Current research is investigating new methods to enhance tissue healing to speed up recovery time and decrease the risk of failure in Anterior Cruciate Ligament (ACL) reconstructive surgery. Biological augmentation is one of the most exploited strategies, in particular the application of Platelet Rich Plasma (PRP). Aim of the present paper is to systematically review all the preclinical and clinical papers dealing with the application of PRP as a biological enhancer during ACL reconstructive surgery. Thirty-two studies were included in the present review. The analysis of the preclinical evidence revealed that PRP was able to improve the healing potential of the tendinous graft both in terms of histological and biomechanical performance. Looking at the available clinical evidence, results were not univocal. PRP administration proved to be a safe procedure and there were some evidences that it could favor the donor site healing in case of ACL reconstruction with patellar tendon graft and positively contribute to graft maturation over time, whereas the majority of the papers did not show beneficial effects in terms of bony tunnels/graft area integration. Furthermore, PRP augmentation did not provide superior functional results at short term evaluation. Anterior cruciate ligament (ACL) tears are among the most common sport-related injuries and therefore ACL reconstructive surgery is one the most frequently performed procedures in the field of sport medicine . EpidemiACL surgery (and the related research) is a classic example of integration between biomechanics and biology: the progress made in recent decades can be attributed to the big steps forward in the knowledge of both the mechanical and biological properties of ACL and its healing process –8. AlthoIn light of such potential, the possibility of applying this biological product to enhance ACL reconstructive surgery appears attractive: PRP not only might promote a better and faster ligamentization of the graft used for ACL reconstruction and reduce the proinflammatory factors released immediately after surgery, but might also contribute to a better integration of the graft within the bone tunnels, thus avoiding their enlargement and failure over time. Furthermore, PRP could be used to accelerate healing and reduce donor-site morbidity at the harvest site of the tendon graft.The aim of the present paper is to review systematically the current preclinical and clinical evidence concerning the application of PRP as a biological augmentation, to determine safety and efficacy of this biological approach to improve ACL reconstruction surgery.A systematic review of the literature was performed on the use of PRP in ACL reconstruction. The search was conducted on the PubMed database on February 20th, 2014 using the following parameters: ((ACL) OR (ACL reconstruction) OR (ACL lesion)) AND ((PRP) OR (platelet rich plasma) OR (platelet gel) OR (platelet derived) OR (platelet concentrate)). The guidelines for Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) were used. Screening process and analysis were conducted separately by 2 independent observers (BDM and LA).First, the articles were screened by title and abstract. The following inclusion criteria for relevant articles were used during the initial screening of titles and abstracts: clinical and preclinical reports of any level of evidence, written in English language, with no time limitation, on the use of PRP in ACL reconstruction, and reporting results on PRP effects. Exclusion criteria were articles written in other languages, reviews, or studies analyzing other applications of PRP in knee surgery not related to ACL procedures. In the second step, the full texts of the selected articles were screened, with further exclusions according to the previously described criteria. Moreover, the articles not reporting clinical, MRI, or histologic results were excluded. Reference lists from the selected papers were also screened. A flowchart of the systematic review is provided in The database search identified 60 records, and the abstracts were screened and selected according to the inclusion/exclusion criteria. As shown in Fifteen clinical trials were selected according to the inclusion criteria: 11 were randomized controlled trials, 3 were prospective comparative studies, and 1 was a retrospective comparative trial .With regard to the graft used to perform ACL reconstruction, in 9 studies, authors used hamstring tendons (gracilis and semitendinosus), in 4 studies bone-patellar tendon-bone (BPB) graft, in 1 study allograft, and in 1 paper authors both hamstring tendons and BPB graft. Fourteen papers described a single-bundle reconstruction technique, whereas just in one trial a double-bundle procedure was used. Concerning PRP delivery methods, in 2 papers  PRP was used to promote harvest site healing, in 1 paper PRP was applied intra-articularly by a suprapatellar injection at the very end of the surgical procedure, in 1 paper PRP was used just to coat the intra-articular portion of the graft, and in 11 papers it was administered both on the graft and in the bony tunnels to enhance the graft-bone integration. Among the delivery techniques used, in 2 studies peculiar approaches were applied. While most of the authors applied PRP on the graft surface, Sánchez et al.  placed PIn all but one case PRP was activated before administration. Details regarding PRP preparation methods, number of patients included, patients' characteristics, and follow-up evaluations are included in harvest site healing;tendon graft maturation and bony tunnel/graft integration;clinical results.To make the results more understandable, they will be discussed separately according to the specific aspects analyzed in the clinical trials:Two papers were specifically aimed at assessing the contribution of PRP in the healing of the harvest site after ACL reconstruction with BPB graft, and both of them reported positive outcomes with PRP.In the study led by de Almeida et al.  two grouTwelve papers examined outcome regarding (a) graft maturation over time and (b) graft integration in the bone tunnels.Among the 6 studies reporting data about graft maturation, 4 of them were in favour of PRP augmentation, whereas 2 reported no intergroup difference. With respect to bony tunnels/graft area, 9 studies focused on the integration, documenting in 7 cases no advantage after PRP administration. The evolution over time was investigated more specifically in 3 trials focused on the bony tunnel widening: none demonstrated that PRP was able to prevent tunnels' enlargement over time.P = 0.051) and even biopsies revealed a superior tissue quality for PRP, with newly formed synovial-like tissue enveloping the graft in 77% of cases versus 40% of the control group. Seijas et al. [Looking in more detail at the available literature, the first paper was published by Orrego et al.  in 2008:s et al.  were thes et al.  they shos et al. , the samAmong the studies documenting no beneficial effects of PRP administration, the first one was authored by Silva et al.  who evalSeven studies reported clinical outcomes after ACL reconstructive surgery with or without PRP augmentation, focusing on the short-term outcome, with followup reported from 6 months to 2 years. None of them showed any statistical inter-group difference. Only Magnussen et al. in a comparative retrospective trial  observed In vitro studies and also animal trials have highlighted overall encouraging results (The reduction of recovery time and prevention of reinjury after ACL reconstruction are the main goals of sport medicine surgeons. In recent years a number of studies have been published aimed at clarifying the best surgical techniques and considering the different types of grafts, the fixation devices, and other biomechanical and technical variables. Biological augmentation through PRP administration is one of the latest topics for researchers, with the aim of enhancing ACL reconstruction via the help of powerful biological