Immunolocalization of hordein synthesis and transport in developing barley endosperm.

The spatial accumulation of hordeins in the developing endosperm of barley grains was examined by immunofluorescence microscopy (immunolight microscopy [iLM]) and immunoelectron microscopy (iEM) to establish the timing and subcellular pattern of hordein synthesis and deposition. The pattern seen for hordeins was compared to other abundant grain proteins, such as serpin Z4 and lipid transfer protein 1 (LTP1). Hordein accumulates throughout grain development, from 6 to 37 days post-anthesis (DPA). In contrast, serpin Z4 was present at 6 DPA, but the greatest synthesis and accumulation occurred during the middle of seed development, from 15 to 30 DPA. LTP1 accumulated later in seed development, from 15 to 30 DPA. Hordeins accumulated within the lumen of the endoplasmic reticulum (ER), were exocytosed from the ER membrane, and accumulated in protein bodies, which then fused either with the protein storage vacuoles or with other protein bodies, which also later fused with the protein storage vacuoles. iEM showed hordein, and LTP1 appeared not to traverse the Golgi apparatus (GA). Hordein, LTP1, and serpin Z4 colocalized to the same protein bodies and were co-transported to the protein storage vacuole in the same protein bodies. It is likely that this represents a general transport mechanism common to storage proteins in developing grains.

Digestibility of wheat alpha-amylase/trypsin inhibitors using a caricain digestive supplement.

Wheat is a major source of nutrition, though in susceptible people it can elicit inappropriate immune responses. Wheat allergy and non-celiac wheat sensitivity are caused by various wheat proteins, including alpha-amylase trypsin inhibitors (ATIs). These proteins, like the gluten proteins which can cause celiac disease, are incompletely digested in the stomach such that immunogenic epitopes reach the lower digestive system where they elicit the undesirable immune response. The only completely effective treatment for these immune reactions is to eliminate the food trigger from the diet, though inadvertent or accidental consumption can still cause debilitating symptoms in susceptible people. One approach used is to prevent the causal proteins from provoking an immune reaction by enhancing their digestion using digestive protease supplements that act in the stomach or intestine, cleaving them to prevent or quench the harmful immune response. In this study, a digestive supplement enriched in caricain, an enzyme naturally present in papaya latex originally designed to act against gluten proteins was assessed for its ability to digest wheat ATIs. The digestion efficiency was quantitatively measured using liquid chromatography-mass spectrometry, including examination of the cleavage sites and the peptide products. The peptide products were measured across a digestion time course under conditions that mimic gastric digestion in vivo , involving the use of pepsin uniquely or in combination with the supplement to test for additive effects. The detection of diverse cleavage sites in the caricain supplement-treated samples suggests the presence of several proteolytic enzymes that act synergistically. Caricain showed rapid action in vitro against known immunogenic ATIs, indicating its utility for digestion of wheat ATIs in the upper digestive tract.

Three-dimensional cardiac print assisted percutaneous closure of left ventricular pseudoaneurysm in patient with Behçet's disease.

Spontaneous left ventricular pseudoaneurysms are very rare and can have catastrophic consequences if unrecognized. A case of combined spontaneous left ventricular aneurysm and pseudoaneurysm in Behcet's disease (BD) has been reported. The case emphasizes advanced techniques for percutaneous closure of the defects with the use of an ex-vivo three-dimensional cardiac printed model as a tool to facilitate the procedure.

Relative Rates of Gluten Digestion by Nine Commercial Dietary Digestive Supplements.

