is the common effector of a vast range of RNA metabolic pathways functioning in mRNA quality control, regulation of gene expression and small RNA processing. Although not discussed here, the ability of the exosome to handle such a diversity of substrates is down to a network of auxiliary factors which interact with exosome to recruit it to particular substrates . The folmRNA degradation in eukaryotes involves a number of complex and interconnected pathways that all converge on three common mechanisms. mRNAs must first be either deadenylated and de-capped or internally cleaved to allow access for either the exosome and paralogues of DIS3 or the 5'-3' exoribonuclease, XRN1 . DeadenyDrosophila, suggests the exosome may function co-transcriptionally in vivo as part of a checkpoint that monitors proper pre-mRNA processing [Aberrant and faulty transcripts must be detected by the cell to prevent the production of potentially toxic protein products. Surveillance mechanisms exist both in the nucleus and in the cytoplasm to detect errors at all stages of mRNA production and maturation. In the nucleus mRNAs that are faulty due to errors in transcription, export or processing are degraded. Both the 3' to 5' and 5' to 3' pathways are involved in nuclear mRNA turnover but which is used depends on the substrate. The exosome is known to specifically degrade un-spliced pre-mRNAs  and mRNAocessing .Surveillance pathways that take place in the cytoplasm are translation dependent and include nonsense mediated decay , non-stoThe most recently discovered RNA surveillance pathway, no-go decay, prevents translation of transcripts with a strong secondary structure. Ribosomes stalled by the secondary structure are detected and the mRNA is endonucleolytically cleaved. The endonuclease responsible has not been identified but seems to require Dom34 and HSB1, proteins which are related to the eukaryotic translation release factors, eRF1 and eRF3 . Once thAlternative to the degradation of aberrant mRNAs, mRNAs may be subjected to regulated degradation as a means of controlling gene expression. This can occur through cis-encoded destabilising elements in the 3' UTRs or by the RNA-induced silencing complex (RISC).Two types of cis-encoded destabilising elements exist: AU-rich elements (ARE) and GU-rich elements (GRE), both found in the 3' UTRs of a number of mRNAs. AREs are found in short-lived mRNAs coding for proteins that mediate regulatory responses in the cell, such as inflammatory or stress responses  . AREs exMiRNA-mediated degradation of mRNAs provides another means of modulating gene expression. miRNAs are short RNA molecules which generally act to downregulate target expression by either repressing translation or causing degradation of their target mRNA by the RNA-decay machinery. In plant cells, miRNAs typically base-pair with their targets with almost perfect complementarity which results in cleavage of the target mRNA by RISC (RNA-induced silencing complex) and subsequent degradation of the 3' section by AtXRN4. The 5' section is thought to be degraded by the exosome . In animNot all RNA substrates that are targeted by the RNA decay machinery are destined for complete degradation. DIS3 and the exosome were originally discovered in yeast to be involved in the processing of ribosomal RNAs (rRNAs)  and wereFor example, rRNA synthesis in yeast begins with the synthesis of a 35S precursor-rRNA in the nucleolus. The pre-rRNA gets internally cleaved in a series of steps to produce a number of smaller fragments including a 7S intermediate. In the nucleus, the Dis3-exosome complex is required for the 3' end processing of the 7S intermediate into the mature 5.8 S rRNA and for the degradation of the 5' external transcribed spacer removed from the full length pre-rRNA transcript . Final mS. cerevisiae tRNA methyltransferase mutants that produce hypomodified tRNAs [Dis3 has been found to specifically degrade tRNA species in yeast. This function was first discovered in ed tRNAs ,74. Thesed tRNAs . This phAs mentioned previously, miRNA-mediated degradation of mRNAs is an important means of modulating gene expression. The unique combination of miRNAs contributes to a cell’s specific array of protein expression and their misexpression is associated with many types of human cancer . For thiDrosophila wing imaginal discs, Dis3 knock-down has been found to increase the expression of the mature form of miR-252-5p but not the precursor, suggesting Dis3 may be functioning to specifically degrade the mature miRNA as a means of regulation. Another miRNA, miR-982-5p, decreases in expression in Dis3 knock-down discs, suggesting Dis3 may be involved in processing the precursor miRNA into its mature form [Drosophila, a family of miRNAs have been discovered that are encoded in introns, which are processed