agents. results , thus coAccording to the clinical trials currently available, it is possible to highlight some important findings. The first one regards the safety of this approach. The intraoperative use of PRP proved to be safe: in none of the clinical trials considered adverse events related specifically to its use could be identified, and no infections or other complications were reported after PRP administration. PRP actually proved to even reduce the surgical morbidity in two papers , 25 specMore controversial are the results in terms of biological efficacy on the reconstructed ACL. The application of PRP has been hypothesized to improve graft integration within the tunnels. Concerning the osteoligamentous integration, Rupreht et al. showed increased vascular density and microvessel permeability in the proximal tibial tunnel at 1 and 2.5 months' MRI evaluation, as well as reduced bone edema around the tunnel and increased cortical bone formation around the tibial tunnel wall at 6 months followup , 47. VogAlso with respect to the graft maturation process, the literature is not univocal, but in this case PRP seems to have a more positive influence. The studies led by Orrego et al., Radice et al., and Seijas et. al proved that PRP augmentation is able to determine a faster and better graft maturation with respect to the control groups , 44, 53.Finally, another important aspect regarding the clinical benefit that could be related to the observed tissue changes in terms of graft integration and maturation. The literature analysis on the clinical outcome of ACL reconstruction did not show any superior results with PRP augmentation. However, the majority of the published clinical studies did not consider clinical results as the primary outcome of the biological augmentation. Clinical evaluations were carried out in only a few studies and were limited to short-term followup, which prevents an accurate analysis of difference in failure rate and revision surgeries between the treatment groups, and further studies at longer followup should focus the real overall clinical benefit of PRP. in situ and might influence the overall regenerative potential of PRP. In light of these aspects, it is even harder to summarize results of clinical trials: each author uses his own formulation and so it is impossible, at present, to establish the best PRP for improving ACL reconstruction, as well as the real potential of this biological approach for ACL augmentation. Considering the great interproduct variability and the various applicative strategies, further high quality studies are needed to determine the best formulation, administration modalities, and the best responding targets of PRP augmentation in ACL reconstructive surgery.The present literature analysis is affected by some limitations. First of all each trial involves a different technique for ACL reconstruction: different types of graft were used, different tunneling, and also different fixation devices, so comparison of results is difficult and, at least hypothetically, it should be considered that some techniques might have a better response to biological stimulation than others. Furthermore, PRP has been administered in different ways: some authors applied it by a simple intra-articular injection after the procedure, others used it to cover the graft before or after its intra-articular fixation, and others injected it into the graft, and PRP could be even combined with substrates to increase ACL augmentation. Moreover, besides the intra-articular area of the graft, some authors also applied PRP inside the bone tunnels: some in both the tibial and femoral one and some only in the femoral one. Therefore, different applicative methods on different specific targets might determine different outcomes. Finally, another confounding factor is directly linked to PRP itself, that is, a well-known and debated topic, that is, the dramatic variability among different PRP formulations . CurrentThis systematic review underlines that clinical results on PRP use for ACL augmentation are controversial. The intraoperative use of PRP proved to be safe, and PRP actually showed to even reduce the surgical morbidity promoting graft harvest site healing. Based on current evidence, PRP seems to play a positive role in the healing mechanisms after ACL surgery for what regards graft maturation, whereas the majority of the studies showed no benefit in terms of graft integration, especially in preventing bone tunnel widening. Finally, PRP did not provide a superior clinical outcome at short-term followup, whereas data at longer followup are lacking to address the overall clinical benefit of PRP augmentation.


Serum adipokines have roles in the development of arterial stiffness. Our aim was to investigate the relationship of leptin and the surrogate marker carotid-femoral pulse wave velocity (cfPWV) in coronary artery disease (CAD) patients.Fasting blood samples were obtained from 105 CAD patients. cfPWV was measured with the SphygmoCor system. A cfPWV > 10 m/s was defined as high arterial stiffness, and ≤ 10 m/s as low arterial stiffness.P = 0.001), hypertension (P = 0.010), older age (P = 0.001), and higher systolic blood pressure (SBP) (P < 0.001), diastolic blood pressure (DBP) (P = 0.021), pulse pressure (P = 0.014), and serum leptin level (P = 0.002) compared to patients with low arterial stiffness. Serum leptin levels correlated with the number of angiographically documented stenotic coronary artery vessels (P < 0.001). After adjusting for factors significantly associated with arterial stiffness, multivariate logistic regression analysis showed that leptin  was a significant independent predictor of arterial stiffness.Thirty-seven patients (35.2 %) had high arterial stiffness, and had a higher percentage of diabetes (Increasing serum concentration of leptin correlated positively with the total number of stenotic coronary arteries, and serum leptin level may predict the development of arterial stiffness in CAD patients. Arterial stiffness is one of the multiple characteristics of cardiovascular disease (CVD), and the mechanisms of arterial stiffness most often include abnormal regulation of elastin fibers and collagen, re-organization of cellular elements, and low-grade inflammation –3. One mStudies have shown a relationship between CVD and adipokines such as leptin, adiponectin, and resistin , 8. LeptThe Protection of the Human Subjects Institutional Review Board of Tzu-Chi University and Hospital approved this study. Between March and December 2012, 105 CAD patients  in a medical center in Hualien, eastern Taiwan were enrolled in this study. Trained staff measured blood pressure (BP) in the morning for all participants using a standard mercury sphygmomanometer with appropriate cuff size after the patients had been sitting for at least 10 min. Systolic BP (SBP) and diastolic BP (DBP) were taken at the points of appearance and disappearance, respectively, of the Korotkoff sounds. SBP and DBP were taken three times at 5 min intervals, and were averaged for analysis. Pulse pressure was calculated by subtracting DBP from SBP. HTN was defined as SBP ≥ 140 mmHg, and/or DBP ≥ 90 mmHg, or prescription of antihypertensive medication in the past 2 weeks. A person was regarded as having diabetes mellitus (DM) if the fasting plasma glucose was either 126 mg/dl or more, or if he/she was using diabetes medications  . ParticiBody weight was measured in light clothing and without shoes to the nearest 0.5 kg, and body height was measured to the nearest 0.5 cm. Body mass index (BMI) was calculated as the weight in kilograms divided by the height in meters squared , 19.g for 10 min. Serum levels of blood urea nitrogen (BUN), creatinine (Cre), fasting glucose, total cholesterol (TCH), triglycerides (TG), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), total calcium, and phosphorus were measured using an autoanalyzer . [Fasting blood samples (approximately 5 ml) were immediately centrifuged at 3000 erland). , 