The data information provided in this article relate to our research article “Using patient serum to epitope map soybean glycinins reveals common epitopes shared with many legumes and tree nuts”   Specifications TableValue of the data•Better understanding of soy storage protein allergens may contribute to allergy management strategies.•It may also contribute to the generation of hypoallergenic soybean cultivars.•Provide risk assessment tools for the evaluation and characterization of the allergenicity of novel foods.1The data presented here show the western blot detection of A2 or A3 subunits by soy-sensitive human sera  and ELIS22.1Soy-sensitive human sera used in the western blots and epitope mapping are previously described 2.2Western blotting of human sera was conducted as previously described 2.3http://www.mimotopes.com) via parallel array platform. Quality Control Assurance was provided for both peptide synthesis and biotinylation by reverse phase HPLC (RP-HPLC), and by mass spectrometry (MS) respectively. The biotinylated 12-mer peptides, frame-shifted by three residues were used as per manufacturer׳s instructions (Application/Method PT3013). DMSO was used to resuspend the dry peptides and streptavidin-coated high capacity plates (Pierce #15500) pre-blocked with SuperBlock™ buffer were used to capture the biotinylated peptides. Serum was diluted at 1/50 in TBS-BSA 2% except for Patients 4 (1/100) and 5 (1/50 or 1/100). The secondary mouse anti-human IgE-HRP  was diluted at 1/4000 in TBS-BSA 2%. SureBlue Reserve TM TMB microwell peroxidase substrate  was added to the plate, the reaction was stopped by acidification and colorimetric detection was performed on a Tecan Sunrise microplate reader with Magellan™ data analysis software  at 450 nm. Each experiment was performed in duplicate. Negative controls were performed using the same protocol, but the addition of human sera was omitted. The data was normalized by calculating the ratio of experimental to negative control and graphed.Two peptide sets representing the mature amino acid sequences of glycinin A2  and A3  were synthesized and biotinylated by Mimotopes  using fixed length patterns


EXP50) and 95% (CEXP95) exposure levels. Benzene levels were estimated to pose a significant risk with HQ50 > 1 and HQ95 > 1 for workers exposed to benzene as base estimates for petroleum refinery workers (Scenario 1), petroleum refinery workers evaluated with personal samplers in Bulgarian refineries (Scenario 2B) and evaluated using air inside petroleum refineries in Bulgarian refineries (Scenario 3B). HQ50 < 1 were calculated for petroleum refinery workers with personal samplers in Italian refineries (Scenario 2A), air inside petroleum refineries (Scenario 3A) and air outside petroleum refineries (Scenario 4) in India and Taiwan indicating little possible adverse health effects. Also, HQ95 was < 1 for Scenario 4 however potential risk was evaluated for Scenarios 2A and 3A with HQ95 > 1. The excess Cancer risk (CR) for lifetime exposure to benzene for all the scenarios was evaluated using the Slope Factor and Overall Risk Probability (ORP) methods. The result suggests a potential cancer risk for exposure to benzene in all the scenarios. However, there is a higher cancer risk at 95% (CEXP95) for petroleum refinery workers (2B) with a CR of 48,000 per 106 and exposure to benzene in air inside petroleum refineries (3B) with a CR of 28,000 per 106.The health risk resulting from benzene exposure in petroleum refineries was calculated using data from the scientific literature from various countries throughout the world. The exposure data was collated into four scenarios from petroleum refinery environments and plotted as cumulative probability distributions (CPD) plots. Health risk was evaluated for each scenario using the Hazard Quotient (HQ) at 50% (C Petroleum refineries and petrochemical plants are major sources of Volatile Aromatic Hydrocarbons (VAHs) in the environment . BenzeneWhere possible, the use of benzene in manufacturing processes has been reduced by replacement with less hazardous compounds. Hence, benzene is now generally regarded as almost exclusively a product of petroleum refining . Workers3, but investigations of petroleum refinery workers in Bulgaria found benzene concentrations levels higher than 3 mg/m3 [Occupational exposure limits (OELs) have been introduced by various organizations for the management of benzene exposure. As reported in  benzene  3 mg/m3 . ExposurHealth risk assessment for exposure to toxic pollutants is usually carried out to evaluate the adverse effects using single data points to quantify the risk. However, risk assessment using probabilistic techniques utilizes probability distributions to estimate the risk thereby giving an evaluation of variability ,13,14,15The aim of this study was to collect and collate exposure data for benzene in petroleum refineries environment on a global scale and conduct a risk assessment to evaluate the possible adverse health effects.EXP50) and 95% (CEXP95) cumulative probability of benzene exposure levels. The CEXP50 level gave an evaluation relevant to most of the exposed population while CEXP95 was relevant to the 5% most exposed group. On the other hand, Overall Risk Probability was an estimated value relative to the whole population.The strategy used in this research involved collection and collation of benzene exposure data and guideline values from the scientific literature. Like data sets presented in Data sets for benzene exposure in petroleum refinery environments used in this study were gathered from the scientific literature using various search engines such as Google, Web of Knowledge, PubMed, Toxnet, Medline and Science Direct. Each reference provided one or more sets of benzene measurements, with each set representing measurements for a sampling location, activity or occupation  1,2,10,,20,21,23The health risk was focused on evaluating exposure data on benzene concentrations in the ambient air of petroleum refineries. Only data sets reported as individual concentrations and base estimates concentrations were utilized for consistency since a number of data sets were reported as mean concentrations. These data sets (mean data) were not included in the risk assessment analysis since they cannot be combined and interpreted with the datasets on individual measurements and base estimates.i, ith point; n, total number of data points. The linear regression equations of the CPD plots were usually calculated between approximately 10%–90% of the Cumulative Probability distribution since this represents the approximately linear part of the CPD plots when a normal distribution occurs.The data sets for benzene exposure were used to develop Cumulative Probability Distribution (CPD) plots by using Microsoft Excel. CP (%) was calculated from Equation (1):The exposure limits for occupational exposure to benzene from various organizations such as European Commission, OSHA, NIOSH, ACIGH and SWA and Air Quality Guidelines (AQGs) and United Kingdom are summarized in 3, ppm and ppb to a uniform unit of µg/m3. The data sets that were used to develop CPD plots for exposure to benzene were categorized into Scenarios as outlined below.The data sets were obtained from the publications listed in This scenario represents benzene concentrations collected as base estimate concentrations for retrospective benzene exposures in petroleum industries from studies using similar methods in deriving the base estimates from benzene measurements. The studies were for early 1940 to 1996 for the Australian study ; 1902 toThis scenario was for petroleum refinery workers in different occupation within the petroleum refineries exposed to benzene. The concentrations of benzene in air were collected by the workers wearing personal air sampling pumps. The data sets used in this scenario were obtained from ,11.The data sets were derived from air samples of benzene taken within various work locations inside the petroleum refineries. Measurements of benzene concentration levels were obtained by using air sampling pumps positioned at various locations inside the petroleum refineries. The data sets used in this scenario were obtained from ,2,10.The data sets obtained for this scenario were for emissions of benzene from petroleum refineries to the immediate surroundings giving exposure to people living near the petroleum refineries. Benzene concentrations were obtained around the petroleum refineries at a maximum distance of 2 km by using air sampling pumps at different sampling locations near the petroleum refineries. The data sets used in this scenario were obtained from ,10,22.EXP50 (the median level which represents the main group) and CEXP95 representing the highest exposed group in the population levels of exposure of the population within each scenario. The benzene concentrations (Scenario 1 to 4) were converted from µg/m3 to µg/kg/day in terms of Lifetime Average Daily Dose (LADD) using values summarized in The data sets for exposure to benzene were categorized into related Scenarios 1 to 4 see  and convEXP is exposure concentration (µg/m3); IR, Inhalation Rate (m3/day); EL, Exposure Length (day/day); ED, the Exposure Duration (days); BW, Body Weight (kg); LT, Lifetime (days).The Lifetime Average Daily Doses (LADD) (µg/kg/day) for exposure to benzene concentrations were calculated for all Scenarios using the default values in EXP50) which represents the main group of individuals and the 95% level (CEXP95) representing the highest exposed group in the population. This highly exposed group occurs at a level of 5% in the population and the median group represents over 50% in the population. Benzene concentrations at CEXP50 and CEXP95 were obtained from the CPD plots (EXP50 and CEXP95 using Equation (3):The HQ method of risk characterization was used to estimate the adverse health effects for exposure to benzene. The USEPA Reference Dose (RfD) derived for benzene was used to estimate the HQ for all Scenarios by using Equation (3). Benzene exposures were estimated at the median level (CPD plots  and conv/kg/day) .Cancer risk is expressed as excess risk of developing cancer over a lifetime of exposure (70 years).EXP50 and CEXP95 in terms of lifetime exposure (LADD) in the various scenarios by using Equation (4):The USEPA inhalation slope factor derived for benzene was used to quantitatively estimate the excess cancer risk at C benzene .The ORP method is based on the use of Overall Risk Probability (ORP) curve. The ORP curve is the plot of exposure exceedence values (1—CP) against the corresponding CP values for dose-adverse effects . A detailed description of overall risk probability in risk assessment has been discussed in . The CPDR2) of 0.97 indicating a normal distribution. At CEXP50, exposure to benzene was higher than NIOSH REL but lower than ACIGH TLV, OSHA PEL, EC LV and SWA OEL. However, at CEXP95 exposure to benzene was higher than NIOSH REL, ACIGH TLV, OSHA PEL, EC LV and SWA OEL. The workers in the highly exposed group reported by the high exposure concentrations in the CPD plots were workers involved in activities such as drum fillers, large terminal operator, gauging, line pigging, rail car loading, refueling, tanker loading and cleaning.The CPD plot as shown in R2) > 0.94 for both CPD plots indicating normal distributions. The CPD plots of Scenario 2A and 2B have almost identical slopes of 65 and 60, respectively. This implies that there