Endopeptidases containing supplements may digest gluten and reduce the impact on celiac and gluten-sensitive subjects who inadvertently consume gluten. We investigated the relative rate of disappearance of coeliac relevant epitopes in extracts of nine commercial supplements, using two competitive enzyme-linked immunosorbent assays (ELISAs)-Ridascreen (detects QQPFP, QQQFP, LQPFP, and QLPFP) and Gluten-Tec (detects Glia-α20 and PFRPQQPYPQ). All epitopes are destroyed by cleavage after P and Q amino acids. Rates at pH 3.5 and pH 7.0 were measured. These experiments were designed to measure relative rates of epitope digestion not to mimic in vivo digestion. The supplements were: 1 GluteGuard, 2 GlutenBlock, 3 GliadinX, 4 GlutnGo, 5 GlutenRescue, 6 Eat E-Z Gluten+, 7 Glutenease, 8 Glutezyme, and 9 Gluten Digest. The mean initial rate and half-lives of epitope digestion were deduced and extrapolated to rates at the recommended dose of one supplement in a fasting stomach volume. At pH 7, supplement 1 was the fastest acting of the supplements, with Ridascreen ELISA, more than twice as fast as the next fastest supplements, 5, 6, 7, and 8. Supplements 2, 3, and 4 showed little activity at pH 7.0. Supplement 1 was also the fastest acting at pH 7 with Gluten-Tec ELISA, more than three times the rate for supplements 2 and 3, with supplements 4-9 showing minimal activity. At pH 3.5, supplement 1 acted more than five times as fast as the next fastest supplements, 2 and 3, when measured by Ridascreen, but supplements 2 and 3 were over two times faster than supplement 1 when measured by Gluten-Tec. Supplements 4-9 demonstrated minimal activity at pH 3.5 with either ELISA. Supplement 1 most rapidly digested the key immuno-reactive gluten epitopes identified by the R5 antibody in the Codex-approved competitive Ridascreen ELISA method and associated with the pathology of celiac disease.

The Web of Legal Protections for Participants in Genomic Research.

The identification and arrest of the Golden State Killer using DNA uploaded to an ancestry database occurred shortly before recruitment for the National Institutes of Health's (NIH) All of Us Study commenced, with a goal of enrolling and collecting DNA, health, and lifestyle information from one million Americans. It also highlighted the need to ensure prospective research participants that their confidentiality will be protected and their materials used appropriately. But there are questions about how well current law protects against these privacy risks. This article is the first to consider comprehensively and simultaneously all the federal and state laws offering protections to participants in genomic research. The literature typically focuses on the federal laws in isolation, questioning the strengths of federal legal protections for genomic research participants provided in the Common Rule, the HIPAA Privacy Rule, or the Genetic Information Nondiscrimination Act. Nevertheless, we found significant numbers and surprising variety among state laws that provide greater protections than federal laws, often filling in federal gaps by broadening the applicability of privacy or nondiscrimination standards or by providing important remedies for individuals harmed by breaches. Identifying and explaining the protections these laws provide is significant both to allow prospective participants to accurately weigh the risks of enrolling in these studies and as models for how federal legal protections could be expanded to fill known gaps.

Hordein Accumulation in Developing Barley Grains.

The temporal pattern of accumulation of hordein storage proteins in developing barley grains was studied by enzyme-linked immunosorbent assay (ELISA), western blot and liquid chromatography tandem mass spectrometry (LC-MS/MS). Hordein accumulation was compared to the pattern seen for two abundant control proteins, serpin Z4 (an early accumulator) and lipid transferase protein (LTP1, a late accumulator). Hordeins were detected from 6 days post-anthesis (DPA) and peaked at 30 DPA. Changes in fresh weight indicate that desiccation begins at 20 DPA and by 37 DPA fresh weight had decreased by 35%. ELISA analysis of hordein content, expressed on a protein basis, increased to a maximum at 30 DPA followed by a 17% decrease by 37 DPA. The accumulation of 39 tryptic and 29 chymotryptic hordein peptides representing all classes of hordein was studied by LC-MS/MS. Most peptides increased to a maximum at 30 DPA, and either remained at the maximum or did not decrease significantly. Only five tryptic peptides, members of the related B1- and γ1-hordeins decreased significantly by 21-51% at 37 DPA. Thus, the concentration of some specific peptides was reduced while remaining members of the same family were not affected. The N-terminal signal region was removed by proteolysis during co-translation. In addition to a suite of previously characterized hordeins, two novel barley B-hordein isoforms mapping to wheat low molecular weight glutenins (LMW-GS-like B-hordeins), and two avenin-like proteins (ALPs) sharing homology with wheat ALPs, were identified. These identified isoforms have not previously been mapped in the barley genome. Cereal storage proteins provide significant nutritional content for human consumption and seed germination. In barley, the bulk of the storage proteins comprise the hordein family and the final hordein concentration affects the quality of baked and brewed products. It is therefore important to study the accumulation of hordeins as this knowledge may assist plant breeding for improved health outcomes (by minimizing triggering of detrimental immune responses), nutrition and food processing properties.

Biomechanical Comparison of 3 Inferiorly Directed Versus 3 Superiorly Directed Locking Screws on Stability in a 3-Part Proximal Humerus Fracture Model.