19 Seruerland). . CalculaR-timing reference. Pulse wave recordings were performed consecutively at two superficial artery sites . Integral software was used to process each set of pulse wave and ECG data to calculate the mean time difference between R-wave and pulse wave on a beat-to-beat basis, with an average of 10 consecutive cardiac cycles. The cfPWV was calculated using the distance and mean time difference between the two recorded points. Quality indices, included in the software, were set to ensure uniformity of data. A cfPWV > 10 m/s was defined as high arterial stiffness, and ≤ 10 m/s as low arterial stiffness according to the European Society of Hypertension and the European Society of Cardiology (ESH-ESC) 2013 Guidelines [Measurement of cfPWV was performed using a pressure tonometer to transcutaneously record the pressure pulse waveform in the underlying artery , as previously described , 19. Allidelines .U test . Data expressed as the number of patients were analyzed by the χ2 test. Differences of leptin levels between numbers of occluded vessels was analyzed by the Kruskal-Wallis analysis of variance (AVONA) test. Variables that were significantly associated with arterial stiffness were tested for independence by multivariate logistic regression analysis . Data were analyzed using SPSS for Windows . Values of P < 0.05 were considered statistically significant.Normally distributed data were expressed as mean ± standard deviation (SD), and comparisons were performed using the Student’s independent t-test (two-tailed). Data not normally distributed were expressed as medians and interquartile ranges, and comparisons were performed using the Mann–Whitney n = 28; 26.7 %), angiotensin-converting enzyme inhibitors , calcium channel blockers , β-blockers , statins , and fibrate . Thirty-seven patients (35.2 %) were defined as high arterial stiffness, and this group of patients had a higher percentage of DM (P = 0.001) and HTN (P = 0.010) as compared to the low arterial stiffness group. There was no statistically significant difference in sex and use of ACEi, ARB, β-blockers, CCB, statins, or fibrate between the two groups. Age (P = 0.001), SBP (P < 0.001), DBP (P = 0.021), pulse pressure (P = 0.014), and serum leptin level (P = 0.002) were higher in the high arterial stiffness group compared with the low arterial stiffness group.Demographic, biochemical, and clinical characteristics of the 105 CAD patients are shown in Tables P < 0.001). Figure r = 0.390; P < 0.001).Fasting serum leptin levels based on the number of stenotic coronary artery vessels are shown in Fig. P = 0.037), age (P = 0.002), and DM (P = 0.023) were independent predictors of arterial stiffness  showed that leptin P = 0.07, age (PThe results of this study showed that fasting leptin level was higher in CAD patients with high arterial stiffness than those with low arterial stiffness, and was an independent predictor for the development of arterial stiffness. In addition, serum concentration of leptin correlated positively with the number of stenotic coronary arteries.Classic CV risk factors, including DM, hyperlipidemia, elevated BMI, and smoking, have been implicated in accelerating arterial stiffness. Aortic stiffness can affect aortic function and reduce baroreceptor responsiveness, and arterial stiffness one of the leading causes of increased blood pressure , 21. TheLeptin is a peptide product of the obese gene, and is predominantly secreted by white adipose tissue , 25. In In the current study, serum leptin correlated with the total number of diseased coronary arteries. This finding is similar with those of previous studies which revealed that serum leptin plays an important role in the occurrence, severity, and extent of CAD –34. PatiReports have shown that several types of medications can affect arterial stiffness –40. ACEiThe limitation of this study is that there was a lack of data regarding abdominal obesity to analyze the possible influence on arterial stiffness in our patients and it was a cross-sectional study with a limited number of CAD patients conducted at a single center. Therefore, the findings of this study should be confirmed by further longitudinal studies before a cause-effect relationship between serum leptin and arterial stiffness can be established in the CAD population.In conclusion, in this study which analyzing the role of leptin in angiographically diagnosed CAD patients, we found that serum leptin level correlated positively with cfPWV and is a predictor for the development of arterial stiffness. In addition, serum concentration of leptin correlated positively with the total number of stenotic coronary arteries in our CAD patients.This study was approved by The Protection of the Human Subjects Institutional Review Board of Tzu-Chi University and Hospital. All participants gave written informed consent after thorough explanation of the procedures involved.Not applicable.All relevant data supporting the conclusions of this article is included within the article.


Clostridium thermocellum to rapidly consume cellulose and produce ethanol makes it a leading candidate for a consolidated bioprocessing (CBP) biofuel production strategy. C. thermocellum also synthesizes lactate, formate, acetate, H2, and amino acids that compete with ethanol production for carbon and electrons. Elimination of H2 production could redirect carbon flux towards ethanol production by making more electrons available for acetyl coenzyme A reduction to ethanol.The native ability of 2 production in C. thermocellum is encoded by four hydrogenases. Rather than delete each individually, we targeted hydrogenase maturase gene hydG, involved in converting the three [FeFe] hydrogenase apoenzymes into holoenzymes. Further deletion of the [NiFe] hydrogenase (ech) resulted in a mutant that functionally lacks all four hydrogenases. H2 production in ∆hydG∆ech was undetectable, and the ethanol yield nearly doubled to 64% of the maximum theoretical yield. Genomic analysis of ∆hydG revealed a mutation in adhE, resulting in a strain with both NADH- and NADPH-dependent alcohol dehydrogenase activities. While this same adhE mutation was found in ethanol-tolerant C. thermocellum strain E50C, ∆hydG and ∆hydG∆ech are not more ethanol tolerant than the wild type, illustrating the complicated interactions between redox balancing and ethanol tolerance in C. thermocellum.HThe dramatic increase in ethanol production suggests that targeting protein post-translational modification is a promising new approach for simultaneous inactivation of multiple enzymes.The online version of this article (doi:10.1186/s13068-015-0204-4) contains supplementary material, which is available to authorized users. A sustainable future will likely be dependent on large-scale production of fuels, chemicals, and products from renewable resources. One of the most promising approaches to this end is microbially catalyzed conversion of lignocellulosic biomass . AbundanClostridium thermocellum, a thermophilic, cellulolytic member of the Firmicutes phylum, is a potential platform to engineer into a CBP organism due to its native ability to efficiently solubilize cellulose and produce ethanol as a fermentation product. However, wild-type C. thermocellum is limited by its low ethanol yield and titer, producing acetate, lactate, H2, formate, and free amino acids as additional fermentation products  hydrogenases, which are thus far only found in methanogens, [FeFe] hydrogenases, and [NiFe] hydrogenases. C. thermocellum encodes three putative [FeFe] hydrogenases and one ferredoxin-dependent [NiFe] energy-converting hydrogenase (Ech)  (see below), produced 63% more ethanol than the parent strain  relative to the wild type (OD600 = 0.80), and the growth rate was also slower . The growth yield and rate of ΔhydGΔech were between these values, with a maximum OD600 = 0.76 and a growth rate of 0.22 h−1  was discovered in C. thermocellum ∆hydG, converting aspartate 494 into glycine (D494G). This exact same