in an exosome mediated biogenesis pathway. These mirtrons bypass normal Drosha cleavage and are processed into pre-miRNAs by the spliceosome. After splicing the 3' tail is trimmed by the exosome [Many miRNAs that are known to be degraded in different organisms have as yet undefined nucleases. Nevertheless, the exosome and sometimes DIS3 specifically have been found to be involved in the turnover of several miRNAs. In ure form . Also in exosome . AdditioOne of the major classes of nuclear exosome substrates in humans is PROMoter uPstream Transcripts (PROMPTs). Similar to cryptic unstable transcripts (CUTs) in yeast, PROMPTS are short-lived RNA species, between 200 and 600 nucleotides in length, transcribed upstream of the promoters of active protein-coding genes. PROMPTs are transcribed by any of the three RNA polymerases and have 3' poly-A tails as well as 5' cap structures. Evidence suggests that most, if not all, actively transcribed RNA pol II genes have associated PROMPTs, but they seem to be especially prominent at TATA-less, CpG-rich promoters with broad transcription start site (TSS) regions . PROMPTsDrosophila which pertain to the biological activity of DIS3  to chromatin in mammalian B-cells, where DIS3 may be functioning specifically in degrading nascent RNA during the DNA repair process . AID funIn vivo knockdown, ChIP, and physical association studies by Basu et al. provide evidence that the RNA exosome functions in recruiting AID to both strands of transcribed duplex DNA substrates [et al propose a model whereby the exosome functions on stalled Pol II units that backtrack to reveal a free 3' end [bstrates . To do tbstrates . Howevere 3' end . DIS3 orThe earliest study linking DIS3 to cancer identified it as a metastasis-related gene. Two independent gene expression profiling studies of colorectal cell lines and human tissues identified overexpression of DIS3 as high as 38-fold in primary and metastatic tumours relative to normal colonic mucosa ,19. AnotDIS3 may also be biologically relevant in melanoma. In superficial spreading melanoma (SSM) cells, DIS3 has reduced expression compared to normal melanocytes because one chromosomal copy is deleted. In contrast, DIS3 is overexpressed in nodular melanoma (NM) cells . FurtherWhole genome sequencing studies have identified missense mutations in DIS3 to occur in ~4% (4/106) of Acute Myeloid Leukaemia (AML) patients . In all The most striking association between DIS3 and cancer is probably the finding that DIS3 is recurrently mutated in multiple myeloma (MM). Multiple myeloma is defined by a malignant proliferation of monoclonal antibody -secreting plasma cells and counts for 20% of deaths related to haematological malignancies ,102. MM To date 34 of 306 (11%) myeloma patients analysed by whole genome or exome sequencing studies have been found to have missense mutations in DIS3 that may be functionally relevant  25,26,2,28. In mSimilar to many other cancers, multiple myeloma has been found to develop as a consequence of a clonal evolution of cells ,105,106.High throughput studies have provided semi-quantitative analysis of the size of the clonal populations carrying a particular mutation within an individual tumour. It was anticipated that mutations arising in all the clones would take part in initiating myeloma whereas mutations present only in some subclones would be potentiators of the disease. However, it appears that the situation is not quite that simple. Mutations in DIS3 were found to be both clonal in some patients and sub-clonal in others meaning they are functioning sometimes as the former and sometimes as latter ,28. Thiset al. have found its expression to be highly correlated to the expression of a set of ribosomal proteins and translation initiation factors [Within patient samples, some patterns of cooperation and exclusion can be identified between mutations in DIS3 and other genes. DIS3 mutations seem to be mutually exclusive with mutations in FAM46C. Collectively, DIS3 and FAM46C mutations are observed in 21% of patients . The pre factors . DIS3 ismiR-15a/16). This raises the possibility that mutation and selection of DIS3 as a driver mutation in myeloma is dependent on deletion of 13q14. However, more NGS studies are needed to increase the sample size in order determine whether this correlation is significant.Conversely, DIS3 mutations mostly seem to occur in parallel with a hemizygous  deletion of the RB1 region (13q14), either as del (13q) or as an interstitial deletion of the RB1 locus. The gene of interest at 13q14 may be RB1 (retinoblastoma tumour suppressor protein), or one of the miRNAs at this locus which are under-expressed in CLL and MM (Although there is very little data on the clinical impact of DIS3 mutations, one very recent study has identified a trend towards