OBJECTIVE: To quantify the stability of 3 points of inferiorly directed versus 3 points of superiorly directed locking screw fixation compared with the full contingent of 6 points of locked screw fixation in the treatment of a 3-part proximal humerus fracture. METHODS: A standardized 3-part fracture was created in 10 matched pairs (experimental groups) and 10 nonmatched humeri (control group). Osteosynthesis was performed using 3 locking screws in the superior hemisphere of the humeral head (suspension), 3 locking screws in the inferior hemisphere (buttress), or the full complement of 6 locking screws (control). Specimens were tested in varus cantilever bending (7.5 Nm) to 10,000 cycles or failure. Construct survival (%) and the cycles to failure were compared. RESULTS: Seven of 10 controls survived the 10,000-cycle runout (70%: 8193 average cycles to failure). No experimental constructs survived the 10,000-cycle runout. Suspension and buttress screw groups failed an average of 331 and 516 cycles, respectively (P = 1.00). The average number of cycles to failure and the number of humeri surviving the 10,000-cycle runout were greater in the control group than in the experimental groups (P ≤ 0.006). CONCLUSION: Data support the use of a full contingent of 6 points of locking screw fixation over 3 superior or 3 inferior points of fixation in the treatment of a 3-part proximal humerus fracture with a locking construct. No biomechanical advantage to the 3 buttress or 3 suspension screws used in isolation was observed.

Classification of instability after reverse shoulder arthroplasty guides surgical management and outcomes.

BACKGROUND: Revision of unstable reverse shoulder arthroplasty (RSA) remains a significant challenge. The purpose of this study was to determine the reliability of a new treatment-guiding classification for instability after RSA, to describe the clinical outcomes of patients stabilized operatively, and to identify those with higher risk of recurrence. METHODS: All patients undergoing revision for instability after RSA were identified at our institution. Demographic, clinical, radiographic, and intraoperative data were collected. A classification was developed using all identified causes of instability after RSA and allocating them to 1 of 3 defined treatment-guiding categories. Eight surgeons reviewed all data and applied the classification scheme to each case. Interobserver and intraobserver reliability was used to evaluate the classification scheme. Preoperative clinical outcomes were compared with final follow-up in stabilized shoulders. RESULTS: Forty-three revision cases in 34 patients met the inclusion for study. Five patients remained unstable after revision. Persistent instability most commonly occurred in persistent deltoid dysfunction and postoperative acromial fractures but also in 1 case of soft tissue impingement. Twenty-one patients remained stable at minimum 2 years of follow-up and had significant improvement of clinical outcome scores and range of motion. Reliability of the classification scheme showed substantial and almost perfect interobserver and intraobserver agreement among all the participants (κ = 0.699 and κ = 0.851, respectively). DISCUSSION: Instability after RSA can be successfully treated with revision surgery using the reliable treatment-guiding classification scheme presented herein. However, more understanding is needed for patients with greater risk of recurrent instability after revision surgery.

Total shoulder arthroplasty with minimum 5-year follow-up: does the presence of subchondral cysts in the glenoid increase risk of failure?

BACKGROUND: This study evaluated the effect of cystic changes in the glenoid on postoperative outcomes and implant survival after total shoulder arthroplasty (TSA). MATERIALS AND METHODS: From 2004 to 2012, 75 patients underwent TSA for primary osteoarthritis with minimum 5-year follow-up. Preoperative 3-dimensional models based on computed tomography imaging were created for all patients. A qualitative evaluation of cystic osteoarthritis was performed through survey grading by 3 fellowship-trained shoulder surgeons. The extent of cyst formation in the glenoid (no cysts, small, medium, or large) was assigned for every patient. In addition, quantitative evaluation was performed on 3-dimensional glenoid models. Functional outcomes, radiographic findings, and the need for revision were compared between group 1 (large and medium cysts) and group 2 (small and no cysts). RESULTS: Qualitative evaluation of cyst formation resulted in the following distribution: no cysts in 8 patients (11%), small cyst formation in 27 (36%), medium cysts in 19 (25%), and large cysts in 21 patients (28%; κ = 0.605). The difference in total cyst volume between group 1 and group 2 was significant (P = .004). The overall revision rate was 7% (5 of 75). All revised patients were in the groups with medium or large cysts. There were no statistical differences in American Shoulder and Elbow Surgeons (ASES) Standardized Shoulder Assessment scores or presence of radiographic loosening among the study groups. CONCLUSION: Qualitative computed tomography evaluation of cystic osteoarthritis correlates with quantitative analysis of cyst volume. Severe cyst formation portends a higher risk of failure at midterm follow-up. Cystic disease did not affect functional outcome or the presence of radiographic glenoid loosening.