mutation was identified in the ethanol-tolerant C. thermocellum mutant E50C  hydrogenases), and Clostridium beijerinckii (six predicted [FeFe] hydrogenases)  hydrogenases alone increased ethanol yield substantially, the additional removal of the Ech hydrogenase was needed to further increase yield, clearly demonstrating the importance of Ech in this system. This result might be unexpected, because previous proteomics studies showed a lack of Ech protein in the proteome of related strain C. thermocellum ATCC27405 [C. thermocellum strains ATCC27405 and DSM1313, or differences in physiology due to the deletion of hydG in our study.Production of acetic acid plus COTCC27405 . The cauldh). In C. thermocellum Ldh is known to be allosterically activated by fructose-1,6-bisphosphate (FbP) [Thermoanaerobacter ethanolicus [Caldicellulosiruptor saccharolyticus [C. thermocellum Ldh, deletion of the hydrogenases would likely result in an accumulation of reduced ferredoxin via pyruvate: ferredoxin oxidoreductase (PFOR). This reduced ferredoxin plus NADH could then be used as substrates by NfnAB (Clo1313_1848 - 1849) to transfer electrons to NADPH [The hydrogenase mutants also stopped synthesizing lactate via lactate dehydrogenase (te (FbP) , presentanolicus  and inhiolyticus . While tto NADPH , potentiC. thermocellum revealed that it contained, among many other mutations, two point mutations in adhE, resulting in amino acid changes P704L and H734R. This mutation was reconstructed in an otherwise wild-type strain, resulting in altered alcohol dehydrogenase (ADH) enzyme cofactor specificity from NADH-dependent to NADPH-dependent and increased ethanol tolerance [C. thermocellum strains E50C and E50A also acquired independent mutations in adhE, resulting in amino acid changes to D494G and G553R, respectively [hydG mutant independently acquired the exact same D494G mutation as strain E50C. Unlike the previous P704L-H734R mutant, NADH-dependent activity was not eliminated. Instead, the hydG deletion strain contained high levels of both NADH- and NADPH-dependent ADH activities. Interestingly, ΔhydG was not ethanol tolerant, illustrating the complexity of the C. thermocellum ethanol tolerance phenotype. While it is possible that the D494G mutation does not contribute to the ethanol tolerance phenotype of strain E50C, a more likely explanation is that the additional redox imbalance(s) caused by deletion of hydG prevent the D494G mutation from rebalancing metabolism. Regardless, this gain of function mutation that allows use of either NADH or NADPH for reduction of acetaldehyde to ethanol will be useful for further metabolic engineering efforts by minimizing NADH/NADPH redox imbalances. Indeed, C. thermocellum ΔhydGΔech, with its high ethanol yield, near wild-type growth rate, and ADH capable of utilizing both NADH and NADPH, is now an ideal platform for further engineering of C. thermocellum for production of cellulosic fuels and chemicals.Previous characterization of an ethanol-tolerant mutant of olerance . This suectively . HoweverClostridium thermocellum degrades cellulose well, efforts to engineer this strain are laborious and time-consuming. In addition to making ethanol, C. thermocellum converts cellulosic to lactate, formate, acetate, H2, ethanol, amino acids, and other products that must be eliminated to achieve high yield. Here, we developed a methodology to simultaneously inactivate multiple enzymes by targeting a common post-translational modification system, simplifying strain construction by minimizing the necessary genetic modifications. We eliminated H2 production and successfully redirected electron and carbon flux towards desired product. This strain will serve as a platform for further engineering to economically produce fuels and chemicals from lignocellulosic biomass.While Escherichia coli TOP10 and BL21 were grown in LB medium supplemented with 12 μg ml−1 chloramphenicol when appropriate. Clostridium thermocellum DSM1313 and mutant strains were grown in a modified DSM122 medium similar to that described by Tripathi et al. [−1 thiamphenicol , 50 μg ml−1 5-fluoro-2′-deoxyuridine , and 500 μg ml−1 8-azahypoxanthine  as needed and MTC minimal medium [−1): 3 g sodium citrate tribasic dehydrate, 1.3 g ammonium sulfate, 1.43 g potassium phosphate monobasic, 1.8 g potassium phosphate dibasic trihydrate, 0.5 g cysteine-HCl, 10.5 g 3-morpholino-propane-1-sulfonic acid (MOPS), 6 g glycerol-2-phosphate disodium, 0.41 g sodium acetate, 5 g cellobiose, 4.5 g yeast extract, 0.13 g calcium chloride dehydrate, 2.6 g magnesium chloride hexahydrate, 0.0011 g ferrous sulfate heptahydrate, and 0.0001 g resazurin, adjusted to pH 7.0. The minimal medium consisted of (L−1): 2 g sodium citrate dehydrate, 1.25 g citric acid monohydrate, 1 g sodium sulfate, 1 g potassium phosphate dibasic trihydrate, 2.5 g sodium bicarbonate, 1.5 g ammonium chloride, 2 g urea, 1 g magnesium chloride hexahydrate, 0.2 g calcium chloride dehydrate, 0.1 g ferrous chloride tetrahydrate, 1 g L-cysteine hydrochloride monohydrate, 5 g cellobiose, 0.001 g resazurin, 5 g MOPS, 20 mg pyridoxamine dihydrochloride, 1 mg riboflavin, 1 mg nicotinamide, 0.5 mg DL-thioctic acid, 4 mg 4-amino benzoic acid, 4 mg D-biotin, 0.025 mg folic acid, 2 mg cyanocobalamin, 4 mg thiamine hydrochloride, 0.5 mg MnCl2.4H2O, 0.5 mg CoCl2.6H2O, 0.2 mg ZnSO4.7H2O, 0.05 mg CuSO4.5H2O, 0.05 mg HBO3, 0.05 mg Na2MoO4.2H2O, and 0.05 mg NiCl2.6H2O.Standard molecular methods were used for plasmid cloning ,31. Eschi et al.  supplemel medium  preparedl medium . The modC. thermocellum ∆hpt, which is a derivative of strain DSM1313 and is herein referred to as wild type. Plasmid pAMG278 , resulting in strain C. thermocellum ∆hpt ∆hydG, hereafter referred to as C. thermocellum ∆hydG. In the C. thermocellum ∆hydG background, plasmid pAMG275  and echABCDEF (Clo1313_0570 to 0575), resulting in strain C. thermocellum ∆hpt ∆hydG ∆hyp-ech, hereafter referred to as C. thermocellum ∆hydG∆ech. All genetic manipulation was carried out using the modified DSM122 medium. Briefly, the plasmids were each isolated from a dcm− strain of E. coli  [C. thermocellum ∆hpt via electroporation, and plated in medium supplemented with TM. Colonies were picked into liquid medium with TM, grown at 51°C, and plated in medium supplemented with TM and FUdR. Colonies were single colony purified and picked into liquid medium with TM. These cultures were then subcultured without TM and plated with 8AZH to select for the final deletion, followed by single colony purification to obtain pure cultures [Strains were constructed as previously described -10 in st−1 of cellobiose under a N2 headspace sealed with butyl rubber stoppers. The tubes were inoculated with 0.5% v/v culture and incubated at 55°C. The fermentation products were determined after 53 hours of growth. The final cellobiose concentration was usually less than 0.5 mM, suggesting that fermentation activity was complete. Fermentations were performed at least two times with three independent biological replicates each. Growth rate was calculated using the change in absorbance during mid-log phase, from an OD600 of approximately 0.1 to 0.3.The inoculum for batch fermentation was prepared by growing the mutants in MTC medium overnight at 55°C in an anaerobic chamber . The fermentation was carbon limited and carried out in 27-ml Balch tubes with 10 ml of MTC media containing 5 g LEthanol tolerance was tested in Balch tubes containing minimal medium with 0, 1, 2, 3, 4, and 5% v/v added ethanol, inoculated with 0.5% of overnight grown culture, and incubated at 55°C. Growth was monitored by measuring the optical density at 600 nm on a Unico 1200 spectrophotometer . Growth experiments were performed at least two times with three independent biological replicates each.2 was measured using an Agilent Technologies 6850 Series II Gas Chromatograph  using a thermal conductivity detector at 190°C with a N2 reference flow and a Carboxen 1010 PLOT  column.Fermentation products, including ethanol, acetate, lactate, and formate, were analyzed on a Breeze 2 High Performance Liquid Chromatograph system using an Aminex-HPX-87H column with a 5 mM sulfuric acid mobile phase. HC. thermocellum cells were grown anaerobically to OD600 = 0.3 and harvested by centrifugation at 2800 × g for 30 minutes at 4°C and stored at −80°C. Before the assay, the pellet was resuspended anaerobically in 0.5 ml Assay Buffer . The cells were lysed with 2 μl Ready-Lyse Lysozyme from Epicentre Technologies , and 1 μl DNase I from New England Biolabs  was added to reduce viscosity. The resulting solution was centrifuged at 17,000 × g for 10 minutes at room temperature, and the supernatant was used as cell-free extracts for enzyme assays. For the ADH  reactions, the anaerobic reaction mixture contained 0.25 mM NAD(P)H, 18 mM acetaldehyde, 1 to 10 μl cell-free extract, and 800 μl of Assay Buffer. Decrease in absorbance at 340 nm caused by NAD(P)H oxidation was monitored by an Agilent 8453 UV–vis spectrophotometer with Peltier controlled heating set at 55°C inside an anaerobic chamber. Protein concentration was determined by using the Bradford method [50-ml cultures of d method .