Iron, vitamin B12, and folic acid deficiencies are among the most common deficiencies occurring after laparoscopic Roux-en-Y gastric bypass (LRYGB). The present study evaluates the effectiveness of a specially designed multivitamin supplement  specifically developed for LRYGB patients.A triple-blind, randomized, 12-month study was conducted comparing WLS forte with a standard multivitamin supplement (sMVS) containing approximately 100% of the recommended daily allowance (RDA) for iron, vitamin B12, and folic acid. WLS Forte contains vitamin B12 14000% RDA, iron 500% RDA, and folic acid 300% RDA.P < 0.05). Iron deficiency (ID) was reduced by 88% after WLS Forte compared with sMVS. Adverse events related to supplement use did not occur.In total, 148 patients (74 in each group) underwent a LRYGB procedure. Baseline characteristics were similar for both groups. Per protocol analysis demonstrated that sMVS treatment was associated with a decline in ferritin (−24.4 ± 70.1 μg/L) and vitamin B12 (−45.9 ± 150.3 pmol/L) over 12 months, whereas in WLS Forte patients, ferritin remained stable (+3.2 ± 93.2 μg/L) and vitamin B12 increased significantly (+55.1 ± 144.2 pmol/L). The number of patients developing ferritin or vitamin B12 deficiency was significantly lower with WLS Forte compared with sMVS (An optimized multivitamin supplement is safe and reduces the development of iron and vitamin B12 deficiencies after LRYGB. Most commonly diagnosed postsurgical deficiencies are iron (47%–66%), vitamin B12 (37%–50%), folic acid (15%–38%), vitamin D (20%–51%), and calcium (± 10%).25 To prevent these postsurgical deficiencies, daily use of a multivitamin and mineral supplements are generally recommended26; however, guidelines vary and RYGB-specific, multivitamin supplements (MVS) are currently not available.27Bariatric surgery has proven itself as an effective treatment to establish sustained weight loss in morbidly obese patients, and to reduce obesity-related comorbidities such as type 2 diabetes mellitus, hypertension, and sleep apnea syndrome.Based on the literature and pilot studies performed in our hospital, a customized MVS for RYGB patients was developed . The present study evaluates the effectiveness and safety of WLS Forte compared with standard MVS  after RYGB in a triple-blind randomized controlled trial.The study protocol was approved by the National Medical Ethics Review Committee of the Radboud University Medical Centre and Local Ethical Committee of the Rijnstate Hospital Arnhem (RHA), and was conducted in concordance with the principles of the Declaration of Helsinki. The study protocol was registered at the clinical trials registry of the National Institutes of Health .The present study was a triple-blind, randomized, clinical trial. The patients were randomized in 2 groups, receiving 2 different multivitamin supplements (standard multivitamin supplement and WLS Forte) after a primary laparoscopic Roux-en-Y gastric bypass (LRYGB) operation. Patients were included by bariatric surgeons, (supervised) specialized obesity nurses, or researchers at the outpatient surgery department of the RHA. RHA is a teaching hospital for surgical residents and a center of excellence in bariatric surgery.Patients who were scheduled for a LRYGB operation between June 2011 and March 2012 were invited for participation in the study and written informed consent was obtained from each patient.2with an obesity-related comorbidity) who met the criteria for bariatric surgery according to the National Institutes of Health Consensus Development Conference Panel for bariatric surgery28 were eligible for the study. Exclusion criteria were a secondary LRYGB operation or other type of a bariatric procedure.Adult patients (age >18 years) with morbid obesity (body mass index [BMI] of >40 or >35 kg/mAll procedures were performed by 1 of 3 experienced bariatric surgeons (>750 procedures each). They performed an antecolic antegastric LRYGB, with a proximal gastric pouch of 30 mL, a biliopancreatic limb of 50 cm, and a Roux limb of 150 cm. All patients received low-molecular heparin (nadroparin 5700 IU daily) for 6 weeks and proton-pump inhibitor (omeprazol 40 mg daily) for 6 months, as part of our standard postoperative protocol.WLS Forte, a customized multivitamin supplement for RYGB patients, contains high doses of multiple vitamins and minerals, in particular iron , folic acid (3 times RDA), and vitamin B12 (140 times RDA). A standard multivitamin supplement  served as control and contains the compounds of interest in a dose equivalent to the RDA. The composition of both supplements is shown in Table The allocation sequence was computer generated. Variable block schedule was used for randomization and conducted by the independent trial pharmacist. Allocation concealment was achieved by generating a randomization list that was only available to the pharmacist. All supplements were packaged in a nonmarked sealed box at the hospital pharmacy, which was similar for both supplements, and numbered according to the randomization list. This was performed before the inclusion of the participants. The included participants received the sealed box with the supplements according to the list, which was delivered by the pharmacist. Both supplements were similar in color, size, and taste. Therefore, patients, surgeons, and researchers were unaware of the type of the supplements. After the last visit of the last study patient, the randomization list was available for the research team. No earlier unblinding occurred. Patients with a nutritional deficiency before or during the study were treated with a predefined medication, independent of the trial group.All patients followed a strict postoperative schedule consisting of 17 visits in the first year, and on each visit patients were encouraged to keep taking their supplements. Standard laboratory blood tests were performed at baseline, and at 6 and 12 months. This included a complete blood count, calcium, phosphate, magnesium, zinc, albumin, iron, total iron-binding capacity, ferritin, folic acid, vitamin B12, 25-hydroxyvitamin D (25-OHD), parathyroid hormone (PTH), vitamin B1, and vitamin B6 .corr), according to the following equation: Cacorr = Total Calcium –  + 1.Primary outcome variables were the percentage of iron, folic acid, and vitamin B12 deficiencies developed during the 12 months after LRYGB. Iron deficiency (ID) was defined as a serum ferritin <20 μg/L, folic acid deficiency if the level was < 9.0 nmol/L, and vitamin B12 deficiency if the level was < 150 pmol/L. Anemia was defined as hemoglobin level of <7.4 mmol/L for females and <8.4 for males. Vitamin D deficiency was diagnosed if 25-OHD was <50 nmol/L, hypocalcaemia if serum total calcium was <2.1 mmol/L, and zinc deficiency of the serum level was <9.2 μmol/L. Calcium data are shown as calcium levels corrected for albumin  was calculated with the formula: 40 ×  × body weight in kilograms, as described previously.30 Thereafter, a maintenance dosage of 25,000 IU cholecalciferolFNA per week was prescribed for a period of 3 months. For other deficiencies the endocrinologist was consulted.ID with or without anemia was treated with ferrous gluconate 695 mg 3 times daily for 3 months. Vitamin B12 deficiency was corrected with intramuscular injection of 1000 μg hydroxocobalamin once every 2 months for 12 months. Vitamin D deficiency was corrected with oral solubilized cholecalciferolIntention-to-treat analysis was conducted to present the outcomes. Additionally, per protocol analysis was performed, excluding patients who received additional medication because of a nutritional deficiency during the study.t-test for continuous data and chi-square test for ordinal/nominal data. A P value <0.05 was considered statistically significant.All data were analyzed using IBM SPSS Statistics  20 for Windows. Data are expressed as mean (±standard deviation), unless otherwise specified. Differences between groups were calculated using Student Sample size calculation was performed by the epidemiologist of the Research Department of RHA using Openepi.com. Sample size calculation was based on the number of patients developing ID. To detect a 25% reduction of ID 12 months after surgery, with 95% sensitivity and a power of 90%, a minimum of 56 patients per group were needed. Taking into account a 10% dropout and 15% of cases excluded because of ID diagnosed and treated at 6 months, it was decided to include 75 patients per treatment group.P = 0.04).Two patients were excluded after randomization: 1 patient in the WLS Forte group because he underwent a sleeve gastrectomy instead of a LRYGB because of multiple adhesions during surgery, and 1 patient in the sMVS group because he cancelled the scheduled operation procedure. In total, 148 patients (74 in each group) underwent a LRYGB and were included for analysis. Baseline patient characteristics are shown in Table Weight LossP = 0.24), respectively. Percentage excess weight loss, defined as weight loss divided by excess weight based on ideal body weight at BMI 25 kg/m2, were after 12 months 72.5 ± 20.9 kg/m2 versus 72.1 ± 23.2 kg/m2 for MVS and WLS Forte (P = 0.92), respectively.The degree of weight loss over 12 months was similar in both groups. Weight dropped to 90.6 ± 17.4 kg in the sMVS group versus 93.8 ± 16.9 kg in the WLS Forte group (P = 0.24), and did not change over 12 months. Mean cell volumes also did not change (data not shown). In total, 10 (6.8%) patients had anemia preoperatively: 6 (8.1%) patients in the sMVS and 4 (5.4%) in the WLS Forte group (P = 0.50). Three of them (1.4%), 2 patients in sMVS and 1 patient in WLS Forte, had ID anemia. After 12 months postoperatively, 3 patients (4.3%) versus 5 patients (7.4%) had anemia (P = 0.75), respectively. Of these patients, 2 patients had anemia de novo in both groups. Two patients (1.5%), 1 patient in each group, had ID anemia.In Table P = 0.03). In total, 55 (37.2%) patients, 28 (37.8%) in the sMVS group and 27 (36.5%) in the WLS Forte group, received additional iron medication at any time during the 12 month follow-up. Results after exclusion of these patients are shown in Table P = 0.08).The total number of patients developing ferritin deficiency during follow-up was 8 (10.7%) in sMVS and 1 (1.3%) in WLS Forte (P = 0.14). The results obtained after exclusion of these patients receiving vitamin B12 injections are shown in Table P<0.001) after 12 months, and as a result mean vitamin B12 blood serum levels at 6 months and 12 months were significantly higher with WLS Forte compared with sMVS (P < 0.05). After 12 months, vitamin B12 deficiency had developed in 5 (7.9%) patients receiving sMVS versus 1 patient (1.6%) in the WLS Forte group (P = 0.207).At baseline, vitamin B12 deficiency was diagnosed in 9 (6.1%) patients, that is, 5 (6.8%) patients in the sMVS group and 4 (5.4%) patients in the WLS Forte group. These patients received vitamin B12 injections by protocol. In total, 27 (18.2%) additional patients were treated with vitamin B12 injections at any time during the 12-month follow-up: 17 (23%) in the sMVS group and 10 (13.5%) in the WLS Forte group (P = 0.83) for sMVS and WLS Forte, respectively. In both groups, 1 patient (1.4%) had folic acid deficiency at baseline. During the study period, folic acid deficiency developed in 5 (6.8%) patients taking sMVS and 2 (2.7%) patients in the WLS Forte group (P = 0.441). After 6 and 12 months, mean serum folic acid levels were significant higher in the WLS Forte group (P < 0.05).Mean serum folic acid levels were similar at baseline: 17.6 ± 7.3 nmol/L and 17.9 ± 7.4 nmol/L (P = 0.56), had vitamin D deficiency before surgery. Mean serum vitamin D levels were 42.5 ± 17.9 nmol/L and 44.3 ± 20.6 nmol/L at baseline (P = 0.58), respectively. Vitamin D deficiencies were corrected according to the protocol and this resulted in mean 25-OHD levels close to the target of 75 nmol/L in both groups. The mean loading dose was 226,087 ± 60,442 IU with a maintenance dose of 25,000 IU/month. At 12 months, 7 (10.1%) patients in the sMVS and 12 (18.5%) patients in the WLS Forte group had vitamin D deficiency (P = 0.168). Excluding the patients who received extra vitamin D