Development of a Saw Bones Model for training pedicle screw placement in scoliosis.

OBJECTIVE: The purpose of this study was to determine if a Sawbones Scoliosis Model could be used as a simulator to train residents in placing pedicle screws-a complex procedure with a steep learning curve. Surgical simulation, a common tool teaching residents complex procedures in a safe environment, was staged using a Sawbones Scoliosis Model. Ten junior and ten senior residents out of 25 total possible residents (80%) were instructed how to place pedicle screws using the free-hand technique. They were then asked to place them unilaterally from T4 to L4 and were assessed on completion time, accuracy placement accuracy, and overall competency using an objective rating scale. RESULTS: Senior residents had an average time to completion of 38.9 ± 4.7 min vs. junior's 50.1 ± 11.7 min, and a pedicle screw accuracy of 43.6 ± 6.4% vs. junior's 44.4 ± 17.4%. Overall competency scores were similar for both groups; however, senior residents scored higher in the time and motion subdomain. Senior residents had a faster completion time and were more efficient, suggesting greater experience in spine surgery. The low rate of screw accuracy in both groups validates that simulation is a safe way for trainees to learn complex tasks.

Comparing Multiple Reaction Monitoring and Sequential Window Acquisition of All Theoretical Mass Spectra for the Relative Quantification of Barley Gluten in Selectively Bred Barley Lines.

Celiac disease (CD) is a disease of the small intestine that occurs in genetically susceptible subjects triggered by the ingestion of cereal gluten proteins for which the only treatment is strict adherence to a life-long gluten-free diet. Barley contains four gluten protein families, and the existence of barley genotypes that do not accumulate the B-, C-, and D-hordeins paved the way for the development of an ultralow gluten phenotype. Using conventional breeding strategies, three null mutations behaving as recessive alleles were combined to create a hordein triple-null barley variety. Proteomics has become an invaluable tool for characterization and quantification of the protein complement of cereal grains. In this study multiple reaction monitoring (MRM) mass spectrometry, viewed as the gold standard for peptide quantification, was compared to the data-independent acquisition strategy known as SWATH-MS (sequential window acquisition of all theoretical mass spectra). SWATH-MS was comparable (p < 0.001) to MRM-MS for 32/33 peptides assessed across the four families of hordeins (gluten) in eight barley lines. The results of SWATH-MS analysis further confirmed the absence of the B-, C-, and D-hordeins in the triple-null barley line and showed significantly reduced levels ranging from <1% to 16% relative to wild-type (WT) cv Sloop for the minor γ-hordein class. SWATH-MS represents a valuable tool for quantitative proteomics based on its ability to generate reproducible data comparable with MRM-MS, but has the added benefits of allowing reinterrogation of data to improve analytical performance, ask new questions, and in this case perform quantification of trypsin-resistant proteins (C-hordeins) through analysis of their semi- or nontryptic fragments.

Creation of the first ultra-low gluten barley (Hordeum vulgare L.) for coeliac and gluten-intolerant populations.

Coeliac disease is a well-defined condition that is estimated to affect approximately 1% of the population worldwide. Noncoeliac gluten sensitivity is a condition that is less well defined, but is estimated to affect up to 10% of the population, and is often self-diagnosed. At present, the only remedy for both conditions is a lifelong gluten-free diet. A gluten-free diet is often expensive, high in fat and low in fibre, which in themselves can lead to adverse health outcomes. Thus, there is an opportunity to use novel plant breeding strategies to develop alternative gluten-free grains. In this work, we describe the breeding and characterization of a novel ultra-low gluten (ULG) barley variety in which the hordein (gluten) content was reduced to below 5 ppm. This was achieved using traditional breeding strategies to combine three recessive alleles, which act independently of each other to lower the hordein content in the parental varieties. The grain of the initial variety was shrunken compared to wild-type barleys. We implemented a breeding strategy to improve the grain size to near wild-type levels and demonstrated that the grains can be malted and brewed successfully. The ULG barley has the potential to provide novel healthy foods and beverages for those who require a gluten-free diet.