ΔhydG and ΔhydGΔech samples. As the ninhydrin method detects primary and secondary amines, the interfering peak presumably represents an as yet unidentified amino compound.Amino acid analysis was carried out by AminoAcids.com . Samples were prepared for analysis by appropriate dilution with a deproteinizing solution , L-2-amino-3-guanidinopropionic acid hydrochloride, and eluent lithium buffer. The mixture was vortexed, micro-centrifuged, and filtered through a 0.2-μm filter. Amino acid analysis was performed on a Hitachi Model L-8900 HPLC with 10-cm cation exchange columns, four sequential lithium-based eluents (Hitachi and Pickering Laboratories), and lithium hydroxide for column regeneration. Absorbance was measured at 440 and 570 nm following post-column color development by ninhydrin reagent at 135°C. Data are an average of three independent biological replicates. AminoAcids.com reported that an unknown peak overlapped with the asparagine peak, preventing quantification in both the adhE region was PCR amplified as 982-bp product using primers XD520 and XD521 (Additional file The


Unfortunately, the original version of this article  containehttp://www.saglik.gov.tr/TR/dosya/1-97020/h/saglik-istatistik-yilligi-2013.pdf)” should have read “The Turkish neonatal mortality rate is 0.4 % according to the Ministry of Health of Turkey (http://www.saglik.gov.tr/TR/dosya/1-97020/h/saglik-istatistik-yilligi-2013.pdf)”.The sentence “The Turkish neonatal mortality rate is 0.04 % according to World Health Organization Reports and the Ministry of Health of Turkey (The corrected sentence has been included in full in this erratum.


The spin diffusion and drift at different excitation wavelengths and different temperatures have been studied in undoped InGaAs/AlGaAs multiple quantum well (MQW). The spin polarization was created by optical spin orientation using circularly polarized light, and the reciprocal spin Hall effect was employed to measure the spin polarization current. We measured the ratio of the spin diffusion coefficient to the mobility of spin-polarized carriers. From the wavelength dependence of the ratio, we found that the spin diffusion and drift of holes became as important as electrons in this undoped MQW, and the ratio for light holes was much smaller than that for heavy holes at room temperature. From the temperature dependence of the ratio, the correction factors for the common Einstein relationship for spin-polarized electrons and heavy holes were firstly obtained to be 93 and 286, respectively. Much attention has been given to semiconductor spintronics for the promising applications in information technology , 2. One Neff) flowing through the region of x=0 has a Gaussian distribution [g,τs,c, x, and σ are the generation rate of spin-polarized carriers, the spin relaxation time, an arbitrary constant, the spot coordinate along the x axis, and the standard deviation of the Gaussian distribution, respectively. On the one hand, under normal incidence, the gradient of the spin density will induce a diffused spin polarization current (SPC): Ds is the spin diffusion coefficient of the photoinduced carriers, nz(r) the spin density along the z direction, and r the radial direction in the x−y plane. According to the RSHE [e, γ are the elementary charge, the spin-orbit interaction coefficient based on RSHE, respectively. As a result, a swirling electric current will be induced around the light spot, which will further generate an observed ACPGE current jACPGE/e=−γDs∇Neff  SI-GaAs substrate, followed by ten periods of 100-Å In 0.15Ga0.85As/ 100-Å Al 0.3Ga0.7As QWs. Then, a 500 Å Al 0.3Ga0.7As layer and 100 Å GaAs cap layer are deposited. The sample is cleaved into a narrow strip along the GaAs [1y and x direction, respectively, by indium deposition and annealed at about 420 °C in nitrogen atmosphere.The sample studied here is an undoped In λ/4, to yield a modulated circularly polarized light with a fixed modulating frequency at 50 KHz. By using an optical chopper with the rotation frequency of 223 Hz, the spectra of common photoinduced currents (IPC) are also measured for comparison, which show clearly the energy positions corresponding to 1hh-1e  and 1lh-1e  transitions , of which the peak retardation is set to be In the experiment, the total spin-related currents at electric fields of 0, + 4, and −4 V/cm are measured as a function of the spot location at three different wavelengths. As shown in Fig. 222Next, we extracted the extreme values of ACPGE and AHE currents from Fig. 233332Neff=τeN0 for electrons and Neff=τhhN0 for heavy holes, where g0 is the generation rate for 1hh-1e transition), τe is the spin relaxation time of electrons, and τhh is the spin relaxation time of heavy holes. For 1lh-1e transition, Neff can be simplified as Neff=τeN1 for electrons and Neff=τlhN1 for light holes, where g1 is the generation rate for 1lh-1e transition), and τlh is the spin relaxation time of light holes. So, the total spin-polarized electric current corresponding to 1hh-1e and 1lh-1e can be expressed as From the fitted data in Fig. and respectively.Thus, the ratios of the spin diffusion coefficient to the mobility of the photoinduced spin-polarized carriers can be defined as for 1hh-1e transition and p-type quantum wells has been demonstrated by several authors [p-type quantum wells. Thus, the reciprocal spin Hall coefficient  of light holes (γlh) is probably far larger than that of electrons (γe). Assuming μlh is comparable with μe, while Dlh is far smaller than De, and τe is at the same order with τlh, only then can we get μlhγlhτlh≫μeγeτe. As a result, for 1lh-1e transition. The Rashba effect which is stronger for the first valence band than that for the first conduction band and is stronger for the first light hole subband than that for the first heavy hole subband in  authors –18. Suppsee Fig. . Around T−2, i.e., τe=AeT−2, where Ae is a constant, which is quiet different from the assumption of a narrow quantum wells in [T−1, i.e., τhh=AhhT−1, where Ahh is a constant [χe and χhh are correction factors for the common Einstein relationship for the spin-polarized electrons and holes, respectively. At high temperatures (≥80 K), the mobility of GaAs/AlGaAs two-dimensional electron and hole gases are respectively proportional to T−2.4 and T−2 given by former studies [T (there were few relevant reports on the temperature dependence of spin mobility), i.e., λ0 is a constant. Then, Eq. At last, we studied the temperature dependence of ACPGE and AHE currents. From Fig. 666entclass1pt{minimawells in , 19; andconstant . Assumin studies , 22. Forγe and γhh, one can use Eq. χe=93 and χhh=286, respectively. The factor of spin-polarized heavy holes is almost 3 times larger than that of spin-polarized electrons. According to the Einstein relationship for electron transport, the value of Ignoring the temperature dependence of ervative . We beliIn conclusion, the spin diffusion and drift at different wavelengths and different temperatures have been studied in undoped InGaAs/AlGaAs MQW. By using the AHE and ACPGE which are all derived from RSHE, we obtained the ratio between the spin diffusion coefficient and the mobility of spin-polarized carriers. From the wavelength dependence of the ratio, we found that the spin diffusion and drift of holes became as important as electrons in this undoped MQW, and the ratio for light holes was much smaller than that for heavy holes at room temperature. From the temperature dependence of the ratio corresponding to the 1hh-1e transition, we believed the ratio is contributed by the combined effect of spin-polarized electrons and spin-polarized heavy holes. The correction factors for the common Einstein relationship for spin-polarized electrons and heavy holes are firstly obtained to be 93 and 286, respectively. It is worth noting that the AHE and ACPGE measurements used in this study are conducted under ambient conditions with a simple setup and operation, which provides a good method for the study of spin-related diffusion and drift.