supplementation additional to the standard suppletion, mean vitamin D levels at baseline were 61.8 ± 10.8 versus 66.8 ± 19.3 nmol/L (P = 0.29) for sMVS and WLS Forte; after 12 months 84.6 ± 20.8 nmol/L versus 83.4 ± 24.8 nmol/L (P = 0.87), respectively. There were no differences in PTH levels and calcium levels between the 2 groups (P ≥ 0.05), although PTH levels rose by 1.26 ± 2.39 and 1.75 ± 3.1 pmol/L. Table In total, 96 65.3%) patients, 46 (63.0%) patients in the sMVS group and 50 (67.6%) patients in the WLS Forte group (.3% patieP = 0.13), respectively. There were no vitamin B6 deficiencies observed at baseline. In total, 15 (10.1%) patients, 5 (6.8%) patients in sMVS versus 10 (13.5%) patients in WLS Forte (P = 0.173), had elevated serum vitamin B6 levels at baseline. After 12 months, there were no deficiencies in either group and elevated serum vitamin B6 levels were observed in a total of 52 (40.6%) patients, that is, in 21 (31.8%) patients in the sMVS group and 31 (50%) patients in the WLS Forte group (P = 0.036). In 1 patient, an extremely high level of vitamin B6 (2777 nmol/L), without any neurological symptoms, was detected in the WLS Forte group; however, this patient had used additional multivitamin supplements on advice of his general practitioner in addition to the study medication. Two months after withdrawal of these additional medications, the serum level had dropped to 110 nmol/L.Results of monitoring vitamin B1, vitamin B6, zinc, and magnesium are shown in Table For vitamin B1, zinc, phosphate, and magnesium, no differences were observed between the groups.The present study illustrates that a multivitamin supplement specifically developed to prevent deficiencies in LRYGB patients has advantages over standard supplementation. sMVS was associated with a substantial decline in serum ferritin and vitamin B12 levels over 1 year, whereas, in patients on WLS Forte, ferritin remained stable and vitamin B12 increased significantly. Moreover, the numbers of patients developing ferritin or vitamin B12 deficiencies were significantly fewer with the LRYGB-specific multivitamin. These results were achieved by raising the iron content from 1 to 5 times RDA and vitamin B12 from 1 to 140 times RDA. No benefit was found with respect to folic acid, despite a difference between the supplements in folic acid content of 1 and 3 times RDA.31 ID in the first year after LRYGB has been reported to occur in 44% to 66% of patients not using supplements. These figures should be interpreted with caution because of marked differences in the definition of iron deficiency between these studies. Sometimes serum iron levels are erroneously used to diagnose iron deficiency. Serum iron levels do not reflect the body iron store. In the absence of inflammation, serum ferritin is the gold standard. Standardized supplementation of 14 mg iron daily reduced the incidence of iron deficiency in the first year after LRYGB to 11%. The American Society of Metabolic and Bariatric Surgery (ASMBS) guidelines advise a minimum of 18 mg iron per day for patients after RYGB. An additional minimum of 18 to 27 mg per day elemental iron is advised for high-risk patients including menstruating women.32 However, Vargas-Ruiz et al22 demonstrated that 18 mg iron per day was insufficient in LRYGB patients. ID was diagnosed in 20% of the patients after 12 months and rose to 55% after 3 years. This is in line with the observation of Aarts et al8 where ID was diagnosed in 21% of the patients despite a daily intake of a minimum of 21 mg iron. In addition, female patients had a higher risk of developing ID than men . Brolin et al11 demonstrated, in a double-blind, randomized controlled trial, the effectiveness of 320 mg ferrous sulphate twice daily  in young women who underwent a RYGB for prevention of ID.Decreased acid production in the small gastric pouch, exclusion of the duodenum and proximal part of the jejunum, use of proton-pump inhibitors during the first months of surgery, and intolerance of iron-rich foods, such as red meat, are the main factors increasing the risk of iron deficiency.34 In the present study with a male to female ratio of 1 : 2.3, this was sufficient to maintain serum ferritin at baseline level, whereas, in patients on sMVS, ferritin steadily decreased over the year. The fact that the difference in ferritin levels at 12 months did not reach statistical significance is attributed to a lack of power in this study.Based on the literature that advises 40 to 65 mg intake of iron in men and 100 mg in female patients after RYGB, the iron content in WLS Forte was increased to 70 mg, that is, 5 times RDA.24 Vargas-Ruiz et al22 reported a post-RYGB incidence of vitamin B12 deficiency of 10% after 12 months and 18% after 3 years, despite the use of a multivitamin containing cobalamin 6 μg daily. Brolin et al11 reported that 37% of the patients had vitamin B12 deficiency after a mean follow-up period of 42 months. ASMBS guidelines advise vitamin B12 supplements of 350 to 500 μg daily, and as-needed addition of intramuscular injections of 1000 μg per month.32 WLS Forte contains high doses of 350 μg vitamin B12, which is 14,000% ADH. After 12 months, vitamin B12 hypervitaminosis was observed in 9 patients on WLS Forte compared with 7 patients in sMVS (P = 0.58). No adverse events of vitamin B12 hypervitaminosis were observed during the study period.Vitamin B12 in food is bound to proteins, and needs to be liberated by the action of hydrochloric acid, pepsin, and pancreatic enzymes. This process is compromised after RYGB. In addition, intrinsic factor release is also reduced, which further compromises vitamin B12 absorption. Finally, preoperative vitamin B12 deficiencies are not uncommon in the morbidly obese, probably because of their dietary habits. In the present study, 9% of patients had vitamin B12 deficiency preoperatively. Published data of postoperative vitamin B12 deficiencies diagnosed after variable periods of time vary from 10% to 50%.22 A previous study of our group demonstrated high incidence of deficiencies for iron (66%), vitamin B12 (50%), and folic acid (15%) in patients with anemia after LRYGB.8 Based on these results, minimal daily intake of 65 mg of iron in males and 100 mg in female patients, 350 μg of vitamin B12, and 400 μg of folic acid were advised after LRYGB. In our current study, the prevalence of anemia was, in total, 11.7% after 12 months, that is, in 4.3% on sMVS versus 7.4% on WLS Forte (P = 0.75). However, only 1 patient had an ID anemia in the sMVS group. The fact that 23% of the patients received additional oral iron medication at baseline and 10% received vitamin B12 injections at baseline or 6 months may have resulted in lower nutrient-related anemia. A previous study of Brolin et al35 showed also reduction in ID with additional iron supplements; however, incidence of anemia was not reduced compared with placebo. Other pathophysiological aspects play probably a role in anemia after RYGB surgery.ID, vitamin B12 deficiency, and folic acid deficiencies are associated with anemia after LRYGB.37 It is metabolized in the liver into 25-OH-D, the best parameter for patients’ vitamin D status.33 Several studies claimed high incidence of vitamin D (34%–73%) deficiency after RYGB40; however, the exact mechanisms of postoperative vitamin D deficiency are not completely clear yet. Obesity itself seems to be a risk factor of developing vitamin deficiency.Vitamin D, a fat-soluble vitamin, can be produced in the skin under influence of sunlight, and ingested through the diet  or by dietary supplements.32 In contrast to these guidelines, reports in the literature advise postoperative vitamin D supplements varying between 320 IU and 2000 IU per day,42 and a calcium intake of 1000 to 1500 mg daily.42Despite the high incidence of postoperative vitamin D deficiency, optimal prevention strategies are lacking. The ASMBS guidelines advise daily intake of 2000 IU vitamin D. In addition, 1500 to 2000 mg per day of calcium was recommended.43 demonstrated that despite daily intake of 800 IU vitamin D, 44% of the population still remained insufficient for vitamin D. Goldner et al41 conducted a prospective, randomized controlled trial comparing 3 different doses of vitamin D supplementation of 800 IU, 2000 IU and 5000 IU daily following RYGB. The postoperative increase of mean serum vitamin D level at 12 months was higher with higher doses (800–5000 IU) of vitamin D supplement. Therefore, a minimum of 2000 IU daily intake of vitamin D was advised. However, Signori et al44 reported that daily use of prophylactic vitamin D of 1200 to 2000 IU did not prevent postoperative vitamin D deficiency.Carlin et alP = 0.168).In the current study, all patients received 1500 mg calcium carbonate and 1200 IU vitamin D daily as standard postoperative protocol. Furthermore, sMVS (control group) contained 160 IU (4 μg) vitamin D, whereas WLS Forte contained 500 IU (12.5 μg). In total, 29% of the cohort had developed vitamin D deficiency after 12 months, without significant differences between the 2 supplementation regimes. At 12 months, 10% in the sMVS group and 19% of patients in the WLS Forte group had developed vitamin D deficiency  RDA in the WLS Forte supplements.In our study, no differences were found in vitamin B1, vitamin B6, zinc, magnesium, or phosphate serum levels between standard MVS and WLS Forte. A notable finding is the high incidence (10%) of hypervitaminosis of vitamin B6 before the surgery. In addition, in total, 41% had high levels of vitamin B6 after surgery, which was significantly higher for WLS Forte compared with sMVS at 12 months. Increased levels of vitamin B6 were also observed in the literature,48 demonstrated a serum folic acid concentration of five-fold compared with baseline, 4 hours after oral intake of standard MVS containing 1.6 mg folic acid. Therefore, folic acid can be used as a marker for compliance of MVS intake.Vitamin and mineral deficiencies are common both before and after LRYGB surgery. Therefore additional supplements are advised after weight-loss surgery. However, life-long compliance of daily supplement intake is hard to achieve. To measure adequate intake of MVS, one can monitor the serum concentration of highly absorbable vitamins. Navarro et alThe capsules in both groups had a cherry flavor with powder, nevertheless, several patients complained about vitamin B aftertaste for a period of 30 minutes after ingestion. This had no influence for the compliance of the patients. After the end of the study, commercially available capsules have a bubblegum taste with microcapsulation to prevent this aftertaste.∗∗50 For example, excess vitamin B12 intake may be associated with prostate cancer.50Despite the double-blind, prospective, randomized character of the study, several limitations of the study should be considered. High prevalence of preoperative nutritional and mineral deficiencies, which are linked to morbid obesity, required additional treatment, and some patients received additional supplements after surgery outside of protocol. This will have obscured differences of the suppletion regimens to some extent. In Table The compliance of patients was inquired during the follow-up interviews; however, the answers depended on the honesty of the patients and could only be compared with their folic acid levels. On the contrary, patients were blinded for the nature of their supplements and, thus, it is plausible that any noncompliant patients were equally distributed in both groups.In summary, prevention of mineral and vitamin deficiencies after LRYGB is hard to achieve with standard multivitamin supplements, especially when deficits before the operation are present. WLS Forte was introduced as a customized multivitamin supplement for post-RYGB intake, including 14,000% RDA (350 μg) vitamin B12, 500% RDA (70 mg) iron, and 300% RDA (600 μg) folic acid. Using WLS Forte after LRYGB surgery results in fewer deficiencies in iron, vitamin B12, and folic acid compared with sMVS. Despite the decreased risk of vitamin deficiencies with WLS Forte supplements after LRYGB, a strict follow-up regime remains necessary to prevent patients becoming vitamin deficient.