a shorter median overall survival for patients with DIS3 mutations. Patients carrying DIS3 mutations in minor subclones of their tumours also showed a significantly worse response to therapy compared to patients with DIS3 mutations in the major subclone . NeverthWhile the cohort sizes in these genome-wide sequencing studies are large, the absolute number of patients with DIS3 mutations is nonetheless relatively small. For this reason, more patient sequencing studies need to be conducted in order to fully determine the clinical implications of DIS3 mutations to myeloma prognosis.DIS3 has been proven to be far more than simply an RNA disposal machine, with essential roles in both gene regulation and small RNA processing. The phenotypes of DIS3 knockdowns and mutants have led to both mechanistic and biological insights about the roles of this enzyme in the cell. Mutants often reveal specific developmental phenotypes, suggesting DIS3 may be targeting particular functional subsets of RNAs such as those involved in cell-cycle regulation, spindle assembly and kinetochore function. While a large body of structural and biochemical data exists, it is only in recent years that the functional diversity of DIS3 has been fully appreciated and DIS3 has been connected with human disease.Although DIS3 has been found to be associated with multiple types of cancer including colorectal, melanoma and three types of blood cancers, there is no consensus about whether DIS3 is a tumour suppressor or oncogene. It seems to be over-expressed in some cancers and under-expressed in others, whilst displaying loss-of-function mutations in AML, suggesting the role of DIS3 might differ within the context of different tissues and tumourigenic pressures. However, the most significant association of DIS3 and human disease is the recurrent loss-of-function mutations in multiple myeloma patients. Although it is an extremely heterogenous disease, multiple myeloma is currently, for the most part, treated as a single entity with the result that therapeutic success is varied amongst individual patients. As the characterisation of the PML-RARA fusion showed, a lot of progress can be made by the identification of a single molecular event regarding disease definition. Therefore, identifying the role of DIS3 in oncogenesis may help to develop new targeted therapies for affected patients.The mutant and knock-down phenotypes we have discussed in lower organisms and cell lines have the potential to yield insight into the role of DIS3 in myelomagenesis. The observations that DIS3 causes cell cycle arrest in many mutants may at first appear illogical in relation to its role in MM as a tumour suppressor gene. If DIS3 plays an important role in myeloma progression, we may expect DIS3 mutants to display over-proliferation or de-differentiation, phenotypes more consistent with those observed in tumours. However, when we consider that cancers are a result of a clonal evolution of cells that have acquired not just one, but many cooperating mutations, it is not surprising that mutation of just one of these genes in a model organism produces a different phenotype. This is demonstrated in single yeast Dis3 mutants that display elongated metaphase spindles. The production of faulty spindles leads to a block in metaphase to anaphase transition. In these single mutants, the intact Mad2 protein restrains mitotic progression because the chromosomes cannot be segregated properly, leading to eventual apoptosis. However, in Dis3 Mad2 double mutants, cells proceed to anaphase despite having elongated spindles meaning chromosomes are not properly segregated, leading to the production of aneuploid cells. This phenotype may have relevance to the pathogenesis of multiple myeloma of which 90% of patients display aneuploidy . A combii.e., those defined by IgH translocations [Another scenario by which DIS3 could be contributing to the development of myeloma is through the interaction between the RNA exosome and AID during the process of class switching and hypermutation in B-cells. Multiple myeloma is a malignancy of mature antibody-producing B-cells. DIS3 mutations could indirectly, through disruption of an interaction with AID, cause mistargeting of somatic hypermutation leading to chromosomal translocations. This fits with DIS3 mutations being almost exclusively observed in non-hyperdiploid subtypes ocations . ExactlyThe preferred method by which to experimentally verify these hypotheses is to use true myeloma models. The cell line OPM2 for instance, carries a DIS3 mutation in the PIN domain paired with cnLOH ; howeverAs well as performing functional assays to elucidate the role of DIS3, important information can be derived from mutational patterns revealed by genome-wide sequencing studies. Many mutations arising in myeloma