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We also expected the settled cyanobacteria with their associated microheterotrophic community and relatively high nitrogen content to increase the isotopic niche area, trophic diversity and dietary divergence between individuals (estimated as the nearest neighbour distance) in the benthic fauna after the bloom. The three surface-feeding species  showed significantly lower δ15N values after the bloom, while the sub-surface feeder Pontoporeia femorata did not. The effect of the bloom on isotopic niche varied greatly between stations; populations which increased niche area after the bloom had better body condition than populations with reduced niche, regardless of species. Thus, cyanobacterial nitrogen is efficiently integrated into the benthic food webs in the Baltic, with likely consequences for their functioning, secondary production, transfer efficiency, trophic interactions, and intra- and interspecific competition.Benthic communities below the photic zone depend for food on allochthonous organic matter derived from seasonal phytoplankton blooms. In the Baltic Sea, the spring diatom bloom is considered the most important input of organic matter, whereas the contribution of the summer bloom dominated by diazotrophic cyanobacteria is less understood. The possible increase in cyanobacteria blooms as a consequence of eutrophication and climate change calls for evaluation of cyanobacteria effects on benthic community functioning and productivity. Here, we examine utilization of cyanobacterial nitrogen by deposit-feeding benthic macrofauna following a cyanobacteria bloom at three stations during two consecutive years and link these changes to isotopic niche and variations in body condition (assayed as C:N ratio) of the animals. Since nitrogen-fixing cyanobacteria have δ Temporal changes in trophic interactions can be considerable, particularly in temperate aquatic environments, where primary production is strongly pulsed. In these systems, growth and population dynamics of deposit-feeders is tightly coupled to sedimentation of algal blooms Both phytoplankton and benthic communities in the Baltic Sea have recently undergone large-scale changes due to a combination of eutrophication, biological invasions, and climate change. The biomass of the spring bloom, which is the most important input of high quality organic matter to the sub-thermocline soft-bottom community 15N to 14N (expressed as δ15N) exhibit stepwise enrichment with each trophic transfer, whereas ratios of carbon isotopes (δ13C) change little with trophic transfer and can be used to determine ultimate sources of the dietary carbon. The isotopic composition of primary producers may vary over a season and influence the isotopic composition of primary consumers and predators 15N signal of about −2‰ 13C usually becomes more enriched later in the season, due to discrimination against the heavy isotope 13C than other phytoplankton 13C-signal has therefore been proposed as a tracer of cyanobacterial input in benthos 13C and carbon processing by microbial communities Stable isotopes are commonly used as tracers when studying trophic relationships. They provide time-integrated information on diet and habitat use 13C- δ15N bi-plot space; for example, the distance between the most enriched and most depleted individual (or species) in δ13C is the niche width of the group (population or community) with regard to the carbon sources. Recently, Tecchio et al. Stable isotopes can also be used to quantify subtle changes in community- or population-level trophic structure This study uses stable carbon and nitrogen isotope data from macrobenthic communities sampled at several stations during the summer season to test four interrelated hypotheses addressing incorporation of cyanobacteria-derived nitrogen and carbon in the benthic food web and the importance of cyanobacterial blooms for feeding and nutritional condition of the deposit-feeders. The tested hypotheses were:15N values will become more depleted after the cyanobacteria bloom, reflecting uptake of diazotrophic nitrogen. In addition, their δ13C will be also more depleted after the bloom, since cyanobacteria tend to have more depleted δ13C than other phytoplankton.In deposit-feeders, δAfter the bloom, the isotope niche area will increase in deposit-feeders because microheterotrophic communities supported by the bloom will become more abundant and diverse, which will increase and diversify the food available to consumers.Increased isotope niche-area will be positively related to the body condition of the animals, reflecting complementarity in the food sources, higher resource availability and lower intra- and interspecific competition.A decreased niche overlap among the species will be observed after the bloom, due to resource diversification and differential utilization of the bloom material by co-occurring competitors. This, together with the species-specific niche increase will widen the isotope niche of the total community.Our results show that cyanobacterial nitrogen is utilized by deposit-feeders, and indicates that the settling bloom material induces trophic changes in the community.www.smhi.se) and stn Mörkö right by the phytoplankton stn H3 , 14 km from stn B1. No specific permission is required for sampling invertebrates in the Baltic Sea and the field studies did not involve any endangered or protected species. We sampled sediment and deposit-feeding macrofauna at three coastal stations (stn) in the north-western Baltic proper ; HåldämmMacoma balthica and the non-indigenous spionid polychaete Marenzelleria cf. arctiaMonoporeia affinis was less abundant and absent from stn Mörkö in May and July 2009. The amphipod Pontoporeia femorata was never found at stn Uttervik and absent from stn Håldämman in May and July 2009. On each sampling event ∼10 individuals of each species of approximately 2 mg dry weight  were collected for isotope analyses. Due to the unbalanced sampling design and the different timing of the bloom between years and stations, we pooled the two pre-bloom sampling occasions and those after the bloom (in 2009) in the statistical analyses . The stable isotope ratio, denoted by δ, is defined as the deviation in ‰ from the international reference standard . Higher δ indicates a higher proportion of the heavy isotope. Samples were run in continuous flow with a standard deviation of <0.2‰ among replicate standard samples both for C and N. The δ13C values in animals were corrected for lipid content according to Post et al. Animals and bulk sediment  from each sampling occasion were oven-dried (60°C), packed individually into tin capsules and analysed for elemental and stable isotope content (carbon and nitrogen) at the UC Davis Stable Isotope Facility, USA. The C and N isotope ratios