Saccharomyces cerevisiae strain carrying deletions in all three pyruvate decarboxylase (PDC) genes  represents a non-ethanol producing platform strain for the production of pyruvate derived biochemicals. However, it cannot grow on glucose as the sole carbon source, and requires supplementation of C2 compounds to the medium in order to meet the requirement for cytosolic acetyl-CoA for biosynthesis of fatty acids and ergosterol.A −1, respectively. Several genetic changes were identified in the evolved Pdc negative strains by genomic DNA sequencing. Among these genetic changes, 4 genes were found to carry point mutations in at least two of the evolved strains: MTH1 encoding a negative regulator of the glucose-sensing signal transduction pathway, HXT2 encoding a hexose transporter, CIT1 encoding a mitochondrial citrate synthase, and RPD3 encoding a histone deacetylase. Reverse engineering of the non-evolved Pdc negative strain through introduction of the MTH181D allele restored its growth on glucose at a maximum specific rate of 0.053 h−1 in minimal medium with 2% glucose, and the CIT1 deletion in the reverse engineered strain further increased the maximum specific growth rate to 0.069 h−1.In this study, a Pdc negative strain was adaptively evolved for improved growth in glucose medium via serial transfer, resulting in three independently evolved strains, which were able to grow in minimal medium containing glucose as the sole carbon source at the maximum specific rates of 0.138, 0.148, 0.141 hMTH1 alleviated the glucose repression by repressing expression of several hexose transporter genes. The non-synonymous mutations in HXT2 and CIT1 may function in the presence of mutated MTH1 alleles and could be related to an altered central carbon metabolism in order to ensure production of cytosolic acetyl-CoA in the Pdc negative strain.In this study, possible evolving mechanisms of Pdc negative strains on glucose were investigated by genome sequencing and reverse engineering. The non-synonymous mutations in The online version of this article (doi:10.1186/s12934-015-0305-6) contains supplementary material, which is available to authorized users. Saccharomyces cerevisiae is an important cell factory widely used for the production of beer, bread, wine, bioethanol, nutraceuticals, chemicals and pharmaceuticals [S. cerevisiae. The only strategy to eliminate ethanol production that has worked so far is removing pyruvate decarboxylase activity so that pyruvate cannot be converted to acetaldehyde, the direct precursor of ethanol [S. cerevisiae, pyruvate decarboxylase is encoded by three structural genes, PDC1, PDC5 and PDC6 [pdc1∆ pdc5∆ pdc6∆, also called Pdc negative strains) cannot grow on glucose as the sole carbon source [GLY1) over-expression all suggested that the growth defect of Pdc negative strain on glucose was due to the lack of cytosolic acetyl-CoA for biosynthesis of cellular biomolecules, especially lipids.euticals –5. When  ethanol . In S. cand PDC6 –9. Howevn source . The met2 source or with GLY1 over-expression [2-independent, glucose-tolerant, and pyruvate-hyperproducing’ strain TAM [MTH1 allele with a 225 bp internal deletion (MTH1-∆T) was identified, and was attributed with restoring growth of the Pdc negative strain on glucose [MTH1-∆T allele into an un-evolved Pdc negative strain, the growth rate (μmax = 0.10 h−1) was slower in minimal medium with 2% glucose, compared to the TAM strain (μmax = 0.20 h−1), indicating the possible presence of additional advantageous genetic changes in the TAM strain besides MTH1-∆T.Interestingly, the Pdc negative strains are still sensitive to high glucose concentrations even when supplemented with a Cpression , 11. vanrain TAM . In the  glucose . HoweverMTH1 alleles have been identified in selections of glucose or catabolite repression suppressors using glucose sensitive mutants [MTH1 alleles seemed to be able to resolve the sensitivity to glucose in these mutants. Previous studies have shown that these MTH1 alleles reduced glucose transport by repressing the transcription of several hexose transporter genes (HXTs) [MTH1 had similar effects [MTH1-∆T resulted in a decreased degradation of Mth1 [MTH1-∆T allele could therefore repress the transcription of hexose transporter genes even during growth on glucose.Mth1 functions as a negative transcriptional regulator in the glucose signaling pathway together with other regulators, i.e. Snf3, Rgt2, Std1, Rgt1. Several other  mutants –18. The s (HXTs) , 16, 17, effects . It has  of Mth1 , which c of Mth1  situated of Mth1 , 21. TheMATaura3-52 his3-Δ1 pdc1Δ pdc5Δ pdc6Δ) was evolved in glucose medium via serial transfer in 3 independent culture lines. Genomic DNA sequencing of these three evolved Pdc strains identified mutations in MTH1 as well as in HXT2 [CIT1 [RPD3 [MTH1 alleles were also identified in our evolved Pdc negative strains, indicated again that MTH1 might be an important target for relieving high glucose repression. In order to understand the roles of these genetic changes in the evolved strains, the effects of the mutations in mutated genes, MTH1, HXT2, CIT1 and RPD3, were investigated using homology analysis of their protein sequences and published crystal structure models of homologous proteins, and possible mechanisms were proposed and discussed. Although the speculations regarding the possible mechanisms in evolved Pdc negative strains still require further investigations, they may be useful and helpful for metabolic engineering strategies on Pdc negative strains.In this study, a Pdc negative strain PDC-E1  and CEN.PK 110-10C (MATαhis3-Δ1) were used as background strains for the construction of Pdc negative strains  (Table CEN.PK 113-5D (MATaura3-52 his3-Δ1 pdc1Δ pdc5Δ pdc6Δ), E1 for short, was evolved in 3 independent culture lines in YPD medium with gradually reduced ethanol concentration. Once fast-growing, glucose tolerant strains were obtained in YPD media, they were further evolved for C2 carbon source-independent and faster growth in minimal medium with 2% glucose.PDC-E1 . E1A, E1B, E1C could grow in minimal medium with glucose as the sole carbon source, with maximum specific growth rates of 0.138, 0.148, 0.141 hGenome sequencing of the parental strain E1 and its evolved strains  was performed to study the genetic changes that occurred during the adaptive evolution. The raw sequencing data were filtered and trimmed to remove adaptor sequences and sequence ends with a quality score below 20. The filtered reads were mapped to the CEN.PK 113-7D reference genome. In the E1A strain, three single nucleotide variants (SNVs) in coding regions representing non-synonymous mutations were identified, as listed in Additional file MTH1, CIT1, HXT2, were found to carry point mutations in all three evolved strains. And one gene, RPD3, was found to carry point mutations in two of the evolved strains. The mutations of the proteins encoded by the four genes are listed in Table Among all genes with SNVs, three genes, MTH1 was identified in their evolved Pdc negative strain TAM, which was demonstrated to be responsible for relieving glucose sensitivity in the Pdc negative mutant. Among the previously identified MTH1 alleles affecting glucose sensing, e.g. BPC1-1, DGT1-1, HTR1-5, HTR1-19 and HTR1-23, two mutations in codon 85  and one mutation in codon 102 (S102G) were found, and it was also found that the S102G mutation could reinforce the mutations in codon 85 although having no effects by itself [MTH185S allele has already been confirmed to suppress glucose repression, we chose to validate the MTH181D allele through reverse engineering in this study, to investigate if it has similar effects of suppressing glucose repression like other alleles.As previously reported by Oud et al. , a 225 by itself , 17. SinMTH181D allele was integrated into the MTH1 locus of the non-evolved E1 strain, resulting in strain M81-11. The growth profile in minimal medium with 2% glucose is shown in Fig. −1. The fact that MTH181D by itself could restore the growth of the Pdc negative strain on glucose suggested it had the similar effects on glucose repression alleviation. However, the maximum specific growth rate was not as high as those of the evolved strains E1A, E1B, E1C. The MTH181D mutation contributes around 35% of the maximum specific growth rate in the evolved E1 strains, indicating there are likely other genetic changes that contribute to their growth recovery.The Even though the N-terminal part of Mth1 appears to be important in glucose repression it is not yet clear why. To gain sequence-function insight into Mth1 protein, a bioinformatics approach was undertaken.S. cerevisiae and 22 other homologous sequences of Mth1 and Std1 from unicellular fungi revealed that A81 and I85 are positioned on an ‘island’ composed of 22 highly conserved amino acids from codon 71–91  caused the low pH (<3) in the cell culture. Therefore, a lower final OD600 was observed in M81C compared to M81  [S. cerevisiae , where the mutations are located, A81 and I85 may play a critical role in maintaining the alpha helix structure formed within it, and thus probably affect the function of Mth1. However, a crystal structure of Mth1 would be needed to validate these predictions.The growth defect of a Pdc negative strain on glucose  was prev glucose , 11, 30.by ACH1) . Althougiae Fig. a, e.g. vMTH1 alleles seem to decrease glucose uptake transport by alleviated repression of transcribing several HXTs, especially HXT1 and HXT3 [MTH1-∆T allele) by van Maris et al. [HXT3  was also found in the evolved strain E1C, which would undoubtedly destroy its activity to transport glucose. A previous study suggested that the rate of glucose transport