do not co-occur in the same cell progeny suggesting disruption of one gene in a pathway may be sufficient to drive oncogenesis. As discussed, mutations in DIS3 and mutations in genes involved in protein translation and/or homeostasis such as FAM46C appear to be mutually exclusive in MM . ConversIt is useful to note that there appears to be an accumulation of mutations in the RNB domain of DIS3 but not DIS3L in myeloma, suggesting that inhibition specifically of the exonucleolytic activity of the nuclear exosome is what may facilitate oncogenesis. If this is the case, one might ask the question: Why Rrp6, which can act redundantly with DIS3 in the nucleus, does not compensate for the loss of DIS3 activity? This may be explained by a study in yeast which shows that when DIS3 is mutated, Rrp6 in the 11-subunit exosome complex is inhibited . This do


Subacromial pain syndrome (SAPS) accounts for around 50 % of all cases of shoulder pain. The most commonly used treatments are glucocorticosteroid (steroid) injections and exercise therapy; however, despite treatment SAPS patients often experience relapse of their symptoms. Therefore the clinical effect of combining steroid and exercise therapy is highly relevant to clarify. The aim of this randomized controlled trial was to investigate if exercise therapy added to steroid injection in patients with SAPS will improve the effect of the injection therapy on shoulder pain.In this two-arm randomized trial running over 26 weeks, patients with unilateral shoulder pain (> 4 weeks) and thickened subacromial bursa (> 2 mm on US) were included. At baseline all participants received two steroid injections into the painful shoulder with an interval of one week. Subsequently they were randomized (1:1) to either 10 weeks exercise of the involved shoulder (intervention group) or exercise of the uninvolved shoulder (control group). The patients were re-examined after the exercise program (at week 13) and again at week 26. The primary outcome assessed after 26 weeks was change in shoulder pain analyzed using the intention-to-treat principle (non-responder imputation).P = 0.53); pain in activity 2.2 , 26 weeks: rest 5.6 ; activity 2.2 . The reduction in pain was most evident in the control group at all four pain measurements. The only difference between groups was seen by US examination at week 13, where fewer participants with impingement were observed in the intervention group compared with the controls .Ninety-nine SAPS patients  participated (49 intervention/50 control). At both follow up visits (week 13 and 26) no statistically significant between-group differences in pain changes on a visual analog scale (mm) were seen . Registration date 5 May 2011.ClinicalTrials.gov (The online version of this article (doi:10.1186/s13075-016-1002-5) contains supplementary material, which is available to authorized users. Shoulder pain is common and accounts for around 12 % of the contacts in primary care , 4. SAPSThe most commonly used treatments for SAPS are glucocorticosteroid (steroid) injection and exercise therapy. SAPS is often recurrent, so patients often receive an unstructured mix of both treatments , 7–10. EInflammation and pain may impair muscle function . ExperimWe conducted an assessor-blinded, two-arm, parallel-group, randomized controlled trial running over 26 weeks; primary outcome was assessed after 26 weeks (primary end point). A methodological protocol was developed and registered with ClinicalTrials.gov before the trial began (NCT01506804) 05/05-2011. The protocol was also approved by the Regional Health Research Ethics Committee (H-4-2010-022). The trial was conducted in accordance with the Helsinki Declaration and this paper follows the CONSORT reporting guidelines . All parParticipants were recruited from a rheumatology outpatient clinic at a Hospital in Denmark and by advertising on a specific site on the Internet for participants for clinical trials, between May 2009 and April 2011.Inclusion criteria were age 18 to 70 years and unilateral shoulder pain provoked by active shoulder abduction and lasting at least 4 weeks. In addition, an enlarged subacromial bursa (≥ 2 mm) as assessed by ultrasound imaging (US) in the symptomatic shoulder was required.Exclusion criteria included complete or partial ruptures of the biceps or rotator cuff tendons assessed by US , symptomPotentially eligible participants having received oral and written information about the study were invited to a clinical examination by a rheumatologist and an US examination of the shoulder. Upon inclusion, and subsequent to baseline assessments, the participants received two US-guided injections of steroid in the thickened bursa in the painful shoulder, given with an interval of 1 week. Two injections were given to increase the chances of a