was a comparatively wide range of benzene concentration distribution. At CEXP50 and CEXP95 exposure to benzene for Scenario 2A was lower than NIOSH REL, ACIGH TLV, OSHA PEL, EC LV and SWA OEL. While at CEXP50 and CEXP95 exposure to benzene in Scenario 2B was higher than NIOSH REL, ACIGH TLV, OSHA PEL, EC LV and SWA OEL. The high exposure to benzene was for workers in transport and storage of petroleum products facility, benzene manufacturing plant and ethylbenzene—styrene manufacturing plant.The CPD plots  are for R2) > 0.94 for both CPD plots indicating high level of linearity in the distributions. The slope for Scenario 3C is 40 while Scenario 3A has a slope of 117, indicating a relatively wide range of benzene concentration distribution for both CPD plots. At CEXP50 and CEXP95 exposure to benzene for Scenario 3A was lower than NIOSH REL, ACIGH TLV, OSHA PEL, EC LV and SWA OEL. While at CEXP50 and CEXP95 exposure to benzene in Scenario 3B was higher than NIOSH REL, ACIGH TLV, OSHA PEL, EC LV and SWA OEL. The high exposure to benzene was for workers in transport and storage of petroleum products facility, benzene manufacturing plant and ethylbenzene—styrene manufacturing plant.The CPD plots in R2) > 0.96 indicating a high level of linearity in the distribution and a normal distribution. The slope for CPD plot ) and the results were summarized in 50 were < 1 for petroleum refinery workers (Scenario 2A), benzene concentrations in air inside the petroleum refineries (Scenario 3A), and benzene concentrations in air outside the petroleum refineries (Scenario 4). This result suggests minimal risk to the majority of the population in these exposure Scenarios . Also, HQ95 was <1 for Scenario 4 suggesting minimal risk to the high exposed group. However, HQ95 were >1 for Scenarios 2A and 3A indicating possible risk to adverse effects. HQ50 and HQ95 for lifetime exposure to benzene for Scenario 1 (base estimates for petroleum refinery workers), 2B (exposure to benzene for petroleum refinery workers) and 3B (benzene concentrations in air inside the petroleum refineries) were >1 indicating possible adverse health effects for the main group of exposed individuals and the high exposed.The calculated LADD at CEXP50) which represents the main group of exposed individuals and the 95% level (CEXP95) representing the highest exposed group in the population (Scenarios 1 to 4) and the results were presented in EXP50, the excess cancer risk in terms of lifetime exposure to benzene for Scenario 2A (petroleum refinery workers), 3A (benzene concentrations in air inside the petroleum refineries) and 4 (benzene concentrations in air outside the petroleum refineries) are very low in the range of 6 to 18 per 106 as compared to Scenario 1 (base estimates for petroleum refinery workers), 2B (petroleum refinery workers) and 3B (benzene concentrations in air inside the petroleum refineries) that is in the range of 590 to 10,000 per 106. On the other hand, at CEXP95, the highly exposed group that occurs at a level of 5% in the population, the excess cancer risk in terms of lifetime exposure to benzene for Scenario 2A (petroleum refinery workers), 3A (benzene concentrations in air inside the petroleum refineries) and 4 (benzene concentrations in air outside the petroleum refineries) are very low in the range of 200 to 460 per 106 as compared to Scenario 1 (base estimates for petroleum refinery workers), 2B (petroleum refinery workers) and 3B (benzene concentrations in air inside the petroleum refineries) that is in the range of 10,000 to 48,000 per 106. The cancer risk estimated at CEXP95 is only for 5% of the exposed population. The significance difference in the cancer risk estimated is as a result of higher concentration levels of benzene observed in 2B (petroleum refinery workers) and 3B (benzene concentrations in air inside the petroleum refineries) that was 17 to 1400 and 4.6 to 230 times higher than Scenarios 1 to 4 at (CEXP50) and (CEXP95) respectively.The excess CR was calculated for exposure to benzene at the median level (C6) was obtained for Scenario 1 (base estimates for petroleum refinery workers), 0.011% (110 per 106) for Scenario 2A (petroleum refinery workers), 4.8%  for Scenario 2B (exposure to benzene for petroleum refinery workers), 0.015% (150 per 106) for Scenario 3A (benzene concentrations in air inside the petroleum refineries), 1.7%  for Scenario 3B (benzene concentrations in air inside the petroleum refineries) and 0.009% (110 per 106) for Scenario 4 (benzene concentrations in air outside the petroleum refineries).Cancer risk adverse dose—response relationship are shown in EXP95) and the main group of individuals (CEXP50) in the population, while with ORP all of the exposed population were taken into consideration as shown in Overall the ORP and the CR are in reasonable agreement . The dif50 > 1 and HQ95 > 1 for workers exposed to benzene as base estimates for petroleum refinery workers (Scenario 1), petroleum refinery workers evaluated with personal samplers in Bulgarian refineries (2B) and evaluated using air concentrations inside petroleum refineries in Bulgarian refineries (3B). On the other hand HQ50 were <1 for lifetime exposure to benzene in petroleum refinery workers (Scenario 2A), benzene concentrations in air inside the petroleum refineries (Scenario 3A), and benzene concentrations in air outside the petroleum refineries (Scenario 4) suggesting minimal risk to the majority of the population in these exposure Scenarios. HQ95 was <1 for Scenario 4 suggesting minimal risk to the high exposed group however, HQ95 were >1 for Scenarios 2A and 3A indicating possible risk to human health for the high exposed group. The excess cancer risk for lifetime exposure to benzene for all the Scenarios was evaluated using the Slope Factor method at CEXP50 and CEXP95 and also using the ORP method. The two methods showed a reasonable level of agreement. With the ORP method, workers in petroleum refineries in Scenario 2B were observed to have the highest cancer risk 44,000 per 106 followed by those evaluated with data from air inside the petroleum refineries in Scenario 3B with cancer risk of 17,000 per 106 and base estimates for petroleum refinery workers Scenario 1 with cancer risk of 1700 per 106.Benzene levels were estimated to pose a significant risk with HQ


Primary immunodeficiency diseases comprise heterogeneous group of disorders that affect distinct components of the innate and adaptive immune system. The aim of this study was to evaluate the humoral response in children’s patients with recurrent infections.+ for the cases that presented decreased of the serum immunoglobulins.Venous blood samples were collected from 64 children in Pediatric Hospital Professor Heriberto Ferreira Bezerra (HOSPED) - Federal University of Rio Grande do Norte (UFRN), Brazil. The laboratory investigation included measurement of serum IgG, IgM and IgA levels by imunoturbidimetry and the B lymphocyte quantification by flow cytometry with monoclonal anti-CD19Among the 64 children included, 33  were male and 31  were female. The children age ranged from 2 months to 15 years old. According to age, 36 (56%) children were aged 0-5 years; 11 (17%) children were aged 5 years and 1 month to 10 years; 10 (16%) children were aged 10 years and 1 month to 13 years and 11 months and 7 (11%) children were aged 14 years to 15 years. Immunoglobulin concentration below age-appropriate reference values was observed for IgG in 7 (11%) children; for IgM in 3  children and for IgA in 3  children. B lymphocytes were absent in 3  children. The superior respiratory tract infections were the most prevalent in this population.B-cell disorders are the most common type of immunodeficiencies and they are characterized by an increased susceptibility to respiratory tract infections. In this study, 3 patients received the clinical and laboratory diagnostic of X-linked agammaglobulinemia.


Clinical signs and symptoms of pneumoperitoneum are not specific and abdomen radiography is positive in less than 50% of cases. Ultrasonography (US) accuracy for the diagnosis of pneuperitoneum is still unknown.1) define the accuracy of abdominal US for the diagnosis of pneumoperitoneum; 2) define the accuracy of a “2 scan-fast exam” vs a full abdominal exam; 3) compare accuracy of US and abdomen radiographyStudy patients: 11 consecutive adults with a diagnosis of pneumoperitoneum by CT. Control patients: 11 consecutive adults with severe acute abdominal pain with a diagnosis other than pneumoperitoneum by CT. US examination has been performed with a convex and a linear probe using the following scans: epigastrium, right and left hypocondrium, umbelical area and right hypocondrium. All exams were recorded in a video of 5 seconds and each videos reviewed by 2 radiologists and 2 senior physicians blinded to other imaging studies. The reviewers fulfilled a standardized form signing for each scan either presence or absence of pneumoperitonem signs (enhancement of the peritoneum stripe with ring-down artifacts or “comet tails” starting from peritoneum). If one of the signs of pneumopeitoneum was present in at least one scan, the patient was considered to have a US diagnosis of pneumoperitoneum. The reviewers also evaluated abdomen radiography for the presence/absence of pneumoperitoneum. CT was considered the gold standard.1) Accuracy of abdomen US was 88.6%. Sensitivities of convex and linear probes were similar (88.6% vs 84.1%), while specificity of convex was lower than linear probe (81.8% vs 95.5%). 2) Accuracy of a “2 scan-fast exam” was similar to global exam (87.5%). 3) Abdominal radiography sensitivity (72.2%) was lower than US while specificity (92.5%) was higher.Abdominal US has a good accuracy. The 2 scan-fast exam has a similar accuracy of the full abdominal exam. US sensitivity is superior to abdominal radiography thus ultrasonography can be a useful imaging modality for the detection of pneumoperitoneum.


Exsanguination and coagulopathy remain one of the leading causes of preventable trauma related death . Low ionA retrospective cohort analysis was performed on all major trauma patients who had received early blood product in the Emergency Department of a single London Major Trauma Centre over a one year period (January 2013 – January 2014). Ionised calcium levels were taken from venous blood gases from before and after blood product had been transfused. Excel was used to analyse the data.The study included 60 patients aged between 10 and 92 (mean 40), 46 male (77%) and 14 female (23%). Mechanism of injury was predominantly blunt 48 (80%) and penetrating 12 (20%). Patients received between 1 and 16 units of blood product (mode 2). Mean ISS was 26 (5-50) and overall 30 day mortality was 12%.60% were hypocalcaemic on arrival before receiving any blood product (Mean [Ca] 1.1mmol/L 95% CI 1.08 – 1.13) 89% of patients were hypocalcaemic after receiving blood product (Mean [Ca] 0.95mmol/L 95% CI 0.9 – 1.01). There was a statistically significant difference between ionized calcium levels pre and post blood transfusion. A drop in calcium was seen after receiving just one unit of packed red blood cells, with the average drop being 0.05 mmol/L per unit of blood product received.Trauma patients that have sustained blood loss are at risk of hypocalcaemia. Receiving just one unit of blood product further compounds their hypocalcaemic state and the more units that are given the greater the fall that is seen.