determines the strength of glucose repression [MTH1, it is possible that the C2 supply from the mitochondria to the cytosol via Ach1 route was no longer blocked  and CEN.PK 110-10C (MATα his3-Δ1) [ura3-52, his3-Δ1 or ura3-52 his3-Δ1. The mating type of these pdc negative mutants was determined using primers 35–37 listed in Additional file his3-Δ1) . TogetheMTH1, an MTH181D construct was created and used to replace the normal MTH1 gene in strain PDC-E1 (MATaura3-52 his3-Δ1 pdc1Δ pdc5Δ pdc6Δ), as shown in Additional file MTH181D with upstream and downstream sequences was amplified using primers 23–26, and the sequence downstream of MTH181D was amplified using primers 27–28. Two overlapping fragments of the amdSYM marker cassette were PCR amplified from plasmid pUG-amdSYM  using primers 29–32. The two fused PCR fragments for integration were transformed into PDC-E1 using the electroporation method as described in [MTH181D and amdSYM was confirmed using primers 33–34. The amdSYM marker was looped out via selection on counter-selective plates. The replacement of MTH181D was confirmed by sequencing PCR product amplified by primers 33–34.To create a Pdc-negative strain with a point mutation in ribed in . The intCIT1 was deleted in strain M81-11 using the same strategy as described in PDC1 deletion . Selections for transformants carrying the amdSYM marker were performed on SM-Ac plates with 2% ethanol instead of glucose, and the counter selections were performed on SM-Fac plates with ethanol as carbon source, as described in [Cultivations were performed at 30°C in YP medium with 2% glucose (YPD) or 2% ethanol (YPE). Selections for transformants containing the ribed in . A diplo4)2SO4, 14.4 g/L KH2PO4, 0.5 g/L MgSO4∙7H2O, 2 mL/L trace metal solution, 1 mL/L vitamin solution, with the pH adjusted to 6.5 by adding 2 M NaOH. The final concentrations of trace metal elements and vitamins were previously described in [Cultivations for strain characterization were performed at 30°C in triplicate in 100 mL shake flasks with 40 mL defined minimal medium. The defined minimal medium for cultivation was composed of 20 g/L glucose, 7.5 g/L (NHribed in . The min600) measurement at a wavelength of 600 nm with a GENESYS™ 20 Visible spectrophotometer . Glucose, ethanol, glycerol, pyruvate and formate concentrations were determined in culture supernatants by high-performance liquid chromatography  equipped with UV detector and RI detector using a Bio-Rad HPX 87H column . The HPLC was operated at 45°C with 5 mM H2SO4 as mobile phase at a flow rate of 0.6 mL min−1.Biomass was determined by optical density (ODMATaura3-52 his3-Δ1 pdc1Δ pdc5Δ pdc6Δ) towards growth on glucose as the sole carbon source were performed in three independent culture lines in 100 mL shake flasks with 20 mL medium at 30°C, which involved three phases  and QIAGEN Genomic-tip 500/G. The DNA samples were prepared for sequencing using the Illumina DNA TruSeq protocol, with an insert size of 650 bp. The sequencing was performed multiplexed on an Illumina MiSeq using the version 2 chemistry .http://cenpk.tudelft.nl) using the mapper MosiakAligner version 2.1.32  with a hash size of 15. Sites with potential indels were detected and realigned using the tools from the Genome Analysis Toolkit  [http://picard.sourceforge.net). The number of mappable reads after post-processing ranged from 2.9 to 5.2 million resulting in a mapped coverage spanning from approximately 50× to over 100× in the different samples. Variant calling  was performed using GATK UnifiedGenotyper, and annotation of the detected variants was performed using SnpEff version 3.4 (http://snpeff.sourceforge.net). Finally, the alignments were visually inspected for all detected variants, in order to eliminate obvious false positives caused by incorrect alignments.The raw sequencing data was filtered and trimmed to remove adaptor sequences and sequence ends with a quality score below 20. The filtered reads were mapped to the CEN.PK 113-7D reference genome (n 2.3.9) , Realign600 ~ 1.0) by centrifugation at −20°C, quenched by liquid nitrogen, and stored at −80°C for use. Total RNA was isolated using RNeasy Mini Kit (QIAGEN), which was then processed to obtain fragmented cDNA using a QuantiTect Reverse Transcription Kit (QIAGEN). 2 μL of the synthesized cDNA (corresponding to 100 ng RNA) was used as the template for the qPCR reaction to a final reaction volume of 20 μL, using a DyNAmo Flash SYBR Green qPCR Kit . Quantitative RT-PCR was performed on Stratagene Mx3005P . The thermocycling program consisted of one hold at 95°C for 15 min, followed by 40 cycles of 10 s at 95°C and 20 s at 60°C, and a final cycle of 1 min at 95°C, 30 s at 55°C and 30 s at 95°C.Cells were cultured in shake flasks using minimal medium with 2% glucose in triplicate, and harvested for gene expression analysis at exponential growth phase (ODhttp://bioinfo.ut.ee/primer3-0.4.0/) with melting temperature (Tm) around 60°C. ACT1, a housekeeping gene, was selected as the reference gene [HXT6 and HXT7, they used the same pair of primers for real-time PCR. Therefore, the transcription analysis result for HXT6 and HXT7 was their sum-up.Primers for real-time PCR , Hxt2 (UniProt# P23585), Rpd3 (UniProt# P32561) and Mth1 (UniProt# P35198) was separately used to query the NCBI database with the basic local alignment search tool (BLAST). To eliminate sequences with low identity to the S. cerevisiae sequences, the BLAST results were filtered to retain only those with more than 40% identity to the S. cerevisiae enzymes. The 40% cutoff was arbitrary set and is intended to limit the analysis to sequences which can reasonably be said to have the same function as the S. cerevisiae enzyme. Sequences were also filtered such that anything more than 95% identical to anything else in the dataset was removed. This was done to reduce bias, should there be a large number of deposited sequences of the same gene from the same organism. The remaining sequences were aligned using MUSCLE [S. cerevisiae proteins compared to the filtered BLAST results. The computed conservation values were used to color-code sequence representations of the S. cerevisiae proteins. Homology models were generated for three of the four proteins using the Swiss-Model repository [The protein sequence of each of the four g MUSCLE . The respository . For Mth


Osteogenesis imperfecta (OI) is a congenital disorder characterized by increased bone fragility and low bone mass.COL1A1 or COL1A2 mutation was investigated by direct sequencing in 72 patients with OI type I, III, or IV  from 37 unrelated families. The clinical features of these patients were also recorded.The presence of COL1A1 and COL1A2 mutations were identified, including 28 COL1A1 mutations and 9 COL1A2 mutations. Fifteen (41 %) were novel mutations, and twelve (32 %) were familial mutations. A review of their medical records revealed that the 72 patients could be classified into OI type I (n = 42), III (n = 5), and IV (n = 25). Twenty-nine patients had helical mutations (caused by the substitution of a glycine within the Gly-X-Y triplet domain of the triple helix), and 42 had haploinsufficiency mutations . Compared with haploinsufficiency, the patients with helical mutations had more severely impaired skeletal phenotypes, including shorter height, lower bone mineral density, poorer walking ability, more frequent manifestations of dentinogenesis imperfecta and scoliosis (p < 0.05).Thirty-seven Genotype and phenotype databases are expected to promote better genetic counseling and medical care of patients with OI.The online version of this article (doi:10.1186/s13023-015-0370-2) contains supplementary material, which is available to authorized users. Osteogenesis imperfecta (OI)  is a hereditary disease characterized by increased bone fragility, low bone mass, short stature, and other connective tissue manifestations, with a reported incidence of 1:15,000-1:25,000 , 4. In WCOL1A1/COL1A2 mutations have been identified in patients with OI . There are two general categories of type I collagen mutations giving rise to OI types I to IV. The first are haploinsufficiency mutations caused by frameshift, nonsense, and splice-site mutations, which lead to failure to synthesize the products of one COL1A1 allele. The second involves the synthesis of collagen molecules with structural abnormalities, most frequently caused by the substitution of glycine by another amino acid in the Gly-X-Y triplet domain of the triple helix. Previous studies have reported that OI patients with COL1A1 haploinsufficiency mutations have milder bone fragility and damage than those with COL1A1/COL1A2 helical glycine mutations [COL1A1 and COL1A2 genes are very large, and they have rarely been analyzed systematically in Taiwan. The aim of this study was to characterize the correlations of genotype and phenotype for Taiwanese OI patients, with the hope that this may aid in the clinical diagnosis, genetic counseling and prenatal diagnosis of this disease.To date, more than 1,000 different utations , 8. BothCOL1A1 or COL1A2 mutations. This is an analysis of only those patients found to have a molecular genetic finding of a mutation in one of the two type I collagen genes. Clinical manifestations and the results of physical examinations and imaging studies of these patients were also recorded. The hospital’s Ethics Committee approved the study protocol, and all of the participants or their parents provided written informed consent.Seventy-two Taiwanese patients  from 37 unrelated families were diagnosed with OI during the study period (January 1996 through December 2014) at Mackay Memorial Hospital, Taipei, Taiwan. None of the patients belonged to