clinical effect. After the second injection, the participants were randomized. The intervention group received a training program for the painful shoulder. The control group received the same program for the asymptomatic shoulder. Both groups commenced the training program 1 week after receiving the second injection. The exercise program lasted for 10 weeks.The participants were randomized 1:1 in blocks of 4–6 using an envelope-based lottery. The individual allocations were held in sealed opaque envelopes. The leader of the project (KE), who was blinded to all clinical data, made the allocation. Due to the type of treatment in both the intervention and control group neither the participant nor the person performing the intervention were blinded. The persons performing the clinical tests and training sessions in both intervention and control group were blinded to the self-reported shoulder function. The person performing all the statistical analysis was blinded to group allocation.The two injections given (separated by 1 week) each contained 1 mL methylprednisolone  and 2 mL lidocaine (5 %). All injections were given US-guided, as it has been shown that the effect on pain and function is superior to blind injections –21. An ePatients sat in an upright position. The arm was positioned behind the lower back, with the elbow flexed. The US probe was placed on the lateral contour of the shoulder parallel to the underlying supraspinatus tendon. The injection was performed with a lateral approach and real-time US evidence of the bolus being correctly distributed in the enlarged bursa.The exercise intervention followed the descriptors by Toigo & Boutellier . The exeDuring the first 2 weeks of exercise, (phase 1), the emphasis was on scapula muscle control and strength. The following 8 weeks (phase 2) aimed at progressive strengthening of the muscles of the rotator cuff. The program rationale was to target muscle function deficits of the scapula prior to targeting muscle function deficits of the rotator cuff muscles , 23. An Outcomes were measured at baseline (before the first injection), at the week-13 visit (after the exercise program), and at a 26-week follow-up visit (12 weeks after cessation of the exercise program). As the purpose of this study was to assess the effect of stringent combined pharmacological and exercise therapy the co-primary outcomes were changes from baseline in current shoulder pain during active shoulder abduction and at rest at the 26-week follow up (primary end point). These pain measures were assessed on 0–100 mm VAS with anchors being 0 = no pain and 100 = worst imaginable pain. The secondary outcomes were the same pain ratings at week 13; clinical impingement signs (Hawkins-Kennedy test), US impingement test, subacromial bursa size on US , self-reported shoulder function (Shoulder Disability Questionnaire (SDQ)), and isometric muscle strength.In the Hawkins-Kennedy sign the shoulder pain is reproduced by shoulder flexion followed by a forced internal rotation . The tesIMS was assessed unilaterally (painful shoulder) using a handheld dynamometer . This method has been shown to be reliable for assessment of muscle strength in the shoulder and hip , 27. IMSSelf-reported functional status of the shoulder was assessed by the SDQ. The questionnaire consists of 16 items assessing pain during the previous 24 h in the shoulder during activities of daily living , 30. ThrP < 0.025), using the usual F test of the group effect in the general linear univariate model with fixed class effects ) with an additional fixed covariate (baseline pain). To account for attrition we decided to include 100 patients in total, yielding a power of 88.8 %. Although we also investigated effects on other outcomes, we did not power the trial for this because we had no a priori assumptions about effect sizes in our secondary outcomes and thus a larger trial may be needed to reliably detect these.This study was powered as a superiority trial for a comparison between the participants allocated to exercise of the painful shoulder (intervention) and those allocated to exercise of the non-painful shoulder (control). Assuming that the intervention produced a reduction in one of the co-primary outcomes (pain during active shoulder abduction and pain at rest) that was 10 mm larger than the control with a standard deviation of 20 mm and a conjectured correlation of 0.7 between the covariate (baseline pain) and the response (change in pain), we calculated that we would need 42 participants per group to test a two-tailed hypothesis with 80 % power and a 2.5 % significance level . All descriptive statistics and tests are reported in accordance with the recommendations of the Enhancing the Quality and Transparency Of health Research (EQUATOR) network, as per the Consolidated Standards