Helianthus annuus L.) Heat Shock Factor A9 (HaHSFA9) enhanced seed longevity in transgenic tobacco (Nicotiana tabacum L.). In addition, the overexpression of HaHSFA9 in vegetative organs conferred tolerance to drastic levels of dehydration and oxidative stress.We have previously reported that the seed-specific overexpression of sunflower (HaHSFA4a) and HaHSFA9 enhanced all the previously reported phenotypes described for the overexpression of HaHSFA9 alone. The improved phenotypes occurred in coincidence with only subtle changes in the accumulation of small Heat Shock Proteins (sHSP) that are encoded by genes activated by HaHSFA9. The single overexpression of HaHSFA4a in vegetative organs (which lack endogenous HSFA9 proteins) did not induce sHSP accumulation under control growth conditions; neither it conferred thermotolerance. The overexpression of HaHSFA4a alone also failed to induce tolerance to severe abiotic stress. Thus, a synergistic functional effect of both factors was evident in seedlings.Here we found that the combined overexpression of sunflower Heat Shock Factor A4a (in planta function. Our results strongly support the involvement of HaHSFA4a and HaHSFA9 in transcriptional co-activation of a genetic program of longevity and desiccation tolerance in sunflower seeds. These results would also have potential application for improving seed longevity and tolerance to severe stress in vegetative organs.Our study revealed that HaHSFA4a requires HaHSFA9 for  DS10 sequences (a seed-specific promoter). We have also shown that the ectopic overexpression of HaHSFA9 from Cauliflower mosaic virus (CaMV) 35S sequences in tobacco seedlings conferred dramatic resistance of green organs and of whole seedlings to severe dehydration  of PSII after treatments with H2O or with 200 mM H2O2 for 24 h.Click here for fileComparison of the accumulation levels of the tagged HaHSFA4a protein in the 35S:A4a and 355:A9/A4a seedlings. 1D-western analyses using anti-hemaglutinin antibodies.Click here for fileExamples of total protein loading controls for the protein samples analyzed by western blot in this article. Ponceau S stained PVDF membranes.Click here for file


Quiescent Interval Single Shot (QISS) has emerged as a robust technique for non-enhanced angiography of peripheral arteries (1). It has been clinically validated with contrast enhanced magnetic resonance angiography (CE-MRA) and digital subtraction angiography (DSA) . Both of these validation studies were performed at 1.5T field strength. Although the initial experience with QISS at 3T using similar imaging parameters as those at 1.5T were promising, venous suppression was inadequate in the thigh and pelvic regions of some patients . In this work, we present a strategy to improve venous signal suppression with the QISS sequence at 3T.The imaging parameters were similar to those reported earlier for QISS at 1.5T (2) with the following exception: the tracking saturation pulse for venous signal attenuation was replaced with an adiabatic inversion pulse (hyperbolic secant). This enables a more homogenous venous suppression at 3T than is possible with regular sinc pulse due to B1 inhomogeneity. The prototype sequence was tested in three volunteers in a 3T system .The use of adiabatic inversion pulse instead of a sinc saturation pulse improved venous signal suppression, as shown in Figure Although QISS has shown robust performance at 1.5T, initial experience at 3T demonstrated suboptimal venous suppression. Although not established, this could be attributed to increased B1 inhomogeneity at 3T. We have demonstrated an approach to improve venous signal suppression at 3T using QISS. More patient studies are required and are underway to test the robustness and consistency of this approach.The primary author is a full time employee of Siemens Healthcare.


Both GCA1 and GCA2 were loss-of-function gene in low-GCA parent and gain-of-function gene in high-GCA parent, encoding the putative Pseudo-Response Regulators, OsPRR37 and Ghd7, respectively. Overexpression of GCA1 in low-GCA parent significantly increases GCA effects in three traits. Our results demonstrate that two GCA loci associate with OsPRR37 and Ghd7 and reveal that the genes responsible for important agronomic traits could simultaneously account for GCA effects.Artificial selection of high yield crops and better livestock is paramount importance in breeding programs. Selection of elite parents with preferred traits from a phalanx of inbred lines is extremely laborious, time-consuming and highly random. General combining ability (GCA) was proposed and has been widely used for the evaluation of parents in hybrid breeding for more than half a century. However, the genetic and molecular basis of GCA has been largely overlooked. Here, we present two pleotropic QTLs are accounting for GCA of days to heading (DTH), plant height (PH) and spikelet per panicle (SPP) using an F One of the most fundamental functions of living organisms is to pass on to their progeny advantageous traits that ensure positive interactions with their environment, whereas any unfit traits are gradually eliminated by natural environmental pressures. This is the well-known evolutionary theory of natural selectionThe first research regarding the genetic variance of GCA and specific combining ability (SCA) was reported by Griffing 1956) using a diallelic cross-mating design. Since then, it has become possible to produce precise estimates of GCA and SCA6 using a6789Recently, GCA has been used as one of the most reliable ways to predict the performance of hybrids. Several prediction methodologies based on molecular markers and genomic, transcriptomic and metabolic prediction methods have been developed111213et al. identified the genetic loci for combining ability that correspond to agronomic traits through QTL mapping using three testcross populations and a backcross recombination inbred line (BCRIL) in rice. The characteristics of the QTLs for combining ability were found to be similar to those of the QTLs for BCRIL performanceet al. identified several genetic loci of GCA and SCA in maize using four testers from different heterotic groups and introgression lines (ILs)More recent studies have attempted to uncover the genetic basis of GCA by employing a quantitative trait locus (QTL) mapping approach1516GCA1 and GCA2—through the BC3F2 population and a set of nearly isogenic lines (NILs). We revealed that GCA1 and GCA2 encode the putative Pseudo-Response Regulator OsPRR37 and Ghd7, respectively. Furthermore, we confirmed that GCA1 functions as a positive regulator not only of the agronomic traits per se but also of GCA by virtue of the overexpression of GCA1 (GCA1OX) in rice. Additional experiments revealed that GCA1 is located in the nucleus and that the loss of the 234 amino acids at its C terminus does not affect its nuclear localisation. Expression profiling of GCA1 indicated that it was constitutively expressed in all developmental stages and tissues and was highly expressed in leaves, young panicles and stem. Our results revealed that the genes for important agronomic traits could simultaneously account for the GCA effects. These findings provide new insight into the molecular genetic basis of GCA and the relationship between important agronomic traits and the effects of GCA.In the present work, we identified 13 QTLs that account for the GCA of three agronomically important traits associated with rice grain yield by using an F2-based NCII design with five testers. We also finely mapped two major pleotropic QTLs—2 population for genetic mapping. To identify the effects of GCA and SCA in the F2 population, variance analysis of GCA and SCA for the three agronomic traits DTH, PH and SPP was carried out on 139 individual plants in the F2 population. The mean squares for the GCA effects from the F2 individuals and testers as well as the SCA effects from progeny of F2 individuals and testers (Testers×F2) were found to be significant for all the three agronomic traits  exhibited the lowest GCA effects, whereas Teqing (TQ) exhibited the preferred positive GCA effects for three agronomically important traits see  online. c traits . The resc traits . These r2 population. As shown in 2 population ranged from −17.8 to 30.6 for DTH, −10.1 to 9.2 for PH and −22.0 to 15.7 for SPP. These results demonstrated that the values of GCA in the F2 population were typical normal distributions, and the mean values of GCA for the three traits were almost zero, suggesting that the GCA effects are quantitative traits. These results indicated that this F2 population was suitable for further QTL analysis of GCA.Our previous results showed that the GCA effects made a major contribution to the performance of hybrid offspring. To genetically map the GCA loci for the three traits, the GCA values from a segregation population are required as phenotypes for genetic mapping. We first evaluated the GCA effects in 139 individuals derived from the F2 population and 141 molecular markers  were used to construct a linkage map with coverage of approximately 1,763 cM . A total of 13 major QTLs for GCA of the three agronomic traits were identified on five chromosomes based on a cut-off LOD score ≥ 3.0  and GCA2 (BC3F2-GCA2) were developed by continuous backcrossing using GL as the recurrent parent via marker-assisted selection (MAS) with the flanking SSR markers RM3670 and RM1306, respectively  conditions. The DTH of dominant individuals from the BC3F3-GCA1 and BC3F3-GCA2 populations was increased by 23.3 days and 20.9 days compared with those of corresponding recessive individuals under natural long day (NLD) conditions, respectively. Simultaneously, other two traits, PH and SPP, were significantly increased in the dominant individuals of BC3F3-GCA1 and BC3F3-GCA2 populations  and two newly developed InDel markers (ID77 and ID710) were used to screen 3,127 individuals from BC3F2-GCA1 population. A total of 23 recombinant individuals were identified, and their GCA values for three agronomic traits were obtained by crossing to the tester varieties  newly developed were used for genotyping within the 802 kb region. Finally, the location of GCA1 was narrowed to a region of approximately 443 kb extending from S77 to the end of chromosome 7 . The expression patterns of those genes in both parents were obtained by RNA sequencing dataArabidopsis Pseudo-Response Regulator 7 (PRR7). The 8 bp deletion in the OsPRR37 in GL resulted in a 234 amino acid truncation at the C terminus, which led to the loss of the CCT domain, whereas the TQ allele has an entire protein with 742 amino acids  carrying 35S:OsPRR37 were generated. The delayed flowering phenotype was observed in positive transgenic plants. The DTH, PH and SPP of homozygous GCA1OX−5 plants were increased by 32.5 days, 23.9 cm and 62.6 spikelets, respectively, under natural long day (NLD) condition compared with negative plants (OsPRR37 in positive GCA1OX lines was significantly higher than that in NIL (GCA1+/+)  and NILs (GCA2+/+ and GCA2−/−), as well as the parents GL and TQ were evaluated using TC progenies derived from five testers  and NIL(GCA2−/−) lines were significantly lower than that in TQ, NIL(GCA1+/+) and NIL(GCA2+/+) lines. These results confirmed that OsPRR37 was the gene corresponding to GCA1. Again, the GCA effects of NIL (GCA2−/−) and NIL (GCA2+/+) supported that Ghd7 corresponds to GCA2. These results indicated that GCA1 and GCA2 significantly increase the GCA of DTH, PH and SPP.To verify whether the candidate gene e plants  online. GCA1+/+) . The GCA testers . The resGCA1, the transgenic plants carrying a GUS reporter gene driven by the GCA1 promoter were generated. The results revealed that GCA1 was constitutively expressed in all tissues and at all developmental stages  plants exhibited a diurnal pattern (GCA1 in NIL (GCA1+/+) increased from dawn to noon and then gradually decreased until midnight, with a peak expression level at noon under NLD conditions. Surprisingly, the overexpression of GCA1 driven by the 35S promoter in GCA1OX lines also exhibited a diurnal pattern, but the diurnal expression pattern was altered and peaked around dusk (at 20:00 for GCA1OX−5 and 16:00 for GCA1OX−9), with the lowest expression levels observed in the morning (at 8:00 for both GCA1OX lines). These results imply that the mRNA level of GCA1 itself could be regulated by unknown diurnal signal(s).To understand the expression profiles of l stages . The mos pattern . The expAtPRR7, an orthologue of GCA1, has a potential bipartite nuclear localisation signal at the C terminus (amino acids 680 to 696). The localisation of the PRR7-GUS fusion was observed to be nuclear in leek epidermal cellsPrevious research has reported that GCA1 and GCA2—that corresponded to OsPRR37 and Ghd7, respectively, and functioned as positive regulators of GCA for DTH, PH and SPP and these traits per se. Our findings revealed that the genes responsible for GCA effects correspond to the genes controlling important agronomic traits. To take another example in this study, two QTLs for the GCA of DTH were found to be located between RM3431 and RM3438 and between RM584 and RM5815 on chromosome 6. The physical regions represented by these SSR markers are close to the physical positions of the genes Hd1 and Hd3a that control flowering time26Hd1 and Hd3a may be responsible for the QTL loci for corresponding GCA effects. These results suggested that QTLs for GCA are directly associated with QTLs for these important agronomic traits.In this study, we identified 13 QTLs for the GCA of three important agronomic traits that were mainly associated with grain yield in rice. We identified two major pleotropic QTLs—GCA1 and/or GCA2 were widely found in elite rice varieties19212324GCA1 and GCA2 while 93-11 contains GCA2. It is apparently the result of domestic selection during cross-breeding. This pattern explains that high GCA-effect parents could easily pass on the corresponding traits to their hybrid progenies by simultaneously integrating the GCA controlling genes into the genome of hybrid progenies during cross-breeding program. It is well explained why elite varieties could be easily developed from parents with higher GCA effects. Our findings help remove the mystery of GCA that has existed since 1942, even though this trait has been widely used for crop and livestock improvements.Previous studies have reported that GCA1 and GCA2 encode OsPRR37 and Ghd7, respectively, which have both been reported to be associated with agronomic traits for high grain yield and adaptability at different latitudes192123GCA effects have widely been applied by breeders to evaluate breeding parents. The consensus of opinion is that elite breeding parents possess preferred GCA effects, which are characteristics of wide adaptability, high hereditary capacity and elite agronomic traits. Our data show that GCA1+/+) was lower than that of GCA1OX transgenic lines, and the GCA effect of SPP in NIL(GCA1+/+) and NIL(GCA2+/+) was even lower than zero (GCA1+/+) and NIL(GCA2+/+) was partly caused by the extremely high GCA effect of SPP in parent TQ. Alternatively, the negative value of the GCA effect of SPP in NIL(GCA1+/+) does not mean inconsistency between NIL(GCA1+/+) and GCA1OX transgenic lines. As in this respect, the GCA effects of NIL(GCA1+/+) and GCA1OX transgenic lines showed the same trends when comparing to the GCA effects of both NIL(GCA1−/−) or GL. For the other explanation, the GCA difference could be caused by the different expression pattern of GCA1 between NIL(GCA1+/+) and GCA1OX transgenic lines  and LATE ELONGATED HYPOCOTYL (LHY) in Arabidopsis32GCA1 and the nuclear localisation of the GCA1 protein indicated that GCA1 might have a similar function as a circadian-regulated transcription factor in rice. Interestingly, GCA2 also possesses a CCT domain exhibiting a diurnal expression pattern and nuclear localisationArabidopsis PRR5 and target DNA in vivoGCA1 and GCA2 might function as transcription factors of various downstream genes in the nucleus by regulating their diurnal expression patterns or expression levels.Previous studies have reported that 2 population with 139 individuals derived from GL×TQ was used in this study. Five varieties—ZS, AJ, GL, L6 and Aizizhan (AZ)—were used as testers to cross to individual plants from the F2 population based on the NCII mating design in Lingshui, Hainan Province, in the spring of 2009. The 139 F2 plants were crossed with the five testers to evaluate GCA effects and were planted in Wuhan, Hubei Province, in the summer of 2009. Twenty plants of each testcross (TC) progeny were planted in a 16.5 cm × 20 cm area following a randomised complete block design with three replicates.Estimation of the GCA effects of elite rice varieties was conducted with a diallelic cross design using the Yuetai B (YB), Zhenshan 97 B (ZS), Aijiaonante (AJ), Guangluai #4 (GL), 93-11(L6) and Teqing (TQ) varieties. An F2 population containing the QTL region of interest were continually backcrossed to GL. The BC3F1 plants were selected by marker-assisted selection (MAS), and the GCA effects of the recombinant plants from BC3F2 were evaluated according to the agronomic traits of the TC progeny , plant height (PH) and spikelets per panicle (SPP) were collected for between 10 and 15 plants from each plot. For fine mapping, the accurate GCA effects of three traits were measured for five plants of non-segregated lines and for between 10 and 15 plants of segregated lines derived from the TC progeny in each replication. Variance analysis and evaluation of the GCA were carried out as previously describedwhere  t-test of the agronomic traits—was performed in SPSS statistics software version 17.0 .The variance analysis and effect evaluation of GCA were processed with DPS software version 7.052 population, including 123 SSRs (http://www.gramene.org/), 14 SNPs and four InDel markers developed from previous genome sequencing data for GL and TQ2 population as described previously3F3 family and the corresponding TC progeny was used to distinguish the genotype of the GCA locus in each recombinant plant of BC3F2.A total of 141 polymorphic molecular markers were used to construct the linkage map using plants from the FGCA1 was confirmed by PCR-based sequencing of the exons in genomic DNA and cDNA obtained by the mRNA reverse transcription of TQ. The CDS  and the 3′UTR (623 bp) were artificially synthesised by Gene-Script  and were inserted into the binary vector pCAMBIA1300. A CaMV 35S promoter was then fused to the 5′ end of the CDS, resulting in the overexpression construct pOsPMP625. To generate the GCA1 promoter-GUS construct, a 1,884 bp promoter region was amplified by PCR and inserted upstream of the 5′ end of the reporter gene beta-glucuronidase (GUS) in the binary vector pCAMBIA1300, designated pOsPMP626. All transgenic plants were generated by introduction into GL through Agrobacterium-mediated transformation.The coding sequence (CDS) of the candidate gene GCA1 and GCA2 were obtained from homozygous BC4F2 plants with more than 90% of the genetic background derived from the GL line, as determined by genome-wide SSR and InDel markers (see GCA1+/+ or GCA1−/−) and NIL (GCA2+/+or GCA2−/−) were determined by using appropriate molecular markers. The homozygous GCA1OX plants from the T1 generation were determined by qPCR as described previouslyGCA1+/+ or GCA1−/−) and NIL (GCA2+/+ or GCA2−/−)], three independent homozygous GCA1OX lines and the parent lines (GL and TQ) were crossed to five tester varieties  at Lingshui, Hainan Province, in the spring of 2014. The GCA effects and the traits of these lines were evaluated.NILs corresponding to different genotypes kers see  online. Actin1 gene was used as an internal control to normalise the data.The uppermost fully expanded leaves from at least three individual plants were harvested at 45 days after germination under natural long day (NLD) conditions. Total RNA was extracted from the pooled leaf materials using TRIzol Reagent  according to the manufacturer’s instructions and then treated with RNase-free DNase I . First-strand cDNA was synthesised using M-MLV reverse transcriptase and oligo (dT) primer  as described by the manufacturer. Quantitative RT-PCR was carried out in a total volume of 10 μl, including 2 μl of cDNA template (5- to 10-fold dilutions), 0.5 μl of 10 μM gene-specific primer and 5 μl of SYBR Green qPCR Supermix-UDG with ROX Reference Dye . The qRT-PCR was conducted on a StepOne System (ABI) using the following parameters: 95°C for 10 min, followed by 40 cycles of 95 °C for 10 s and 60 °C for 30 s. The relative gene expression levels were calculated according to the ΔΔCT method described previously4 buffer (pH 7.0), 2 mM K3Fe(CN)6, 2 mM K4Fe(CN)6 and 0.2% Triton X-100 on ice and then incubated with staining solution containing 1 mM X-Gluc at 37 °C overnight. The coloured tissues were photographed with a camera .A GUS staining assay was performed as described in a previous studyGCA1 and truncated GCA1 (tGCA1) were amplified using appropriate primers based on the sequences in TQ and GL, respectively (GCA1 and tGCA1 were inserted into the pHBT-sGFP(S65T) construct at the C terminal of sGFP without a stop codon between sGFP and GCA1 or tGCA1 to create the in-frame fusion constructs sGFP-GCA1 and sGFP-tGCA1, respectively. Rice protoplasts were used for determining subcellular localisation. Protoplast isolation and PEG-mediated transfection were carried out as described in a previous studyCaMV 35S promoter was co-transformed with sGFP-GCA1 and sGFP-tGCA1. To avoid fluorescence artefacts that could disturb localisation, an optimised concentration of plasmid DNA  was used for the transient expression assay. Subcellular localisation was visualised on an Olympus Fluoview 1000 laser scanning microscope . Triplicates of each transient expression experiment were performed to obtain a robust result.The CDS of full-length ectively . IsolateHow to cite this article: Liu, C. et al.OsPRR37 and Ghd7 are the major genes for general combining ability of DTH, PH and SPP in rice. Sci. Rep.5, 12803; doi: 10.1038/srep12803 (2015).