a consanguineous family. Molecular analysis was performed to investigate the presence of Diagnosis and classification were based on clinical and radiological characteristics according to the Sillence classification system . In thisCOL1A1 gene and 52 exons of the COL1A2 gene, including the intron-exon boundaries, was performed using a BigDye Terminator cycle sequencing kit . The nucleotide sequence was determined using an Applied Biosystems 3100 DNA sequencer. Sequence traces were aligned with the GenBank reference sequences of COL1A1 genomic DNA (AF017178.2) and cDNA (NM_000088.3), and COL1A2 genomic DNA (AF004877.1) and cDNA (NM_000089.3). DNA mutation numbering was based on the cDNA sequence using the A of the ATG translation initiation start site as nucleotide +1. Novel mutations were identified by their absence from the Osteogenesis Imperfecta Variant Database (https://oi.gene.le.ac.uk/home.php). In addition, identified nucleotide changes were re-examined in 100 control alleles. Polymorphisms were considered if the same nucleotide changes were detected in the control group.Total genomic DNA was isolated from peripheral blood using standard extraction methods. DNA sequencing of all 51 polymerase chain reaction-amplified exons of the r-z transformations. The clinical and radiological data were compared between patients with helical mutations versus those with haploinsufficiency mutations using the Student’s t-test for continuous variable, and Pearson’s chi-squared test and Fisher’s exact test for categorical variables. Two-tailed p-values were computed. SPSS version 11.5  was used for calculations and differences were considered to be statistically significant when the p value was less than 0.05.Relationships between gender, age, BMD SDS, DI and the existence of each clinical feature in the OI patients were tested using Pearson correlation, and significance was tested using Fisher p < 0.01). The BMD SDS was positively correlated with the ability to walk without assistance (p < 0.01). Patients with DI appeared to be prone to developing bone deformities (p < 0.01) and scoliosis (p < 0.01). Girls with OI had a slightly higher BMD SDS than boys with the same mutation type (p < 0.05)  were classified as OI type I, 5 (7 %) as type III, and 25 (35 %) as type IV. None of the patients had OI type II. The SDSs for height, weight, and BMD for all patients were–1.61 ± 2.57,–0.84 ± 1.56, and–2.06 ± 1.62, respectively. Furthermore, when dividing the patients into groups by OI type, the height SDSs were–0.49 ± 1.37,–6.42 ± 5.54, and–2.51 ± 1.85, respectively; and the BMD SDSs were–1.57 ± 1.15,–3.32 ± 1.86, and − 2.69 ± 1.96, respectively, for type I, III, and IV. A triangular face, blue sclera, DI, hearing loss, bone deformity, scoliosis, and walking without assistance were recorded in 18, 89, 43, 19, 58, 36 and 90 % of the patients, respectively. An older age was associated with hearing loss  were novel mutations. Among the 28 COL1A1 mutations, 7 were missense mutations, 4 were nonsense mutations, 6 were splicing mutations, and 11 were frameshift mutations. Eight familial mutations were identified. All 9 COL1A2 mutations were missense mutations, 4 of which were from familial inheritance  were caused by the substitution of a glycine within the Gly-X-Y triplet domain of the triple helix. There were 7 glycine mutations in COL1A1 and 8 in COL1A2. Among the 15 glycine mutations, aspartic acid substitutions were the most common type , followed by serine , arginine , cysteine , and glutamic acid  substitutions. Of the 21 haploinsufficiency mutations in COL1A1, 11 were frameshift mutations, 6 were splicing mutations, and 4 were nonsense mutations.Thirty-seven COL1A2 mutations, COL1A1 mutations were more frequent in the patients with OI type I (46 % vs. 33 %) and III (14 % vs. 11 %), and less frequent in the patients with OI type IV (39 % vs. 56 %). Helical mutations were more frequent in the patients with OI type III (27 % vs. 5 %) and IV (47 % vs. 38 %), and less frequent in the patients with OI type I (27 % vs. 57 %) compared to those with haploinsufficiency mutations  (Table The OI type was correlated with mutated genes and mutation types. Compared with 46 % vs. 3 % and I5) Table  and b.TaCOL1A1/COL1A2 mutations in 72 patients from 37 unrelated families, including 28 COL1A1 mutations and 9 COL1A2 mutations. Among these 37 COL1A1/COL1A2 mutations, 16 (43 %) were missense mutations, 11 (30 %) were frameshift mutations, 6 (16 %) were splicing mutations, and 4 (11 %) were nonsense mutations. These mutations were unique and were not repeated between the families, meaning that there was no hot-spot COL1A1/COL1A2 mutation in the study population. Fifteen (41 %) of the mutations were novel and 12 (32 %) were familial. In Zhang et al’s study [A number of studies have described the clinical and molecular findings for Western patients with OI –17; howe’s study  describe’s study –19, mostGrowth retardation is a notable symptom of OI, and it is extremely severe in patients with OI type III. Moriwake and Seino  reportedPatients with OI have a significantly lower BMD. DEXA can detect a low BMD that may be missed on plain radiographs, even in patients with the milder forms of OI . It may Blue sclera is a distinctive feature of unknown etiology in OI, and most patients with OI type I have blue sclera throughout their lives. In OI type III and IV, the sclera may also be blue at birth and during infancy, however the blue color fades with time during childhood . The majp < 0.01) and scoliosis (p < 0.01).DI is another principle manifestation of OI, and is associated with an abnormal type I collagen molecule. Discoloration and pulpal obliteration are the major characteristics. Lukinmaa et al.  reportedp < 0.01).Progressive hearing loss is an important symptom of OI, with the most common age at onset being in the second to fourth decades of life . In our In patients with OI, the causes of bone deformities include an imperfect healing process after a fracture, and weight bearing itself without an apparent fracture. Bone deformities are frequently observed in the long bones, and scoliosis, spinal deformities, and compression fractures are also commonly seen . In our The pathology of scoliosis is based on vertebral fragility, which progressively deteriorates with age . KarbowsRauch et al.  reportedThere is a tendency to give more weighting to severity in phenotypic grouping whereas recent reviews stress that there is variability in severity encompassing mild to moderate phenotypes within families in patients with both OI type 1 and type 4 . The perCOL1A1 mutations occurred 3.1 times (28:9) more frequently than COL1A2 mutations, and the replacement of glycine residues by other residues within the Gly-X-Y triplet domain of the triple helix in both COL1A1 and COL1A2 was common. Our results are consistent with those of a previous study on Chinese patients [COL1A1 and 15.8 % in COL1A2) [COL1A1 (38.9 %) and COL1A2 (44 %) [In our study cohort of Taiwanese OI patients, 1 times 2:9 more f COL1A2) . The mut2 (44 %) , howeverCOL1A2 mutations, COL1A1 mutations were more frequent in patients with OI type I and less frequent in those with OI type IV. Compared with haploinsufficiency mutations, helical mutations occurred more commonly in the patients with OI type III and IV, and less commonly in the patients with OI type I. Although the Sillence classification is based exclusively on phenotypic criteria and is inevitably used inconsistently among different OI investigators, our results were still similar to previous reports [We found that the OI type was correlated with mutated genes and mutation types. Rauch et al.  and Zhan reports , 8.COL1A1 haploinsufficiency mutations result in a quantitative defect, with synthesis of structurally normal type I procollagen at about half of the normal amount. They initiate nonsense-mediated decay of the mRNA derived from that allele leading to mild bone fragility. COL1A1/COL1A2 helical glycine mutations cause the synthesis of collagen molecules with structural abnormalities, which can result in a clinical severity from mild OI type I to lethal OI type II. Previous studies have reported that OI patients with COL1A1 haploinsufficiency mutations have milder bone fragility and damage than those with COL1A1/COL1A2 helical glycine mutations [utations , 8, 27, Due to the limited sample size in this single-center retrospective study, substitutions by many amino acid residues as well as the position of a mutation occurred too infrequently to draw any conclusions about their phenotypic effects. In addition, our results were limited to BMD of the lumbar spine, which may not be representative of other skeletal involvement, especially the appendicular skeleton. The small sample size also reflects the rare nature of this genetic disorder, and both the age range and degree of disease severity varied widely. Therefore, further multicenter studies with larger cohorts and a longer follow-up period are warranted.COL1A1/COL1A2 genes and were associated with OI type I, III and IV by direct sequencing in our Taiwanese patients, including 28 COL1A1 mutations and 9 COL1A2 mutations. Among them, 15 (41 %) were novel mutations, 12 (32 %) were familial mutations, and 15 (41 %) were caused by the substitution of a glycine within the Gly-X-Y triplet domain of the triple helix. Patients with COL1A1/COL1A2 helical glycine mutations had more severely damaged skeletal phenotypes than those with COL1A1 haploinsufficiency mutations. Genotype and phenotype databases are expected to promote better genetic counseling and medical care of patients with OI.Thirty-seven mutations were identified in the Mackay Memorial Hospital IRB.