of Reporting Trials (CONSORT) statement . In ordeAll patients randomized were assessed for efficacy and safety  population). At 13 weeks (the end of intervention) and after 26 weeks, the exercise and control groups were compared using analysis of covariance (ANCOVA) to analyze mean changes from baseline in the outcomes. The model included the change as the dependent variable, with treatment group as a main effect and the baseline score as an additional covariate.The proportion of patients, who were impingement-positive on US and/or on clinical assessment at the 13-week and 26-week follow up, were analyzed using the chi-square test to evaluate the differences between proportions between the exercise and control group and the group differences are presented as risk differences with 95 % confidence interval.P values, based on the ANCOVA model.The baseline observation carried forward (BOCF) approach was used for patients who did not complete the study, as this method seems conservative in self-help management programs; i.e., using a non-responder imputation. As this trial was designed as a pragmatic trial, baseline data were only imputed when the patient did not attend follow-up visits, and was not based on adherence rate or compliance considerations. Unless stated otherwise, results are expressed as the mean difference between the groups and 95 % confidence intervals (95 % CI) with the associated The flow of patients through the trial can be seen in Fig. n = 65) and the compliance rate was calculated for these participants. In the intervention group 27 of the 33 completers filled in an exercise diary with an average number of exercise sessions of 29 out of 30 (97 %). In the control group, 31 of 32 participants filled in a diary and the number of exercise sessions was 26 out of 30 (87 %).Exercise diaries were only collected from the participants who completed the study (P = 0.62). The group difference in change from baseline in pain at rest was 5.6 mm , potentially in favor of the control group.We found no statistically significant differences between groups in the co-primary pain outcomes at week 26  and the group difference in change from baseline in pain during shoulder abduction was 2.2 mm , potentially in favor of the control group.At the 13-week follow up the group difference in change from baseline in pain at rest was 1.7 mm  , clinical impingement test or isometric muscle strength at the 13-week or 26-week follow up  had US impingement at baseline. This indicates that the result should be interpreted with caution. The difference was not maintained at week 26. No group differences were observed in any of the other secondary outcomes.The equal beneficial change in pain in rest and activity in both groups is in agreement with a previous study of patients with SAPS . In thatIn contrast to other studies investigating the effect of exercise therapy we added no other physiotherapy techniques, such as manual therapy, to the training or control interventions, thus minimising the bias of a mixed intervention , 34.In our study, we observed a minor increase in muscle strength in both groups, but no differences between groups. The intensity of our exercise program is comparable to the standard care offered to patients with SAPS in clinical practice , 35–37. Another study comparing exercise therapy after steroid injection with exercise therapy alone  found noPatients with SAPS form a heterogeneous group and clinical tests used to verify specific diagnoses and guide treatment are not particularly reliable or valid , 38. It Our cohort reflected the general population of persons with shoulder complaints as the mean age was around 50 years and the majority was women , 2. As en = 12 vs. n = 16; missing data, n = 6), indicating that the initial effect of the study participation was not the main reason for leaving the study.It is a limitation of our study that the exercise compliance was only evaluated in the completer participants, and it is not unlikely that the participants lost to follow up had poor adherence to the programs. Nevertheless, the compliance was comparable between groups, which indicates that bias due to difference in exercise compliance was small in our study. Another limitation of the study is that the attrition rate was relatively high. The most common reason for drop-out was lack of time. The reported effect of the steroid injections among drop-out participants was comparable to the compliant subjects . Effects on functional ability, clinical impingement signs, or muscle strength when compared to exercise of the uninvolved shoulder were also comparable between groups. The results indicate a short-term benefit on US-assessed impingement test immediately after the exercise program; however, this effect was not maintained at the 26-week follow up.