Proteomic profiling of 16 cereal grains and the application of targeted proteomics to detect wheat contamination.

Global proteomic analysis utilizing SDS-PAGE, Western blotting and LC-MS/MS of total protein and gluten-enriched extracts derived from 16 economically important cereals was undertaken, providing a foundation for the development of MS-based quantitative methodologies that would enable the detection of wheat contamination in foods. The number of proteins identified in each grain correlated with the number of entries in publicly available databases, highlighting the importance of continued advances in genome sequencing to facilitate accurate protein identification. Subsequently, candidate wheat-specific peptide markers were evaluated by multiple-reaction monitoring MS. The selected markers were unique to wheat, yet present in a wide range of wheat varieties that represent up to 80% of the bread wheat genome. The final analytical method was rapid (15 min) and robust (CV < 10%), showed linearity (R(2) > 0.98) spanning over 3 orders of magnitude, and was highly selective and sensitive with detection down to 15 mg/kg in intentionally contaminated soy flour. Furthermore, application of this technology revealed wheat contamination in commercially sourced flours, including rye, millet, oats, sorghum, buckwheat and three varieties of soy.

Using mass spectrometry to detect hydrolysed gluten in beer that is responsible for false negatives by ELISA.

Gluten is the collective name for a class of proteins found in wheat, rye, barley and oats. Eating gluten triggers an inappropriate auto-immune reaction in ∼70 million people globally affected by coeliac disease, where the gut reacts to gluten proteins and this triggers an immune response, resulting in intestinal inflammation and damage. Gluten-free foods are now commonplace, however, it is difficult to accurately determine the gluten content of products claiming to be gluten-free using current methodologies as the antibodies are non-specific, show cross-reactivity and have different affinities for the different classes of gluten. The measurement of gluten in processed products is further confounded by modifications to the proteins that occur during processing and in some case hydrolysis of the proteins. In this study, LC-MS/MS was used to profile whole beer, and two beer fractions representing hydrolysed hordeins (<30 kDa) and hordein peptide fragments (<10 kDa). Subsequently, multiple reaction monitoring (MRM) MS enabled the relative quantification of selected peptide fragments in beer and revealed that certain classes of hordein were prone to hydrolysis (B- and D-hordein). Furthermore, select beers contained very high levels of gluten-derived fragments. Strikingly, those beers that contained high levels of B-hordein fragments gave near zero values by ELISA. The hydrolysed fragments that persist in beer show a dose-dependent suppression of ELISA measurement of gluten despite using a hordein standard for calibration of the assay. The development of MS-based methodology for absolute quantification of gluten is required for the accurate assessment of gluten, including hydrolysed forms, in food and beverages to support the industry, legislation and to protect consumers suffering from CD.

Measuring hordein (gluten) in beer--a comparison of ELISA and mass spectrometry.

BACKGROUND: Subjects suffering from coeliac disease, gluten allergy/intolerance must adopt a lifelong avoidance of gluten. Beer contains trace levels of hordeins (gluten) which are too high to be safely consumed by most coeliacs. Accurate measurement of trace hordeins by ELISA is problematic. METHODS: We have compared hordein levels in sixty beers, by sandwich ELISA, with the level determined using multiple reaction monitoring mass spectrometry (MRM-MS). RESULTS: Hordein levels measured by ELISA varied by four orders of magnitude, from zero (for known gluten-free beers) to 47,000 µg/mL (ppm; for a wheat-based beer). Half the commercial gluten-free beers were free of hordein by MS and ELISA. Two gluten-free and two low-gluten beers had zero ELISA readings, but contained significant hordein levels (p<0.05), or near average (60-140%) hordein levels, by MS, respectively. Six beers gave false negatives, with zero ELISA readings but near average hordein content by MS. Approximately 20% of commercial beers had ELISA readings less than 1 ppm, but a near average hordein content by MS. Several barley beers also contained undeclared wheat proteins. CONCLUSIONS: ELISA results did not correlate with the relative content of hordein peptides determined by MS, with all barley based beers containing hordein. We suggest that mass spectrometry is more reliable than ELISA, as ELISA enumerates only the concentration of particular amino-acid epitopes; this may vary between different hordeins and may not be related to the absolute hordein concentration. MS quantification is undertaken using peptides that are specific and unique, enabling the quantification of individual hordein isoforms. This outlines the problem of relying solely on ELISA determination of gluten in beverages such as beer and highlights the need for the development of new sensitive and selective quantitative assay such as MS.