Mangifera, consisting of numerous tropical fruiting trees in the flowering plant family, Anacardiaceae. Postharvest treatment by hot water brushing (HWB) for 15–20 s was introduced commercially to improve fruit quality and reduce postharvest disease. This treatment enabled successful storage for 3–4 weeks at 12°C, with improved color and reduced disease development, but it enhanced lenticel discoloration on the fruit peel. We investigated global gene expression induced in fruit peel by HWB treatment, and identified key genes involved in mechanisms potentially associated with fruit resistance to pathogens, peel color improvement, and development of lenticel discoloration; this might explain the fruit’s phenotypic responses.The mango belongs to the genus The mango transcriptome assembly was created and characterized by application of RNA-seq to fruit-peel samples. RNA-seq-based gene-expression profiling identified three main groups of genes associated with HWB treatment: 1) genes involved with biotic and abiotic stress responses and pathogen-defense mechanisms, which were highly expressed; 2) genes associated with chlorophyll degradation and photosynthesis, which showed transient and low expression; and 3) genes involved with sugar and flavonoid metabolism, which were highly expressed.We describe a new transcriptome of mango fruit peel of cultivar Shelly. The existence of three main groups of genes that were differentially expressed following HWB treatment suggests a molecular basis for the biochemical and physiological consequences of the postharvest HWB treatment, including resistance to pathogens, improved color development, and occurrence of lenticel discoloration.The online version of this article (doi:10.1186/1471-2164-15-957) contains supplementary material, which is available to authorized users. Mangifera indica) belongs to the plant family Anacardiaceae, which includes numerous tropical fruiting trees; it is native to South Asia, from where it has been distributed worldwide to become one of the most cultivated fruits in the tropics, with significant economic importance [Mango  for 15–20 s [Control of postharvest development of side rots caused by We recently demonstrated that HWB stresses the fruits and activates processes that could reduce fruit quality after storage . The obj-5. A total of 35,719 transcripts (62.07%) showed significant similarity to known proteins in the NR database. Based on the NR annotations, 53.12% of the annotated sequences showed very high homology , and 20.9% showed high homology . An additional 25.9% showed homology  to available plant sequences , Populus trichocarpa (12%), Prunus persica (7.5%), Fragaria vesca  and Cucumis sativus (1.8%)  fruit tree with a small genome size of about 450 Mbp . A new mn =40 fruP-value <0.05 after false-discovery-rate (FDR) determination , and at 4, 17 and 48 h after the treatment with those of untreated fruits at the same time points. Both HWB and control fruits were stored at 12°C. Two basic criteria were used to define differential gene expression: a twofold difference in transcript levels between treated and control fruits, and a Analysis of gene responses revealed a decrease in the number of differentially expressed genes from time 0 to 48 h: immediately after HWB treatment (10-20 min was considered as 0 h) 827 genes were differentially expressed whereas 48 h later only 87 genes showed differential expression Table  of the dThe expression patterns of 1,225 genes that were differentially expressed in at least one time point were subjected to hierarchical clustering, which resulted in five main clusters Figure A, which Each cluster was analyzed for its GO-enriched profile using BLAST2go and Fisher’s Exact Test  and flavonol synthase flavanone 3-hydroxylase-like (EC:1.14.11) . ClusterAOS) (EC:4.2.1.92) and syntaxin 121 (Syn121) [Cluster 3 was enriched with genes associated with the cellular defense response and its regulation; in this cluster we found genes such as allene oxidase synthase ((Syn121) .LHCIIb) (EC4.99.1.1) [lhca2) [PIRC) (EC1.97.1.12) [Cluster 4 was enriched with genes involved in photosynthesis and chlorophyll catabolism, whose abundance decreased as sampling times increased from 0 through 17 h  , 25 and  [lhca2) , which r97.1.12) . ClusterPAL)  genes involved in chitinase activity and response to wounding were represented in cluster 1  (EC:2.7.11.9) and inositol-tetrakisphosphate 1-kinase 2-like (IT1K2)  genes involved in jasmonic acid stimulus were represented in cluster 2  (EC:1.14.13.78) genes involved in photosynthesis and heme binding were represented in cluster 4  (EC 2.1.1.274) and peroxidase 15-like (peroxidase15) (EC 1.11.1.7) genes were represented in cluster 5  RT-PCR analyses were performed for key genes of interest. Mango genes belonging to the five different clusters of genes that were differentially expressed following HWB treatment were analyzed for their expression levels Figure . ChitinaA. alternata infestation on fruits of cvs. Palmer, Kent, Tommy Atkins, Keitt, Lilly and Shelly . Glutaredoxin gene family is regarded as candidates for controlling the redox state of regulatory proteins [Gutaredoxin is commonly used as a marker for JA-dependent defense responses [HWB treatment of freshly harvested fruits reduced the incidence of natural y Figure A. Hot way Figure A, as alsy Figure C that arn Figure F. This gproteins ; they inesponses .Figure 6Syn121, glutaredoxin and IT1K2 all have a significant effect on the jasmonic acid stimulus [AOS  and resorcinol-5-(pentadecyl) , 15 is aUgft3) (EC 2.4.1.91) – PAL, chalcone-flavanone isomerase-like protein (CFIL) (EC 5.5.1.6) and chalcone synthase (CHS) (EC:2.3.1.74) – related to the anthocyanin accumulation, phenylpropanoid and flavonoid biosynthesis pathways found in cluster 2 were tested by qRT-PCR  PAL,ompounds , 54; 3) entation  4) CHS,  III PKS ; it cata III PKS . Althoug III PKS , suggestLHCIIb encoding for chlorophyll a/b-binding protein, one of the most abundant proteins in the chloroplast, which is important in the structure of photosynthesis reaction centers [Oxepch) (EC1.3.1.74) [PIRC; and thioredoxin-like 1-chloroplastic (Thl1ch) (EC 1.1.1.49), in which the chloroplast thioredoxins have been suggested as mediators in the light-dependent regulation of chloroplast enzyme activity [One of the significant consequences of HWB treatment is the improved and enhanced color development on the fruit skin, which results from both anthocyanin production and inhibition of chlorophyll accumulation. The fruit color index of HWB-treated fruits was higher during all the period of fruit storage at 12 and 20°C Figure . The ind centers , 59, 60 .3.1.74) ; PIRC; aactivity .Figure 8anthocyanin5a) (EC:2.3.1.144) and UDP-glycosyltransferase 85a2-like (85A2) (EC:2.4.1.115) – showed increased expression at the first two time points (0 and 4 h) after HWB, as detected by qRT-PCR  impre Figure .In light of the physiological changes described, the improved fruit quality acquired following heat stress is probably a result of various stress-response mechanisms that act coordinately to improve the fruit quality, prevent pathogen development, prevent cell damage and re-establish cellular homeostasis. Genes identified in the present study that are modified in mango fruits following heat treatment, could have similar functions in other fruits such as citrus or peach.Mangifera indica L. cv. Shelly) were obtained from trees in commercial orchards in the north of Israel. Several experiments to determine the phenotypic response of the fruits to HWB were carried out in each season during three consecutive years. The phenotypic responses obtained in the various years were similar, therefore results from only 1 year are presented. Each treatment comprised 6 replications, each with 15 fruits.Freshly harvested mango fruits  at 12°C, ca. 90% RH [-1 and at a nozzle pressure of 2 atm [Postharvest treatments were carried out at the Department of Postharvest Science of the Volcani Center in Bet Dagan, Israel. HWB treatment at 55°C was applied on the packing line, as a spray (nozzle pressure of 2 atm) above brushes revolving at 60 g, a rate of 100–120 L minDisease severity, measured as the percentage of the fruit surface covered by black lesions, was recorded for 90 fruits (15 fruits from each of six replicates) after about 4 weeks of storage at 12°C and 3 days at 20°C. Fruits were regarded as unmarketable when more than 1% of their surface area exhibited black spots.Lenticel spotting was assessed visually at the end of storage, on a scale of 0 to 3, with values based on a combination of degree of lenticel discoloration and area covered by the symptoms .Skin color development was assessed visually at the end of storage and a color index was calculated on a scale of 1 (green) to 5 (full color) .g, washed with 70% ethanol, and re-suspended at 65°C for 3 min in SSTE buffer  SDS). Samples were extracted with an equal volume of chloroform:isoamyl alcohol (24:1), and with an equal volume of chloroform:isoamyl alcohol:water-saturated phenol (24:1:25), and the phases were separated by centrifugation at 10,000 g for 10 min. The RNA was ethanol-precipitated overnight, and resuspended in diethyl-pyrocarbonate-treated water. RNA was further treated with Turbo DNAse .Total RNA was extracted according to Yang et al. , with miSingle-stranded cDNA was synthesized from 1 μg of total RNA by means of the Verso cDNA synthesis kit . The synthesized cDNA was used as a template for qRT-PCR analysis, to estimate the expression level of the selected genes. The cDNA samples were diluted 1:10 (v/v) to the final template concentration for qRT-PCR. Real-time qRT-PCR was performed with a RotorGene 3000 system . PCR amplification was run with 3.5 μL of cDNA template in 10 μL of reaction mixture containing 5 μL absolute blue qPCR SYBR green ROX mix (Thermo Scientific) and 300 nM primers. PCR conditions were: initial denaturing for 15 min at 94°C; 40 denaturing cycles of 10 s at 94°C; annealing at 60°C for 15 s; extension at 72°C for 20 s (cycling A), 77°C for 6 s (cycling B), or 80°C for 6 s (cycling C), and melting at 72–99°C. The samples were subjected to melting-curve analysis with the RotorGene program. All samples were normalized to actin gene levels in the same qRT-PCR, and the values were expressed as increase or decrease in level relative to a calibration sample. The forward and reverse primers for all of the genes are listed in Table M. indica cv. Shelly was sequenced according to Illumina Hiseq2000 and Trueseq protocols, at the Crown Institute for Genomics, The Nancy and Stephen Grand Israel National Center for Personalized Medicine at the Weizmann Institute of Science, Rehovot, Israel. Eight libraries with total single-end RNA-seq reads 100 nucleotides in length were generated. The eight libraries contained the following sequences: 1) control RNA-seq of mango peel at time 0 h with 20,025,080 reads; 2) RNA-seq of mango peel treated by HWB at time 0 h with 20,202,891 reads; 3) control RNA-seq of mango peel at time 4 h with 21,622,332 reads; 4) RNA-seq of mango peel 4 h after HWB with 20,744,762 reads; 5) control RNA-seq of mango peel at 17 h with 20,916,444 reads; 6) RNA-seq of mango peel 17 h after HWB with 21,410,839 reads; 7) control RNA-seq of mango peel at 48 h with 20,687,358 reads; and 8) RNA-seq of mango peel 48 h after HWB with 21,230,696 reads. The transcriptome datasets are available in the NCBI Sequence Read Archive (SRA) under accession number SRX375390 and BioProject accession PRJNA227243. A new transcriptome was assembled from the 8.6-Gbp sequences by using Trinity software [The transcriptome of software , generatTophat , Bowtie2M. indica cv. Shelly were annotated by using BLASTx [The genes of g BLASTx , after wg BLASTx  was assig BLASTx  by meansg BLASTx . GO-enrig BLASTx .Additional file 1: Table S1: Table of differentially expressed genes and their annotations after HWB treatment versus control at time 0 h. (XLSX 5 MB)Additional file 2: Table S2: Table of differentially expressed genes and their annotations after HWB treatment versus control at time 4 h. (XLSX 5 MB)Additional file 3: Table S3: Table of differentially expressed genes and their annotations after HWB treatment versus control at time 17 h. (XLSX 5 MB)Additional file 4: Table S4: Table of differentially expressed genes and their annotations after HWB treatment versus control at time 48 h. (XLSX 5 MB)Additional file 5: Table S5: GO enrichment analysis of gene clusters . (DOC 10


Maize heat shock protein of 101 KDa (HSP101) is essential for thermotolerance induction in this plant. The mRNA encoding this protein harbors an IRES element in the 5′UTR that mediates cap-independent translation initiation. In the current work it is demonstrated that hsp101 IRES comprises the entire 5′UTR sequence (150 nts), since deletion of 17 nucleotides from the 5′ end decreased translation efficiency by 87% compared to the control sequence. RNA structure analysis of maize hsp101 IRES revealed the presence of three stem-loops toward its 5′ end, whereas the remainder sequence contains a great proportion of unpaired nucleotides. Furthermore, HSP90 protein was identified by mass spectrometry as the protein preferentially associated with the maize hsp101 IRES. In addition, it has been found that eIFiso4G rather than eIF4G initiation factor mediates translation of the maize hsp101 mRNA. Zea mays L.) Maize heat shock protein of 101 kDa (HSP101) mRNA encodes a chaperone essential for thermo-tolerance induction in maize  at its 5′UTR end. The cap structure is recognized by the eIF4F initiation factor consisting of eIF4A, eIF4G and eIF4E Most eukaryotic mRNAs contain a cap structure  translation system in a cap-dependent manner. In parallel, translation of these RNAs was analyzed in lysates expressing the Lb protease of FMDV, as previously done with the pBIC-5 IRES sequence In order to identify the minimal region of the maize hsp101 IRES driving internal initiation of translation, 5′end deletions of the IRES sequence inserted in pBIC-5 construct were generated. The pBIC-5 construct, containing an active maize hsp101 IRES Given that the sequence in 101+1 RNA was identified as the authentic maize hsp101 IRES functional region, we analyzed the secondary structure of this RNA in order to identify potential RNA motifs mediating internal initiation, likely shared with other IRES elements.To characterize the secondary structure in solution of maize hsp101 functional IRES, 101+1 RNA was treated with N-methylisatoic anhydride (NMIA), which reacts with the single-stranded 2′hydroxylic groups of nucleotides to form a stable 2′-O-aduct Once the secondary structure of the functional region of maize hsp101 IRES was elucidated, we set up to identify candidate proteins potentially interacting with this element. For this, poly(A)-tailed RNAs were generated to capture the proteins that bind to maize hsp101 IRES taking advantage of the poly(A) affinity to oligo dT cellulose. In addition, untreated and Lb protease-treated lysates were used to favor cap-dependent or cap-independent conditions, respectively. No differences were observed in the pattern of proteins purified with the RNAs tested using untreated lysates . In contin vitro translation assays. Additionally, two potential inhibitory reagents were added to the translation system. One of them, radicicol is an HSP90 specific ligand To confirm that the HSP90 protein was involved in maize hsp101 IRES-driven translation, we conducted cap-independent in vitro translation assays were performed using the wheat germ system (WGE) depleted from either eIF4F or eIFiso4F  using m7GTP-sepharose. Then, addition of wheat recombinant eIF4G or eIFiso4G to the translation system was conducted to determine whether any of these factors affected hsp101 IRES-directed translation. No significant difference in hsp101 translation efficiency was observed when eIF4G was added to the system compared to the depleted lysate. However, translational efficiency increased by 40% when eIFiso4G was added to the translation system compared to the control. Furthermore, hsp101 translation efficiency increased by 60% when both factors were added to the system  that were coprecipitated with the IRES element. The 75 kDa protein was identified as the molecular chaperone HSP90, a protein belonging to a family of ancient conserved factors found in all organisms including plants and animals Previous evidences has shown that eIF4G initiation factor is required for cap-independent translation driven by various IRES elements Our results raised the possibility that SL1 could provide the binding-site for proteins such as HSP90 or eIFiso4G, a possibility that needs to be tested in the future. Interestingly, the results obtained in the current work suggest that transfer of the IRES hsp101 of maize to other plant mRNAs could confer translational advantage under heat stress and thus, alleviate problems of thermo-tolerance in plants.In summary, this work reports the first experimental secondary structure of the IRES of a cellular mRNA of plants (hsp101), the participation of HSP90 in the maize IRES hsp101 complex and finally, the preference for eIFiso4G for translation initiation under cap-independent conditions.SacI was added to the primers used in the amplification reactions. Construct hsp101+1 was generated with the pair of primers forward hsp101+1 5′-CGAGCTCGCACAACATTTCAACCAGA-3′/reverse RV101* 5′-AGCAGCCATGAATCCGGACAACTCTCGACG-3′. For constructs hsp101Δ17 and hsp101Δ50, the pair of primers forward hsp101-A 5′-CGAGCTCGAAACACTAGCCGAAGCAA-3′/reverse RV101* and forward hsp 101-B 5′-CGAGCTCCACCTGGTGGGATCATCTC-3′/reverse RV101* respectively, were used. PCR products were inserted into the pGEM-T EASY cloning vector to generate pGEM-101+1, pGEM-101Δ17 and pGEM-101Δ50, respectively. The fragments were then excised with SacI to replace the IRES sequence in pBIC-5 generating pBIC-hsp101+1, pBIC-hsp101Δ17 and pBIC-hsp101Δ50 constructs. RNAs derived from pGEM-T constructs were synthesized using T7 RNA polymerase with Pst1 linearized plasmids. Bicistronic RNAs pBIC-5, 101+1, 101Δ17 and 101Δ50 synthesized in vitro using HpaI linearized plasmids and T7 RNA polymerase The IRES sequence amplification reactions were performed as previously reported 35S] methionine  was prepared in a final volume of 35 µl with DEPC-treated water. The mix was incubated 60 min at 30°C; at the end of the reaction, 1µl of each sample was obtained and the synthesized proteins were resolved by SDS-PAGE. Gels were dried and exposed to a phosphorscreen to be analyzed by the QUANTITY ONE software (BIORAD). Values represent the mean (± SD) of three independent assays.Cap-dependent and cap-independent translation was examined either in rabbit reticulocyte lysate (RRL) or Wheat Germ Extract (WGE). For cap-dependent translation analysis, a mix of RRL (17.5 µl), RNasin (1 µl), amino acids mix without methionine (1 µl), the bicistronic RNA (100 ng) and  methionine (10 µCi), 100 ng of eIF4G or eIFiso4G and DEPC-treated water to reach a volume of 25 µl. The mix was incubated for 60 min at 25°C. Newly synthesized proteins were resolved by SDS-PAGE loading 1 µl of each sample. Gels were dried and exposed to a phosphorimager screen.Similarly, WGE was used either complete (cap-dependent) or eIF4E and eIFiso4E-depleted (cap-independent). For cap-independent translation, eIF4E and eIFiso4E were depleted from WGE using mBbuI2, 333 mM NaCl) were added and incubated for 20 min at 37°C. This mix was divided into two tubes (9 µl in each tube); 1.5 µl NMIA (65 mM) 2O, 4 µl 5 M NaCl, 1 µl glycogen (20 mg/ml), 2 µl 100 mM EDTA, pH 8 and 350 µl absolute ethanol were added to each reaction tube. Samples were precipitated and the pelleted RNA was resuspended in 20 µl 0.5X TE. Ten microliters of each RNA sample, NMIA (+) and (−), were transferred to a 200 µl tube; 0.5 µl of the [32P]-5′ labeled oligonucleotide (GGCCTTTCTTTATGTTTTTGGCG) using polynucleotide kinase (PROMEGA) were added and incubated first, for 5 min at 65°C, then for 5 min at 35°C and finally at 4°C for 1 min. Three microliters of reverse transcriptase (RT) Superscript III buffer, 1U RNasin, 0.25 µl 100 mM DTT, 1.5 µl 10 mM dNTPs and 0.25 µl H2O were added to each sample and further incubated for 1 min at 52°C. Then, 0.25 µl Superscript III RT (INVITROGENE) was added and the mix and incubated for 30 min at 52°C. At the end of the reaction, 0.4 µl of 4 M NaOH were added to each sample. Samples were precipitated by adding 1 volume of AcNH4, 1 µl glycogen and 75 µl absolute ethanol. Pellets were resuspended in 10 µl H2O plus 4 µl formamide buffer (90% formamide). Primer extension products were resolved on denaturing 6% polyacrylamide 7 M urea gels, in parallel to a sequence ladder prepared with the same 5′end-labeled primer. Gels were dried and exposed to a radiography film for quantification. SHAPE reactivity data was analyzed as described In order to determine the IRES secondary structure, hsp101+1 RNA was synthesized with T7 RNA polymerase using pBIC-hsp101+1 plasmid linearized with 2) containing 30 µl oligo dT sepharose. Samples were incubated for 30 min at 4°C with gentle shaking and then washed twice with binding buffer to remove unbound RNA. Then, 100 µg of RRL and 10 µg total RNA (nonspecific competitor) were added to a final volume of 100 µl binding buffer and incubated for 60 min at 4°C with gentle shaking. Samples were washed twice with binding buffer followed by washing buffer  to remove unbound proteins to the IRES-oligo dT sepharose complex. IRES-protein complexes were resolved by SDS-PAGE and visualized by silver staining in triplicate experiments. Thin slices containing differential IRES-bound proteins were excised and identified by mass spectrometry (LC/MS/MS) at Quebec Genomics Center.In order to identify the proteins interacting with hsp101 IRES (either sense hsp101+1 or antisense orientation inserted in the bicistronic constructs), 101+1, 101Δ17 and 101Δ50 RNAs (derived from pGEM-T EASY), were synthesized adding a poly-A tail using poly-A polymerase (AMBION) as described by the manufacturer. From each RNA sample, 2.5 µg were transferred to 100 µl of binding buffer (10 mM Tris, pH 7.5, 100 mM KCl, 2 mM MgCl