NEIL1 through promoter hypermethylation was found. Finally, we investigated the reason why an elevated NEIL3 expression level was associated with an increased number of somatic mutations in cancer and found that NEIL3 expression was positively correlated with the expression of APOBEC3B, a potent inducer of mutations, in diverse cancers. These results suggested that the abnormal expressions of NEIL1, NEIL2, and NEIL3 are involved in cancer through their association with the somatic mutation load.The effects of abnormalities in the DNA glycosylases NEIL1, NEIL2, and NEIL3 on human cancer have not been fully elucidated. In this paper, we found that the median somatic total mutation loads and the median somatic single nucleotide mutation loads exhibited significant inverse correlations with the median NEIL1 and NEIL2 expression levels and a significant positive correlation with the median NEIL3 expression level using data for 13 cancer types from the Cancer Genome Atlas (TCGA) database. A subset of the cancer types exhibited reduced NEIL1 and NEIL2 expressions and elevated NEIL3 expression, and such abnormal expressions of NEIL1, NEIL2, and NEIL3 were also significantly associated with the mutation loads in cancer. As a mechanism underlying the reduced expression of NEIL1 in cancer, the epigenetic silencing of NEIL1 , NEIL2 , and NEIL3  are structural human homologues of Escherichia coli (E. coli) Nei and Fpg, the genes encoding a DNA glycosylase that initiates the base excision repair (BER) process. These three homologues also have actual functional activities as DNA glycosylases  were collected from the TCGA data portal (n (RSEM) , excludin (RSEM) . The somμM of 5-aza-dC  for 48 h, as described previously  of the 13 (69.2%) cancer types satisfied the 4 criteria for epigenetic silencing described in activity , these rpression . We nextnk test)  but not Since NEIL1, NEIL2, and NEIL3 have been experimentally shown to have the ability to suppress mutations in human cells and/or in bacterial cells –12, the Using data for 13 cancer types from the TCGA database, we revealed that the median somatic total and SNP-type mutation loads exhibited significant inverse correlations with the median NEIL1 and NEIL2 expression levels and a significant positive correlation with the median NEIL3 expression level. We also showed that a subset of human cancers exhibited reduced NEIL1 and NEIL2 expression levels and an elevated NEIL3 expression level, and these abnormal expressions of NEIL1, NEIL2, and NEIL3 were associated with the mutation load in cancer. We then showed that the reduced NEIL1 expression level observed in various cancers was due to epigenetic silencing by promoter hypermethylation and that such reduction was an independent predictor of a poor outcome among patients with breast invasive carcinoma. Finally, NEIL3 expression was shown to be correlated with the expression of APOBEC3B, a potent inducer of mutations, possibly explaining why an increased NEIL3 expression level was associated with the somatic mutation load in cancer. Thus, our results suggest that the abnormal regulation of NEIL1, NEIL2, and NEIL3 expression is involved in the development of cancer via an increase in the prevalence of somatic mutations, providing a new and important link between abnormalities in the DNA glycosylases NEIL1, NEIL2, and NEIL3 and human cancer.Using a TCGA-based analysis, associations between abnormal NEIL1, NEIL2, or NEIL3 expressions and the somatic mutation load were apparently demonstrated in various cancer types for the first time. The association between reductions in NEIL1 and NEIL2 expressions and the increased number of somatic mutations in cancer is understandable, but the association between an elevation in NEIL3 expression and an increased number of somatic mutations in cancer seems surprising at first glance, since NEIL1, NEIL2, and NEIL3 all have the ability to suppress mutations –12. The  NEIL1 gene via promoter hypermethylation using data from the TCGA database. Among them, the epigenetic silencing of NEIL1 expression in HNSCC, lung adenocarcinoma, lung squamous cell carcinoma, colon adenocarcinoma, and rectal adenocarcinoma was consistent with the findings of previous reports [ NEIL1 gene via promoter hypermethylation in these cancer types, we suspect that the epigenetic silencing of the NEIL1 gene via promoter hypermethylation might be the chief mechanism underlying the downregulation of NEIL1 expression in diverse human cancers. Interestingly, in breast invasive carcinoma, which is one of the cancers that shows the epigenetic silencing of NEIL1, a reduction in NEIL1 expression was shown to be an independent predictor of a poor survival outcome. This novel finding may be useful for the management of breast cancer patients, and if this marker is used in conjunction with other prognosis markers, such as the hormone receptor status [In this study, we found 9 cancer types that showed epigenetic silencing of the reports , 26, 27,r status , the man NEIL1 or NEIL2 mutations have been experimentally demonstrated to actually have reduced or absent repair activity [ NEIL1 or NEIL2 mutations are considered to have a reduced capacity to repair mutagenic bases; thus, similar to cancers with a reduced NEIL1 or NEIL2 expression levels, a higher incidence of mutation is likely to occur in the cells, leading to cancer susceptibility. This scenario is compatible with a previous paper reporting a germline NEIL2 variant that is a marker for risk and the progression of squamous cell carcinomas of the oral cavity and oropharynx [ NEIL1 or NEIL2 mutations should clarify the role of NEIL1 and NEIL2 in the prevention of mutations.So far, several forms of germline nonsynonymousactivity , 29, 30.opharynx  and thatopharynx . Future In this study, the 0.5-fold, 0.5-fold, and 2.5-fold values of the median expression value in noncancerous tissue samples of each organ were used as cut-off values to dichotomize the NEIL1, NEIL2, and NEIL3 expression values in the cancer cases, respectively. If an expression level is downregulated or upregulated in a disease, a fold-change value of 0.5 and 2.5, respectively, has been used to dichotomize disease cases in previous reports , 34; theIn conclusion, our study indicates that the abnormal expressions of NEIL1, NEIL2, and NEIL3 are likely to be involved in mutagenesis in human cancer. Since little is known about gene abnormalities identified by whole-exome sequencing data that induce mutations in cancer, our findings regarding these novel mutagenic factors should contribute to our general understanding of human cancer.Supplementary Table S1: Sample size of TCGA dataset used in this study.Supplementary Table S2: Abnormal NEIL1, NEIL2, and NEIL3 expressions in human cancer.Supplementary Table S3: Incidence of the cancer cases showing abnormal NEIL1, NEIL2, or NEIL3 expression.Supplementary Table S4: Associations between reduced NEIL1 expression levels and increased numbers of somatic mutations in human cancers.Supplementary Table S5: Associations between reduced NEIL2 expression levels and increased numbers of somatic mutations in human cancers.Supplementary Table S6: List of DNA methylation sites used for the analysis of epigenetic silencing of the NEIL1, NEIL2, and NEIL3 genes.Supplementary Table S7: Immunohistochemical score of NEIL1 protein in head and neck squamous cell carcinoma.Supplementary Table S8: Cox proportional hazard analysis of potential predictors of a poor prognosis in breast invasive carcinoma patients  using data from the TCGA database.Supplementary Figure S1: Scatter plots of the median NEIL1, NEIL2, and NEIL3 expression levels and the median mutation loads in 13 cancer types.Supplementary Figure S2: Correlations of PSMB2 and YWHAZ expressions with the expressions of other housekeeping genes in various organs.Supplementary Figure S3: Representative results of abnormal NEIL1, NEIL2, and NEIL3 expressions in human cancer.Supplementary Figure S4: Comparison of the total somatic mutation loads between the group showing abnormal NEIL1, NEIL2, and NEIL3 expressions and the other group in various carcinomas, as performed using a box-plot analysis.Supplementary Figure S5: Inverse correlation between DNA methylation at the NEIL1 CpG site and NEIL1 expression in various human cancers.Supplementary Figure S6: Strong positive relationship between NEIL3 expression and APOBEC3B expression in human cancer.Supplementary Figure S7: Impact of reduced NEIL1 expression in conjunction with hormone receptor status or HER2 status on overall survival in primary breast cancer patients.


We here study a general problem in cellular Ca2+-sensing, namely how similar Ca2+-binding proteins achieve functional selectivity to control finely adjusted cellular responses. We investigated two parameters of critical importance for the trigger and switch function of guanylate cyclase-activating proteins: the myristoylation status and the occupation of Ca2+-binding sites with Mg2+. All zGCAPs can be myristoylated in living cells using click chemistry. Myristoylation does not facilitate membrane binding of zGCAPs, but it significantly modified the regulatory properties of zGCAP2 and zGCAP5. We further determined for all zGCAPs at least two binding sites exhibiting high affinities for Ca2+ with KD values in the submicromolar range, whereas for other zGCAPs (except zGCAP3) the affinity of the third binding site was in the micromolar range. Mg2+ either occupied the low affinity Ca2+-binding site or it shifted the affinities for Ca2+-binding. Hydrodynamic properties of zGCAPs are more influenced by Ca2+ than by Mg2+, although to a different extent for each zGCAP. Posttranslational modification and competing ion-binding can tailor the properties of similar Ca2+-sensors.Zebrafish photoreceptor cells express six guanylate cyclase-activating proteins (zGCAPs) that share a high degree of amino acid sequence homology, but differ in Ca One family of Ca2+-binding proteins named neuronal calcium sensor (NCS) proteins are predominantly expressed in neuronal tissue and are involved in diverse intracellular processes22+-binding motifs, of which in most cases three (sometimes only two) motifs can bind micromolar to submicromolar Ca2+. One group of the NCS proteins is expressed in sensory cells and among them the guanylate cyclase-activating proteins (GCAPs) perform an important function in controlling the membrane bound guanylate cyclases (GCs) in retinal rod and cone cells45Calcium sensor proteins mediate signaling processes that respond to changing concentrations of Ca2+-free, Mg2+-bound form GCAPs activate GCs, but they switch to an inhibitory mode, when all Ca2+-binding sites are filled with Ca2+72+ in rod and cone outer segments are linked to changing levels of the intracellular messenger cGMP. After light activation of the photoreceptor cell the intracellular cGMP level is depleted, leading to a shutdown of cyclic nucleotide gated (CNG) channels in the outer segment of the cell. This stops the influx of Ca2+, which is however still extruded by the continuous operation of a Na+/ Ca2+, K+ exchanger leading to a net decrease of cytoplasmic Ca2+. This decrease is sensed by GCAPs which in turn increase the GC activity, leading to re-opening of the CNG-channels and is a necessary step for the recovery of the photoreceptor to the dark-adapted state45678In their Ca2+-sensitivity, impact on catalytic efficiency of the target GC and different structural implications of the N-terminally