are expressed in the ‰ notation, using the equation:where R is the ratio between the heavy and light isotopes , with higher values reflecting higher fat content For each species, pre-bloom and post-bloom values for individual body weight and C:N ratio were compared using paired t-tests with station as grouping factor. Pearson correlations and t-tests were performed in STATISTICA 12 (StatSoft).15N values were observed at stn Mörkö, due to the nearby sewage treatment plant 15N, δ13C, C:N ratio, weight) were normalized to z-scores  for each station and year to remove the baseline differences in population means between samples collected in different years and at different stations, and to bring the distributions closer to normal.Markedly higher δ15N in the test populations was tested separately for each species in generalized linear models using the GLZ module in STATISTICA 12 (Statsoft) with a log-link error structure and AIC (Akaike information criterion) as the selection criteria. The full model included all categorical variables; bloom , station (3 levels) and year (2 levels), and the three continuous variables: body weight, C:N ratio and δ13C values. Body weight and C:N ratio are indicative of ontogeny and body condition, respectively, and δ13C correlated strongly to δ15N for most species, as frequently observed 13C was tested using the same categorical factors as for δ15N, but with the body weight as the single continuous variable. The response variables, δ15N and δ13C, were Box-Cox transformed prior to analyses. Goodness of fit and residuals were examined to confirm that the selected error distribution and link function were appropriate. Two outliers for M. affinis and M. arctia, respectively, were identified using Grubbs test and removed prior to the analyses. This removal did not affect the analyses outcome but improved normality of residuals.The effect of the bloom on δ15N range (dN) indicates greater trophic diversity in terms of the trophic position and potential for omnivory, whereas a large δ13C range (dC) indicates multiple carbon resources  and potential for niche diversification. Mean nearest neighbour distance (MNND) describes how individuals are distributed relative to one another within the dietary niche space of the population or the community, with higher values indicating greater dietary divergence. The standard ellipse area, SEA or SEAc when corrected for small sample size Isotope niche metrics These niche metrics were calculated both for the populations in question  and for the entire community at the same spatio-temporal resolution. Bloom effects on the niche metrics were tested using paired t-tests, with stations as a grouping factor, or Wilcoxon matched pair tests when assumptions of normality were not met. The relationship between the overlap area between the species sharing the same niche and the community niche area was tested using Spearman rank correlation.Bloom intensity was calculated as the area under the curve for the total cyanobacteria biomass from the mid May to the end of September using linear spline interpolation . The relative change in SEAc following the bloom event was used as a dependent variable in the linear regression with bloom intensity and species as independent variables. We used SEAc because it is less affected by outliers and small sample size Pontoporeia femorata was not included in these analyses since we found no evidence for uptake of cyanobacterial nitrogen by this species. To examine whether there is an exploitative competition between M. balthica and M. affinis (i.e. the species that overlapped most often), we tested if their SEAc values change in concert using Pearson product moment correlation. Residuals were examined to confirm normality prior to the analyses.The mean body C:N ratio was calculated for all species, stations, and years, before and after the bloom. The relative change in the C:N ratio after the bloom event was also used as a dependent variable in the linear regression with bloom intensity and species as the independent variables. Finally, the relative change in C:N ratio after the bloom event was used as a dependent variable in the linear regression with relative change in SEAc (post-bloom to pre-bloom ratio) and species as independent variables. The significance of the categorical factor was tested using a homogeneity-of-slopes model. The amphipod Aphanizomenon constituted 76 and 78% of diazotrophic biomass at stn H3 and stn B1, in 2010 the contribution was higher; 82 (stn H3) and 85% (stn B1). Nodularia spumigena was the second most abundant species at stn B1 , whereas Anabaena spp. contributed more (16 and 13%) at stn H3. At both stations in 2007–2009, seston δ15N mirrored nitrogen fixation, declining from a spring bloom maximum to a minimum (2–4‰) two weeks after the peak of the cyanobacteria bloom (coinciding with the third sampling event in 2009), and then increasing again At stn B1 in 2009, the bloom started in late June, peaking on June 30, while in 2010, the bloom peaked only in mid-August. The bloom at stn H3 occurred in mid-July in both years. Bloom intensity was highest at stn B1 in 2009, with 30–35% lower values observed at stn H3 (both years) and stn B in 2010. In 2009, 15N, C% and N% were lower after the bloom only at stn Uttervik, but in 2010 they were lower at all stations.Elemental content in the sediment varied from ∼3.5% C and 0.5% N at stn Mörkö to ∼5.5% C and 0.8% N at stn Uttervik, with intermediate values at stn Håldämman . In 2009M. affinis, both individual weight and lower C:N ratio were lower after the bloom . In the other species, no change in the individual weight from the pre-bloom to the post-bloom were observed , but their C:N ratios declined . The small sample size for P. femorata precluded meaningful comparisons. Nitrogen content (%) increased after the bloom for the surface-feeding species , with no change in C% . The carbon and nitrogen content for each species and period are shown in Macrofaunal body condition, indicated by the C:N ratio, changed over the season . In M. aP. femorata, the δ15N values became significantly depleted after the bloom . The post-bloom to pre-bloom SEAc ratio explained 25% of the variation in the post- to pre-bloom C:N ratio  - when pooling the three species that were found to respond to the cyanobacteria bloom . Bloom intensity explained 32% of the variation in C:N ratio change following the bloom , with no significant species effect . A significant negative correlation in SEAc change after the bloom was observed between ia bloom . When M.M. arctia was distinct from the other species . As such, post-bloom overlaps were recorded at stations that showed a decreasing or unchanged SEAc after the bloom, but not where the SEAc increased  without significant loss in C%  settling and incorporation of diazotrophically fixed nitrogen, which results in depleted δ15N values, and (2) trophic turnover in sediment microbial loops, which results in enriched δ15N values. Despite the latter process, the δ15N values in the key macrofauna benthic species became significantly depleted after the bloom.Variations in isotopic fractionation can also contribute to the seasonal changes in isotopic signatures of consumers g summer . This sh15N values depleted by several ‰ in late summer at all trophic levels studied; macroalgae, macrobenthos and fish. Similarly, Lesutiene et al. 15N after the cyanobacteria bloom throughout the shallow Curonian Lagoon food web, and estimated that diazotrophically fixed nitrogen may support up to 80% of secondary production during the bloom period. Interestingly, our most depleted values were also found at the shallowest station, indicating a stronger influence of cyanobacteria in these habitats. In shallow waters, the animals have generally higher growth rates  had a better body condition than populations with unchanged or decreasing niches, suggesting that supplemental feeding may convey fitness advantages, which is rarely documented Click here for additional data file.