Quantification of Hordeins by ELISA: the correct standard makes a magnitude of difference.

BACKGROUND: Coeliacs require a life-long gluten-free diet supported by accurate measurement of gluten (hordein) in gluten-free food. The gluten-free food industry, with a value in excess of $6 billion in 2011, currently depends on two ELISA protocols calibrated against standards that may not be representative of the sample being assayed. AIM: The factors affecting the accuracy of ELISA analysis of hordeins in beer were examined. RESULTS: A simple alcohol-dithiothreitol extraction protocol successfully extracts the majority of hordeins from barley flour and malt. Primary hordein standards were purified by FPLC. ELISA detected different classes of purified hordeins with vastly different sensitivity. The dissociation constant (Kd) for a given ELISA reaction with different hordeins varied by three orders of magnitude. The Kd of the same hordein determined by ELISA using different antibodies varied by up to two orders of magnitude. The choice of either ELISA kit or hordein standard may bias the results and confound interpretation. CONCLUSIONS: Accurate determination of hordein requires that the hordein standard used to calibrate the ELISA reaction be identical in composition to the hordeins present in the test substance. In practice it is not feasible to isolate a representative hordein standard from each test food. We suggest that mass spectrometry is more reliable than ELISA, as ELISA enumerates only the concentration of particular amino-acid epitopes which may vary between different hordeins and may not be related to the absolute hordein concentration. MS quantification is undertaken using peptides that are specific and unique enabling the quantification of individual hordein isoforms.

What is in a beer? Proteomic characterization and relative quantification of hordein (gluten) in beer.

The suite of prolamin proteins present in barley flour was characterized in this study, in which we provide spectral evidence for 3 previously characterized prolamins, 8 prolamins with only transcript evidence, and 19 genome-derived predicted prolamins. An additional 9 prolamins were identified by searching the complete spectral set against an unannotated translated EST database. Analyses of wort, the liquid extracted from the mashing process during beer production, and beer were undertaken and a similar suite of prolamins were identified. We have demonstrated by using tandem mass spectrometry that hordeins are indeed present in beer despite speculation to the contrary. Multiple reaction monitoring (MRM) mass spectrometry was used for the rapid analyses of hordein in barley (Hordeum vulgare L.) beer. A selection of international beers were analyzed and compared to the results obtained with hordein deletion beers. The hordein deletion beers were brewed from grains carrying mutations that prevented the accumulation of either B-hordeins (Risø 56) or C-hordeins (Risø 1508). No intact C-hordeins were detected in beer, although fragments of C-hordeins were present in wort. Multiple reaction monitoring analysis of non-barley based gluten (hordein)-free beers targeting the major hordein protein families was performed and confirmed the absence of hordein in several gluten-free commercial beers.

Isovitexin-2'-O-beta-[6-O-E-p-coumaroylglucopyranoside] from UV-B irradiated leaves of rice, Oryza sativa L. inhibits fertility of Helicoverpa armigera.

UV-B irradiated rice leaves (Oryza sativa L.) contained four closely related flavonoids, with either an isoorientin or isovitexin aglycone. These flavonoids have previously been purified and characterized, and were added to artificial diets of the African bollworm (Helicoverpa armigera Hübner) at 0.1x concentration found in irradiated rice leaves. Consumption of different diets had relatively small effects on laval, pupal and adult duration, weight and survival, indicating the insects lived near normal life cycles on all diets. However, one of the compounds, flavonoid IIa, isovitexin-2''-O-beta-[6-O-E-p-coumaroylglucopyranoside], dramatically reduced the number of fertile eggs laid to 7% of control insects (P<0.001) when added to insect diets at 18 nmol gFW(-1) (14 ppm). A similar antifertility effect was observed when only the male partner consumed diet containing flavonoid IIa, indicating that the reduced fertility may be male specific. In contrast, the fecundity and fertility of insects eating diets containing the closely related flavonoids, isoorientin-2''-O-beta-[6-O-E-p-coumaroylglucopyranoside] or isoorientin-2''-O-beta-[6-O-E-p-feruloylglucopyranoside], were not significantly different to control diets.