attached myristoyl group102+-relay mode fashion, where GCAP1 is activated at higher free Ca2+, followed by GCAP2, which becomes active, when Ca2+-levels have fallen to lower levels9102+-relay system seems also to work in zebrafish rod and cone cells, where, however, the system is more complex due to the larger number of GCAP forms132+-binding properties, Ca2+-sensitive GC regulation and spatial-temporal transcription/ expression profiles. Four zGCAPs, namely isoforms 3, 4, 5 and 7 are cone specific1214Bovine and mice photoreceptor cells express two GCAP forms, GCAP1 and GCAP2, which bind to distant regions in the target GC and have different properties with respect to Ca2+-binding sites with Mg2+781016N-myristoyltransferase (NMT) in E.coli15in vivo and in vitro click chemistry in combination with fluorescence microscopy. Revealing that all zGCAPs can exist in a myristoylated and non-myristoylated form we investigated its impact for target regulation and Ca2+-dependent membrane interaction. We further asked whether the presence of physiological Mg2+ can influence the binding of Ca2+ to zGCAPs and how Ca2+-induced conformational transitions in zGCAPs are influenced.Two parameters are of critical importance for the trigger and switch function of NCS proteins in general and GCAPs in particular: the myristoylation status and the occupation of EF-hand Ca2+-dependent membrane binding for all zGCAPs. Further Mg2+ ions control the Ca2+-affinity as well as the Ca2+-induced conformational changes in zGCAPs.Our results indicate that myristoylation has a strong impact on the regulatory properties of two zGCAPs (2 and 5), but it does not facilitate CaGreen-fluorescent protein (GFP) constructs of NCS proteins including all zGCAPs, bovine GCAP2 and recoverin were used to transfect HEK 293 cells, which were also supplemented with azido-dodecyl acid (a myristoyl substitute). This allowed us to incorporate an acyl moiety into an NCS protein in a living cell. Successful attachment of the acyl group was monitored in a subsequent copper (I)-catalyzed azide-alkyne cycloaddition using a biotin alkyne derivative forming a triazole ring. Thus, NCS proteins that were labeled with biotin could be detected via streptavidin coupled to peroxidase. Each transfected cell sample was analyzed by sodium dodecyl-sulfate polyacrylamide gel electrophoresis, blot transfer and presence of biotin in the protein band was visualized by peroxidase staining . Since GN-terminal attached fatty acyl chain and the C-terminal attached GFP to the protein in transfected cells. 192+-concentration, which had been observed in previous studies and is mainly due to hydrophobic/ electrostatic interactions2122In a second alternative approach we employed a copperless cycloaddition suitable for introducing a fluorescent dye (DIBO-TAMRA-dye) to the fatty acyl group of the NCS proteins in living cells. Thus we were able to colocalize the putative 2+-myristoyl switch, which is typically observed in other NCS proteins like recoverin, neurocalcin δ, VILIP or hippocalcin242526E.coli and purified them afterwards. Principal attachment of the myristoyl group was verified by a click chemistry reaction involving azido-dodecanoic acid and the alkyne derivative of biotin as described above. We then incubated myristoylated zGCAPs and recoverin with isolated photoreceptor outer segment membranes in the absence and presence of Ca2+ , which was tested in living and in disrupted cells. This prompted us to ask, whether zGCAPs can perform a Ca of Ca2+ . No zGCA control .2+] at which the GC activity in the presence of a GCAP molecule is halfmaximal denoted as IC50 value and listed in 2+-sensitivity decreased about 4.7-fold for zGCAP2 and increased 5.7-fold for zGCAP5.Nonmyristoylated zGCAPs exhibit different activity profiles when targeting membrane bound GCs2+ under physiological conditions as it was observed for mammalian GCAP1 and 28. So far no precise values of Ca2+-binding to myristoylated zGCAPs are available and furthermore it is not known, whether Ca2+-binding is affected by physiological concentrations of Mg2+. We used a calorimetric approach (ITC) to analyze the energetics of Ca2+-binding to zGCAPs in the presence and absence of 1 mM Mg2+, which allows us to determine precise values of apparent dissociation constants (KD) for multiple binding sites and the associated changes in binding enthalpy (ΔH). For each titration Ca2+-free zGCAP was kept in a temperature controlled compartment, in which a series of small volumes of CaCl2 was injected. Representative examples for all zGCAPs are shown in 2 (zGCAP3) and −17 kcal per mol of CaCl2 (zGCAP7). Small endothermic responses were only observed with zGCAP1  showed high affinity for Ca2+, whereas for other zGCAPs the affinity of KD3 was in the micromolar range.All zGCAPs contain four EF-hand motifs in their primary structure, where the first one is suggested to bind no Cah zGCAP1 a,5a. Howccessful . We inteplicable . In all 2+the multiphasic binding isotherm for Ca2+-binding gave a best fit with a two site model for zGCAP1, 2, and 3. This result can be best interpreted as having two high affinity sites filled with Ca2+, but the lower affinity site being occupied by Mg2+ .Size exclusion chromatography of myristoylated zGCAPs revealed that the monomeric form was the dominant species for all zGCAPs except for zGCAP5 and 7. A Ca2+-myristoyl switch operation in zGCAPs  differed significantly among the proteins exhibiting the following order: zGCAP5 > zGCAP2 > zGCAP7 > zGCAP3 = zGCAP4. No signals were observed for zGCAP1, although the purified protein was functional shown by the GC activation assay.Finally, the maximal amplitudes that were reached at the end of the Ca2+-induced conformational changes, which however have different consequences for the protein hydrodynamic properties indicating differences in the extent of conformational transitions.We conclude from these results that all zGCAPs undergo Ca2+-signaling that result in specific cellular responses can be mediated by rather similar Ca2+-sensor proteins, which are very often expressed in the same cell type. In order to work on these issues we chose to compare zGCAPs representing one subfamily of NCS proteins. These NCS proteins are well suited for a comparative analysis, since previous work has shown 1) that all zGCAPs are expressed in photoreceptor cells in the larval and adult stages of the zebrafish retina1314152+-binding proteins12152+-dependent manner12131516Calcium sensor proteins like the group of NCS proteins are involved in unique patterns of cellular regulatory pathways and therefore mediate various physiological responses including ion channel function, enzyme activity control and cellular trafficking22+-sensitivity of mammalian GCAP1, but less influence on the regulatory properties of mammalian GCAP21029in vitro conditions. We here demonstrate using complementary approaches that the six zGCAPs are myristoylated in cell lysates and in living cells. Further, myristoylation is not involved in reversible Ca2+-dependent membrane association  leading to one group of zGCAPs with high Ca2+-sensitivity  and one with low Ca2+-sensitivities . Myristoylation is catalyzed by a retinal NMT, which is not active before 7 dpf due the myristoylation pattern we reported previously for zGCAP3Our previous work on the six nonmyristoylated zGCAPs revealed that zGCAP1, 2 and 3 display GC-activating response curves that are halfmaximal around 30 nM free  was done exactly as described121515Analysis of purified zGCAP samples by sodium dodecyl-sulfate polyacrylamide gel electrophoresis and determination of GC activity (three to five repetitions) in the presence of zGCAPs as a function of the free [Ca2 stock solution at T = 25 °C. The titration buffer was decalcified using a self-packed gravity flow Chelex 100 column (Bio-Rad). The remaining Ca2+concentration was determined by a BAPTA absorption assay and was found to range between 30 and 100 nM. All buffers were filtered (0.22 μm) and degassed at least 1h before use. At least three independent repetitions were made for each titration set, if not stated otherwise.ITC experiments with zGCAP-isoforms were performed on a VP-ITC from MicroCal  exactly as described previously for mammalian GCAP1 variants2+ into decalcified buffer without any zGCAP was performed, but did not show significant heat changes in the recording cell. Each titration was analyzed by a model implemented in the software Origin  assuming either three or two Ca2+ binding sites. The best fitting results out of these models were used to obtain dissociation constants KDapp and enthalpy changes (ΔH).Reference injections of Ca2+-titration experiments and data analysis were performed as outlined elsewhere30312 of the highest grade available was dissolved in the decalcified buffer to a final concentration of 46 mM. This stock solution was used to obtain Ca2+ samples of 0.4 μM, 0.7 μM, 0.9 μM, 1.1 μM, 1.6 μM, 2.5 μM, 4.8 μM, 14 μM, 37 μM and 46.2 μM. All buffers were filtered (0.22 μm) and degassed for at least 1h before use. Immobilization of protein samples was achieved by attaching them to the carboxy-methyl dextran matrix of CM5 sensorchips  via the terminal amino group or via internal accessible lysine residues. Typical immobilization densities ranged from 3.5 ng to 10.5 ng/mm2.For SPR experiments, we used exactly the same decalcified buffer conditions as for the ITC experiments, except that Tween20 was added to a final concentration of 0.005% (v/v).The CaHow to cite this article: Sulmann, S. et al. Retina specific GCAPs in zebrafish acquire functional selectivity in Ca2+-sensing by myristoylation and Mg2+-binding. Sci. Rep.5, 11228; doi: 10.1038/srep11228 (2015).


To describe the allergenic food ingredients present in complementary feeding and to examine their potential association with persistent eczema.n 1537). An allergenic food was determined present, if labelled as an ingredient and not if stated as ‘may contain’. Eczema was determined if the UK working party diagnostic criteria were satisfied at 6 and 12 months.Prospective data on complementary feeding practices, using a detailed food diary over the first 6-weeks of complementary feeding, were collected as part of Cork BASELINE Birth Cohort Study  were introduced to solids between 17-26 weeks, 18% <17 weeks. At least one allergenic food ingredient was provided to 64% of infants; including cow’s milk (56%), wheat (43%), soy (41%), eggs (10%), fish (8%) and kiwi (7%). Infant breakfast cereals were the main source of exposure to cow’s milk (71%), wheat (92%) and soy (98%). First exposure to an allergen was typically between 17-26 weeks (73%) and 26% were exposed after 26 weeks. Mothers <25 years (84 vs 70%), who were smokers (83 vs 69%) and had no university degree (82 vs 64%) were more likely to give an allergenic food ingredient in the complementary feeding diet . There was no association between exposure to allergenic food ingredients and persistent eczema [OR 0.96 ] or between exposure either before 17 weeks [OR 2.43 ] or after 26 weeks [OR 1.29 ] and persistent eczema, compared with 17 to 26 weeks .Infant breakfast cereals were the main source of cow’s milk, wheat and soy during the first 6 weeks of transition to solid food. Exposure to allergenic food ingredients in the initial stage of complementary feeding had no influence on persistent eczema. Clear guidance on the introduction of allergenic food ingredients in foods labeled as appropriate from 4-6 months is required.