Stage III unresectable non-small cell lung cancer (NSCLC) is preferably treated with concurrent schedules of chemoradiotherapy, but none is clearly superior Gemcitabine is a radiosensitizing cytotoxic drug that has been studied in phase 1 and 2 studies in this setting. The aim of this study was to describe outcome and toxicity of low-dose weekly gemcitabine combined with concurrent 3-dimensional conformal radiotherapy (3D-CRT).2 during 5 weeks of 3D-CRT, 60 Gy in 5 weeks (hypofractionated-accelerated). Overall survival (OS), progression-free survival (PFS), and treatment related toxicity according to Common Toxicity Criteria of Adverse Events (CTCAE) version 3.0 were assessed.Treatment consisted of two cycles of a cisplatin and gemcitabine followed by weekly gemcitabine 300 mg/mBetween February 2002 and August 2008, 318 patients were treated. Median age was 64 years (range 36–86); 72% were male, WHO PS 0/1/2 was 44/53/3%. Median PFS was 15.5 months  and median OS was 24.6 months . Main toxicity (CTCAE grade ≥3) was dysphagia (12.6%), esophagitis (9.6%), followed by radiation pneumonitis (3.0%). There were five treatment related deaths (1.6%), two due to esophagitis and three due to radiation pneumonitis.Concurrent low-dose gemcitabine and 3D-CRT provides a comparable survival and toxicity profile to other available treatment schemes for unresectable stage III. At presentation approximately 30% of patients with non-small-cell lung cancer (NSCLC) have locally advanced (stage III) disease . Median 2 gemcitabine and 3D-CRT [Gemcitabine is among the strongest radiosensitizing drugs available, but its use in lung cancer has been limited due to substantial toxicity when combining full-dose gemcitabine with radiotherapy in treatment of stage III NSCLC , 13. Excd 3D-CRT . These rd 3D-CRT .Here we report survival time and treatment related toxicity of this treatment regimen in a consecutive cohort of patients receiving gemcitabine-based concurrent chemoradiotherapy for stage III unresectable NSCLC over a period of 7 years. A subgroup analysis of patients aged >70 years was planned a priori.18 F-FDG-PET, contrast enhanced chest CT, bronchoscopy, endoscopic ultrasound with fine-needle biopsy and mediastinoscopy. Treatment-related decisions were made during the weekly multidisciplinary meetings. The decision for treating these patients with this scheme, which was regarded as the standard treatment protocol for NSCLC stage III, was based on physical condition (performance status according to WHO), co-morbidity, and expected radiotherapy dose–volume constraints.From February 2002 until December 2009, consecutive patients with stage III unresectable NSCLC who were treated with concurrent chemoradiotherapy with gemcitabine as radiosensitizer referred to one radiotherapy department from 6 hospitals in the northern part of the Netherlands were studied. Staging was performed by whole body 2 on day 1 and gemcitabine 1125 mg/m2 administered intravenously on day 1 and 8 of each 21-day cycle. This dose of gemcitabine provides the same dose intensity as the older 3 out of 4 week schedule. Anti-emetics were ondansetron 8 mg twice daily on days 1 and 2, and dexamethasone 8 mg twice a day on days 1 and 2 of each cycle and aprepitant 125 mg on day 1 and 80 mg on days 2 and 3. The interval between the first dose of induction chemotherapy and the start of radiotherapy was 6 weeks.Chemotherapy consisted of two cycles of cisplatin 75 mg/m2. Gemcitabine was omitted when leucocytes were below 3.109/L or platelets below 100.109/L. The gross target volume (GTV) was delineated on the planning-CT and included the primary tumor and enlarged FDG-avid, or pathologically proven lymph nodes. Until 2005, tumor motion was determined with fluoroscopy and respective margins were added to the GTV, thereafter, an internal target volume (ITV) was individually delineated using a 4D-planning CT. A 5 mm margin was added to arrive at the clinical target volume, another 6 mm were added for the planning target volume (PTV). Radiotherapy was delivered using 6 MV photons, the dose was specified at the isocenter and was corrected for pulmonary heterogeneity. The total radiation dose administered corresponded to an equivalent dose of 62 Gy if it were given in 2 Gy daily fractions.Patients received 3D-CRT to a total dose of 60 Gy, administered over 5 weeks in once daily fractions of 2.4 Gy five days per week, together with once weekly gemcitabine 300 mg/m20  was constrained to 30% .The spinal cord dose was constrained to 50 Gy. The mean lung dose (MLD) should not exceed 20 Gy . The VFor patients with high-volume disease, a proof-planning was made and evaluated for feasibility by the radiation oncologist. If necessary, the radiation dose was adapted. If the pulmonary dose constraints were still considerably exceeded after radiation dose reduction, patients were excluded from this protocol.Complete blood cell counts were performed on days 1, 8, and 22 of each induction chemotherapy cycle. On day 1 of each cycle, patient evaluation also included liver and renal functions, performance status, and toxicity scoring. During radiotherapy, complete blood cell counts and toxicity evaluation were performed. Two months after completion of treatment a response CT scan was obtained and patients were followed every 3 months with physical examination, laboratory tests and chest x-ray. Disease progression was defined according to RECIST 1.0 criteria . ToxicitDescriptive statistics were used to characterize clinical features and toxicity. Using the Kaplan-Meier method, overall survival (OS) and progression-free survival (PFS) were calculated from the date of diagnosis until death, loss to follow-up, or first documentation of progressive disease, respectively. A subgroup analysis in patients aged over 70 years was performed to evaluate the outcome in elderly patients. Cox regression analysis was performed with the variables age, smoking, gender, histology, radiation dose, WHO performance score and PTV. A p-value of < 0.05 was considered statistically significant. All calculations were performed using SPSS 20.0 .Three hundred and eighteen subsequent patients receiving concurrent chemoradiotherapy with gemcitabine as a radiosensitizer were studied. Median age was 64 years (range 36–86); male/female was 72/28%; WHO PS 0/1/2 was 44, 53 and 3%. A total of 93 patients were aged ≥ 70.Two hundred and forty-two patients (76%) received cisplatin and gemcitabine as induction, 42 13%) received carboplatin and gemcitabine, and 9 patients (3%) received other schedules. Twenty five (8%) patients did not receive induction chemotherapy. A total of 259 patients (81%) received all 5 weekly doses of gemcitabine during radiotherapy, 244 (76%) received both full dose gemcitabine and full dose radiotherapy. Fifty-one patients (16%) received between 1 and 4 gemcitabine administrations weekly, mainly due to neutropenia and thrombocytopenia. For the remaining 8, no information was available. Patient characteristics are detailed in Table 3% receivSeven patients (2%) had a tumor resection after chemoradiotherapy.20 and 8 patients who stopped treatment due to radiation related toxicity including 3 patients with CTC grade 3 esophagitis. Nine patients received less than 60 Gy for undocumented reasons.The radiotherapy dose was 60 Gy in 284 patients (89%) while less than 60 Gy  was administered in 34 patients (11%). These 34 patients included 17 patients with increased risk of radiation pneumonitis due to increased Vth percentile at 734 cc. Median V20 was 22.7% and the 90th percentile was 31.0%. In patients aged ≥ 70, median PTV was not different at 411 cc with the 90th percentile at 702 cc. Median V20 was 23%, with the 90th percentile at 30.6%.The median PTV was 431 cc with the 90Median PFS was 15.5 months   of the patients had CTC grade ≥3 radiation pneumonitis. Three patients had a grade 5 event . For these three patients, the PTV was 508, 600 and 578 cc (the 75% percentile of the whole cohort was 576 cc), with a V20 of 16, 34, and 30%, respectively. The first patient had no induction chemotherapy, the two others were treated with induction carboplatin and gemcitabine. In patients who completed the full chemoradiotherapy (N = 244), 7% had CTC grade ≥3 esophagitis (including 1 CTC grade 5 event), and 4% CTC grade ≥3 radiation pneumonitis (including 2 CTC grade 5 events). In patients aged 70 and older (N = 93), 9 patients (9.7%) had CTC grade 3 esophagitis, 4 patients (4.3%) had CTC grade 3 radiation pneumonitis, and three patients (3.2%) died due to pneumonitis. The other patients died due to progressive disease.Most common toxicities were CTC grade 2 esophagitis 13.6%), and CTC grade 2 radiation pneumonitis (17.7%)  combined with a lower radiation dosage (55 Gy) given in a shorter time span (4 weeks) in a study that was prematurely closed due to slow accrual. There were 2 deaths in the gemcitabine arm due to acceleration of pre-existing interstitial lung disease [In 2003 it had been shown that concurrent low-dose gemcitabine and 3D-CRT had acceptable toxicity . Subsequ disease . Of note20 of 40% was associated with significant grade 3–5 pulmonary toxicity [20 was set at a rather conservative 30% (uncorrected for the slightly higher daily fraction dose of 2.4 Gy) and indeed the vast majority (90%) of treated patients had a V20 lower than 30%. Elective nodal irradiation was completely foregone in the first year of this study, with no ensuing statistically significant toxicity differences. However, all three patients in our study who died due to radiation pneumonitis were aged above 70 – and had V20 of 16, 34 and 30%, respectively. The CALGB study also identified older age to be associated with increased pulmonary toxicity [The main factors that are important concerning pulmonary toxicity (i.e. radiation pneumonitis) during chemoradiation with concurrent low-dose gemcitabine are not completely understood. Lung dose, the use of conformal radiotherapy and the timing of the gemcitabine dosage are primary candidates. The CALGB 30105 trial showed that a Vtoxicity , which wtoxicity .Also for esophageal toxicity, radiation technique had been shown to be critical. In a phase 1 study, which initially started using 2D conformal techniques, but halfway switched to 3D techniques, the percentage of the esophagus irradiated to 60 Gy dropped from 68 to 18%, and grade 2 esophagitis dropped from 5/10 patients to 2/14 patients [2) with 3D radiotherapy with elective nodal irradiation of the mediastinum [2, but even after this reduction, still more severe CTC grade ≥ 3 esophageal and pulmonary toxicity was observed compared to our study. Our once weekly schedule featured 3 (for 50 mg/ m2) to 4.3 times (for 35 mg/ m2) the cumulative weekly gemcitabine dose [Administration of gemcitabine more frequently than once weekly was also associated with increased toxicity. One study administered gemcitabine twice weekly  non-hematological grade 5 events in our study, which is comparable to other mainstream treatment regimens % non-hem, 10, 20.Treatment of patients with unresectable stage III NSCLC with cisplatin and gemcitabine followed by concurrent gemcitabine and 3D conformal radiotherapy was safe and yielded effectiveness and toxicity rates comparable with other drugs in our hands. These results are very likely due to conservative radiation dose-constraints and 3D-conformal radiotherapy avoiding irradiation of excessive volumes of uninvolved mediastinal areas. Although age was not a factor influencing survival and the absolute incidence at 3% grade 5 pneumonitis in patients older than 70 years was what could be expected, patients above 70 years of age should be selected with great caution for this regimen.