HER2 status assessment became a mandatory test assay in breast cancer, giving prognostic and predictive information including eligibility for adjuvant anti-HER2 therapy. Precise and reliable assessment of HER2 status is therefore of utmost importance. In this study we analyzed breast cancer samples by a novel technology for concomitant detection of the HER2 protein and gene copy number.Tissue microarrays containing 589 invasive breast cancer samples were analyzed with a double immunohistochemistry (IHC) and silver labeled in situ hybridization (SISH) assay simultaneously detecting HER2 protein and gene copy number in the same tumor cells. This bright-field assay was analyzed using scores according to the modified ASCO guidelines and the results were correlated with patient prognosis.Overall concordance rate between protein expression and the presence of gene amplification was 98%. Fifty-seven of 60 tumors (95%) with IHC score 3+, 6 of 10 tumors with IHC score 2+ (60%) and only 3 of 519 tumors (0.6%) with IHC score 0/1+ were amplified by SISH. Patients with gene amplification despite IHC score 0/1+ had a tendency for worse overall survival  compared to IHC score 0/1+ without amplification. In contrast, there was no difference in overall survival in IHC score 3+/2+ tumors with and without gene amplification.HER2 gene status, especially for the prognostically relevant groups of HER2 protein negative tumors with HER2 amplification and HER2 protein positive tumors without HER2 amplification. Breast cancer without HER2 amplification among IHC score 2+/3+ tumors (10% in our cohort) suggests that other mechanisms than gene amplification contribute to protein overexpression in these cells.The novel double IHC and SISH assay for HER2 is efficient in the identification of breast cancer with discordant HER2 protein and  HER2 status in breast cancer is important in the diagnosis of breast breast cancer by pathologists HER2 protein and/or gene amplification is of enormous importance, as adjuvant or neoadjuvant therapy decisions, made on the assays, all rely on the scores that are given by these tests Determination of HER2 gene amplification.The ASCO guidelines approved several test assays for the HER2 status assessment including immunohistochemistry (IHC) and/or in situ hybridization (ISH) technics with different visualization methods as fluorescence (FISH), silver (SISH) or chromogenic (CISH) labeled probes In this study, we addressed the question whether such tumors with discordant results by IHC and SISH represent prognostically relevant subgroups. We are not aware of any previous studies, correlating results of this bright-field assay with patient prognosis Tissue microarrays (TMAs) containing eligible 652 samples from breast cancer patients were selected for this study. Eligibility was decided upon availability of sufficient tumor tissues in the paraffin blocks. The TMAs were constructed previously as described The cohort was composed of 318 primary breast cancer (49%), 26 local recurrences (4%), 258 axillary lymph node metastases (39%) and 50 visceral metastases (8%). For the TMA analysis there were 589 of 652 interpretable samples available.Histologically, there were 280 invasive ductal (88%), 35 invasive lobular carcinomas (11%) and 5 special types (1%). Grading was assessed by applying the modified Bloom and Richardson grading system after Ellis and Elston on the primary tumors and on most of the metastatic and recurring lesions Details of clinico-pathological parameters are depicted in HER2 dual DNA Probe Cocktail with the UltraView SISH Detection Kit (HER2 locus) and UltraView Red ISH DIG Detection Kit (CEP17 locus) were done on the same tissue micro arrays. Reactions mentioned above in both detection systems (except the hybridization step) were carried out on the automatic Benchmark system . The hybridization step (containing the HER2 and CEP17 gene locus on chromosome 17) was carried out manually according to the instruction of the EnzMettrade mark, Nanoprobes  and on the automatic Benchmark ULTRA system . The HER2 protein was visualized as a purple-red (EnzMet) or DAB brown (Inform) membranous stain, the gene loci (HER2) as confluent black aggregates or singular black spots, the CEP17 region with red spots or aggregates For the simultaneous detection of the HER2 protein and HER2 gene copy number, we used a previously described protocol by Tubbs et al and Downs-Kelly et al. The simultaneous detection of the HER2 protein and HER2 gene was originally scored according to the 2008 guidelines on HER2 scoring in breast cancer SISHHER2 signals were scored as follows: the number of signal copies and the ratios (HER2/CEP17) were calculated, gene copies (>6) or cluster formations  were defined as amplified. Similarly, a ratio >2.2 was set as amplified status; a ratio <1.8 was negative, and a ratio of 1.8–2.2 was referred to as unequivocal (according the 2008 guidelines) and using ratio >2 and > or equal to 6 HER2 gene copies (in >10% of the tumor cells) for a positive status (according to 2013 guidelines).HER2 protein expression was scored as follows: 0 (no staining), 1+ (weak and incomplete membrane staining), 2+  or 3+ (strong complete homogenous membrane staining in more than 30% of the invasive tumor cells). According to the 2013 ASCO/CAP guidelines, the required percentage (10%) for score 3+ and 2+ were used for the re-evaluation of the results based on the 2008 guidelines . Categorical data were analyzed using Chi- Square test. Spearman log rank, Pearson, Breslow and Tarone Ware correlation were used to correlate HER2 protein expression scores and HER2 gene copy number with and clinic-pathological parameters  and overall survival. Non-parametrical tests as Kendall Tau and Spearman rho were used to test correlation between overall survival and HER2 scores with IHC and SISH.There were 589 interpretable spots containing sufficient amount of invasive carcinomas on the TMAs. Sixty of 589 cases were score 3+ by IHC (10%). Fifty-seven of 60 IHC score 3+ cases showed also amplification by SISH (95%). 519 of 589 cases were negative (score 0/1+) by IHC (88%). 3 of 519 IHC negative breast cancers (score 0/1+) showed amplification by SISH (0.6%). Ten of 589 samples were score 2+ by immunohistochemistry (5%), 6 of these cases displayed amplification by SISH (60%). One of the IHC score 3+ cases showed polysomy of both CEP 17 and HER2 gene (<1%) Table 2.. and There was a significantly shorter overall survival among patients whose tumors were either amplified by SISH (p = 0.002) or were IHC score 2+ or 3+ (p = 0.006), when these two parameters were analyzed separately . and Fig 3. and When combining IHC and SISH scores, there was as tendency to shortened overall survival in patients with IHC score 1+ with simultaneous gene amplification, when compared to IHC score 0/1+ tumors without gene amplification , Fig. 4. IHC HER2 scores significantly correlated with hormone receptor status (p = 0.001) and histological grading (p<0.001) and lacked correlation to pathological tumor stage, nodal status or to the presence of distant metastases.HER2 scores also showed a significant correlation to hormone receptor status (p = 0.002) and to histological grading (p<0.001), but not to tumor stadium, nodal status or to distant metastases.SISH Original HER2 status was available in 310 primary tumors performed by IHC and FISH technology. There were 6 of 310 (1.9%) discrepant cases with IHC and 7 of 310 (2.2%) discrepant cases with FISH as compared to double IHC/SISH HER2 testing.HER2 gene copy number. Importantly, the simultaneous detection of HER2 protein and HER2 gene copy number facilitates the identification of relevant prognostic subgroups with poor prognosis especially in the IHC score 0/1+ group with amplification and in the IHC score 3+/2+ group without gene amplification.We analyzed HER2 status in a cohort of invasive breast cancers using a double IHC/SISH assay, enabling the simultaneous detection of the HER2 protein expression and the The novel technology of bright field protein and gene detection assay was developed and described in 2004 by Tubbs et al In our TMA-based study, we demonstrate an excellent correlation between IHC score 3+ and gene amplification by SISH using a dual probe for IHC and SISH. Concordance between IHC and ISH technologies in HER2 status assessment in breast cancer has been the subject of several previous studies Here, we demonstrate that the novel bright-field ISH assay enables improved diagnosis in borderline breast cancer cases. Simultaneous assessment of HER2 status by IHC and SISH shows a significant correlation to overall survival. The use of different ISH assay for the assessment of the HER2 status was assessed in several previous works HER2 gene amplification represent prognostically poor subgroups with shortened overall survival Our study confirms existing data that IHC score 2+/3+ tumors as well as tumors with HER2 gene. This amplification can vary in size, especially in IHC scores 3+ cases The underlying mechanism of protein overexpression in breast carcinomas is preferential amplification or polysomy of the As to the real frequency of IHC score/1+ cases having simultaneous amplification, the literature is inconsistent, mostly due to the fact, that IHC score 0/1+ cases do not need to undergo obligatory reflex testing with an ISH technology Several problematic topics in routine HER2 testing prompted the revision of the 2007 ASCO/CAP guidelines on HER2 diagnostic guidelines. Diversities in the interpretation of chromosome 17 aneuosomy and of HER2/CEP17 ratios may lead to false negative and false positive HER2 status, having an immediate impact on therapy options for the given patient. Tumor heterogeneity, biological characteristics and pre-analytic issues have been also shown to influence accuracy in HER2 status assessment HER2 gene status. Concordance in HER2 status between primary tumors and metastastic sites was the subject of several previous studies, confirming a high consistency in cases with only one metastastic site Our cohort contained also samples from metastases but not from the primary lesions. In metastatic lesions, there was only one case with discordant HER2 protein and HER2 gene amplification independently from HER2 protein expression in IHC score 0/1+ cases correlates with tendency to poorer overall survival. Importantly, our data suggest that the double immunohistochemistry and silver labeled in situ hybridization assay identifies a prognostically relevant IHC 2+/3+ breast subgroup without HER2 amplification, which cannot be detected using a FISH alone approach.We provide data that


The authors have provided a corrected version of ramA was a replication of the image shown for the wild type strain K. pneumoniae Ecl8. The authors have provided a corrected The authors would like to correct The authors confirm that these changes do not alter their findings. The authors have provided raw, uncropped blots for S1 FigybhT and yrbF promoters. The experimental setup was as described previously in 32P-γ ATP. Purified RamA (200 nM) and the labelled DNA probes (2 nM) were incubated on ice. All reactions were performed on ice prior to electrophoresis on 7.5% native gel. Lane 1 of each panel indicates the labelled DNA probe only, Lane 2 is the BSA control and Lane 3 contains RamA+DNA. The dried gel was scanned after overnight exposure to the phosphor screen under default settings on the phosphorimager Typhoon FLA7000IP .Raw data phosphor image scan for the EMSA analyses on the (PDF)Click here for additional data file.


GLIS3 (GLI-similar 3) is a member of the GLI-similar zinc finger protein family encoding for a nuclear protein with 5 C2H2-type zinc finger domains. The protein is expressed early in embryogenesis and plays a critical role as both a repressor and activator of transcription. Human GLIS3 mutations are extremely rare.GLIS3 mutations.The purpose of this article was determine the phenotypic presentation of 12 patients with a variety of GLIS3 gene mutations were sought by PCR amplification and sequence analysis of exons 1 to 11. Clinical information was provided by the referring clinicians and subsequently using a questionnaire circulated to gain further information.GLIS3 who did not present with congenital hypothyroidism. All patients presented with neonatal diabetes with a range of insulin sensitivities. Thyroid disease varied among patients. Hepatic and renal disease was common with liver dysfunction ranging from hepatitis to cirrhosis; cystic dysplasia was the most common renal manifestation. We describe new presenting features in patients with GLIS3 mutations, including craniosynostosis, hiatus hernia, atrial septal defect, splenic cyst, and choanal atresia and confirm further cases with sensorineural deafness and exocrine pancreatic insufficiency.We report the first case of a patient with a compound heterozygous mutation in GLIS3 phenotype, further extending the spectrum of abnormalities associated with GLIS3 mutations and providing novel insights into the role of GLIS3 in human physiological development. All but 2 of the patients within our cohort are still alive, and we describe the first patient to live to adulthood with a GLIS3 mutation, suggesting that even patients with a severe GLIS3 phenotype may have a longer life expectancy than originally described.We report new findings within the  KCNJ11 and ABCC8 genes, encoding the Kir6.2 and SUR1 subunits of the pancreatic ATP-sensitive potassium (KATP) channel involved in regulation of insulin secretion, account for about half of cases of PND , Wolcott-Rallison syndrome (EIF2AK3), and pancreatic agenesis   result in the concomitant presentation of PND and congenital hypothyroidism. GLIS3, a member of the GLI-similar zinc finger protein family encoding for a nuclear protein with 5 C2H2-type zinc finger domains, maps to chromosome 9p24.3-p23 (OMIM 610192)  and congenital hypothyroidism can result from a number of genetic mutations. Mutations in  610192) , 8. In 2GLIS3, providing additional insight into the clinical features associated with this rare condition.We describe a case series of 12 patients with mutations in www.diabetesgenes.org), from clinical notes, and subsequently by using a questionnaire circulated to referring clinicians to gain further information.The study was conducted in accordance with the Declaration of Helsinki principles with informed parental consent given on behalf of children. Clinical information was provided by the referring clinicians via a neonatal diabetes request form (available at GLIS3 gene mutations were sought by PCR amplification (primer sequences are available on request) and sequence analysis of exons 1 to 11 by comparison with the reference sequence NM_001042413. Exon 1 is noncoding (the 5′ untranslated), and the start codon is located within exon 2.−ΔΔtC method.The effect of coding variants on the protein was investigated in silico using the bioinformatic tool Alamut (Interactive Biosoftware). When PCR amplification failed, suggesting a homozygous deletion, parental samples were investigated by real-time quantitative PCR on an ABI 7900 system (TaqMan assay with SYBR Green detection), and the copy number of exons 1 to 11 was determined by the 2Patients 1 and 10 were analyzed for all of the known neonatal diabetes genes using a targeted next-generation assay . MutatioGLIS3 mutations identified in our case series. Deletions of ≥1 of the 11 exons of GLIS3 were observed in most patients. Patients 1, 5, and 10 harbor missense mutations , affecting highly conserved amino acids located in the DNA binding domain and so are likely to be pathogenic, thus severely affecting the function of the GLIS3 protein. Patient 1 is the first patient reported to be a compound heterozygote for 2 mutations in GLIS3 (a deletion and a missense mutation). Patients 3a and 3b are siblings. GLIS3, showing the mutations described in our patient population.GLIS3 mutation to survive into adulthood (aged 36 years). Patient 6 died from liver failure with marked portal hypertension and esophageal variceal bleeding, and patient 9 died of overwhelming measles sepsis and multiorgan failure at 6 months of age. Nine patients in our cohort were born to parents who were first cousins. Patient 3, who was born to apparently not related parents was described previously by Dimitri et al (GLIS3 mutation born to Caucasian parents who are not related (patient 1) and the sibling of patient 3a (patient 3b). A homozygous deletion was confirmed in patient 6; however, consanguinity was denied by the patient's parents.The clinical features of all of the patients are summarized in ri et al . In thisGLIS3 mutations. Age at diagnosis ranged from birth to 23 days. All patients were insulin treated. Patients were initially treated with insulin at 0.4 to 2.0 U/kg/24 h (patient 4 required 2.0 U/kg/24 h), demonstrating a range of insulin sensitivities. Patient 11 had high insulin sensitivity, leading to recurrent hypoglycemic episodes with very small doses of insulin. Others had labile blood glucose (patients 3a and 3b), and one patient clinically demonstrates insulin resistance, particularly during periods of illness (patient 2). In the first year of life, this patient required 0.5 to 0.7 U/kg of insulin per day. However, during times of intercurrent illness, doses of insulin at 3 to 4 times her normal requirement were required to achieve normoglycemia. Despite erratic blood glucose control with periods of insulin resistance, her glycosylated hemoglobin at 1 year of age was 7.8% (62.0 mmol/mol).PND was the only consistent feature of all of our patients with GLIS3 mutations in the first year of life despite normal levels of free T4, which have since normalized. Patients 6 and 10 had normal thyroid anatomy on ultrasonography, and in comparison with other patients with GLIS3 mutations, TSH responded appropriately to conventional doses of levothyroxine with an appropriate increase in the levothyroxine dose with age. Notably, the postmortem examination of the thyroid gland in patient 6 demonstrated a paucity of colloid as well as extensive perifollicular and interstitial fibrosis, explaining the need for thyroxine despite apparently normal thyroid anatomy on ultrasonography.Apart from patient 1, all patients had with congenital hypothyroidism. This is a cardinal feature of the NDH syndrome described previously in all patients with mutations, 9. Althmutations. SimilarLiver disease was documented in 7 of our 12 patients. The hepatic dysfunction presented concomitantly with renal abnormalities and ranged from hepatitis (patients 3b and 4) to hepatic fibrosis and cirrhosis . Nine patients have anatomical kidney changes. Of these, 7 children showed variable renal cystic dysplasia ranging from an isolated cyst observed in patient 10 and bilateral calyceal calcification in patient 11 to extensive cystic renal dysplasia  . PatientGLIS3. She presented with osteopenia, significantly delayed rib fracture healing, and a marked thoracolumbar scoliosis , which subsequently normalized without treatment. Patient 8 was osteopenic by 6 months and despite vitamin D supplementation, her 25-hydroxyvitamin D3 level was 33.5 nmol/L at 6 weeks of age. Adjusted serum calcium in this patient was 2.0 mmol/L in the first week of life and subsequently normalized to 2.5 mmol/L by 12 days of age without calcium replacement. Alkaline phosphatase peaked at 3 months to 2100 U/L but had normalized by 2 years of age. No fractures were identified in patient 8.Patient 2 was the first patient to be described with skeletal manifestations due to a mutation in coliosis , A and Bcoliosis . PatientGLIS3 mutation to present with a splenic cyst (patient 3b).Malabsorption due to exocrine pancreatic insufficiency as demonstrated by low fecal elastase was a feature in patients 2, 3a, 3b, and 7 in our case series. These 4 patients have been treated with pancreatic enzyme supplementation. We report the first patient with a GLIS3 exon affected as suggested by Senee et al  and a deletion of exons 1 to 4 of GLIS3 , have been described previously; the 7.5-kb transcript is strongly expressed in pancreas, thyroid, and kidney with smaller transcripts predominantly expressed in liver, kidney, heart, and skeletal muscle . We thus speculate that the missense mutation may be a hypomorphic change resulting in a mutated protein possessing some residual function.The variability in phenotype observed between our patients and others  has beenmaller 0.–2.0 kb, maller 0.–2.0 kb, GLIS3 plays a key role in pancreatic development, particularly in the embryogenesis of β-cells, which explains why our patients and others with GLIS3 mutations to date have presented with PND (GLIS3 interacts with key regulatory genes in pancreatic embryogenesis including ONECUT1 and NEUROGENIN3 (NEUROG3)  –15. GLISINS gene . Therefodiabetes , 18. ThePAX8 and NKX2-1 (TTF1/NK2 homeobox-1 or thyroid transcription factor 1), involved in thyroid cell differentiation and proliferation and subsequent expression of genes encoding for thyroglobulin, thyroid peroxidase, thyrotropin receptor, and the sodium-iodide symporter  leading to a frameshift and likely degradation of the transcript reported by Senee et al  and smaller (0.8–2.0 kb) transcripts are expressed in the kidney and smaller (0.8–2.0 kb) transcripts are expressed in the liver  were observed in patient 5  (GLIS3 and WW domain containing transcription regulator 1 (WWTR1) overlaps in the kidney, and mutations in both genes result in renal cystic dysplasia with a high glomerular cystic load  . A reductic load , 24. SimGLIS3 mutation to present with sensorineural deafness (patient 3a)  . We now  disease . FurtherSLC1A1 (solute carrier family 1). SLC1A1 is principally expressed in neurons, kidney, and small intestine. Mutations in SLC1A1 are thought to cause dicarboxylic aminoaciduria (In one of our families (patient 2) and in a family reported by Senee et al , the delaciduria  and haveaciduria , 28.GLIS3 gene. Our current study has focused on the clinical manifestations of patients with these mutations, and further in vitro work is required to test the GLIS3 missense variants functionally in biological models.We have presented an extended spectrum of clinical features in relation to patients with mutations in the GLIS3 characteristically present with neonatal diabetes with variable insulin sensitivity and congenital hypothyroidism due to a range of underlying causes. Although mutations in GLIS3 are more common in consanguineous pedigrees, we report 2 patients from apparently unrelated parents with GLIS3 mutations. We also report the first patient with compound heterozygous mutations in GLIS3 with preservation of thyroid function, who is also the first patient reported with a GLIS3 mutation to survive into adulthood. Hepatic and renal disease is common in the patients in our cohort, but the presentation is variable. We report new findings within the GLIS3 phenotype including cardiac disease, hiatus hernia, sagittal craniosynostosis, splenic cystic change, and choanal atresia, further extending the spectrum of abnormalities associated with GLIS3 mutations and providing novel insights into the role of GLIS3 in human physiological development. We report further patients presenting with exocrine pancreatic insufficiency and sensorineural deafness. All but 2 of the patients in our cohort are still alive, suggesting that even patients with a severe GLIS3 phenotype may have a longer life expectancy than originally described.In summary, patients presenting with mutations in