Grain starch phosphorylation and amylose content affect germination and seedling establishment through the combination of direct effects on altered starch granule and molecular structure and indirect effects on amylase activities. per se, is important for germination and seedling establishment. For that purpose, wild-type (WT), and specifically engineered degradable hyperphosphorylated (HP) starch and more resistant amylose-only (AO) starch barley lines were used. The transgenics showed no severe phenotypes and the WT and HP lines degraded the starch similarly, having 30% residual starch after 12 d of germination. However, the AO line showed significant resistance to degradation, having 57% residual starch. Interestingly, protein and β-glucan (BG) degradation was stimulated for both HP and AO lines as compared with the WT. At late seedling establishment stages, specific sugars were rapidly consumed in the AO line. α-Amylase activity was distinctly suppressed in both the HP and the AO lines. Pre-germination β-amylase deposition was low in the AO grains and β-amylase was generally suppressed in both HP and AO lines throughout germination. As further supported by scanning electron microscopy and histochemical analyses on grain and seedlings, it was concluded that inadequate starch granule deposition in combination with the suppressed hydrolase activity leads to temporal and compensating re-direction of starch, sugar, and protein catabolism important to maintain metabolic dynamics during grain germination and seedling establishment.Cereal grain germination is central for plant early development, and efficient germination has a major role in crop propagation and malting. Endosperm starch is the prime energy reserve in germination and seedling establishment. In this study, it was hypothesized that optimized starch granule structure, and not only the endosperm starch content  Germination of grain commences with the uptake of water. Once germination is initiated, the predominant endosperm reserves, starch, cell wall, and storage proteins, are mobilized by the action of hydrolytic enzymes which are synthesized in the aleurone layer and in the scutellum and secreted into the starchy endosperm of germinating barley grains. Starch is the main storage compound synthesized in barley endosperm and serves as the primary source of carbohydrate during germination and seedling growth. α-Amylase enzyme plays a primary role in native starch granule degradation, and its expression is controlled by both gibberellin (GA) and sugar demand/starvation. Sugar or carbon starvation activates the α-amylase promoter . GlucoseNormal starch granules consist of ~25% mostly linear α-1,4-linked amylose and 75% α-1,4 backbone and α-1,6 branched amylopectin deposited as alternating amorphous and crystalline layers. Parallel double helices in the amylopectin align to organize the amylopectin in two different types of crystalline polymorphs: the A-type crystalline polymorph consisting of densely packed double-helical segments typical of cereal endosperm starches; and the B-type crystalline polymorph, typically found in tuberous and leaf starches, and containing a substantial amount of structured water. A third type, the C-type polymorph, is commonly found in, for example, legume seeds, and is a mixture of the A-type and the B-type polymorphs. In high amylose varieties, a single helical polymorph often complexed with lipids is found, termed the Vh-type polymorph . The orgper se is not important for degradability and, for lotus C-type starch granules, α-amylase did not selectively digest A- or B-type polymorphs in this starch  providing weak spots in the starch granule to support cellular starch degradation by local hydration and amorphization . These pSolanum tuberosum GWD overexpressor, 2O) for 4, 8, and 12 d. It is important to note that all of the three types of grains that were compared originate from the same inbred cultivar. The germination was conducted in the dark in a CMP 6010 growth cabinet  at a constant temperature of 23 °C. After germination, the emerging parts, the coleoptiles and radicles, were excised from the grains. Each part was dried for 3 d at 95 °C. The dry weight was determined on a standard laboratory scale to calculate the amount of biomass that has been remobilized from each grain.Grains of the wild type (WT) barley cultivar Golden promise, the hyperphosphorylated  for 5min and the pellet was dried at 40 °C. The pellet was re-suspended using solutions of 2M KOH and 1.2M sodium acetate buffer (pH 3.8) according to the protocol described by the manufacturer (AOAC Method 996.11/AACC Method 76.13); the reaction volumes were scaled down according to the smaller sample weight from Megazyme International Ltd  following the protocol recommended by the manufacturer for samples containing resistant starch.Barley pre-germinated dry grain and seedlings germinated for 4, 8, and 12 d were used for analysis. Coleoptiles and radicles (if present) were cut off and residual grains were freeze-dried. Freeze-dried residual grain samples were ground in a tissue lyser  operating at 30 Hz for 15 s and passed through a 0.5mm screen. Flour samples of 10mg each were used for analysis. Soluble sugars were removed by washing the flour in 80% (v/v) ethanol, in a boiling water bath for 30min and once for 10min, and in 96% ethanol at room temperature for a final wash. The supernatant was removed at the end of every step after centrifugation at 9500 ® PA1 column  corresponding to 1.5 KNU (Kilo Novo Units), followed by incubation at 60 °C overnight with 10 μl of amyloglucosidase  corresponding to 3.0 AGU (Amyloglucosidase Units). The whole reaction was conducted by continuous shaking at 900rpm in a thermomixer . Samples were prepared for chromatography by centrifugation and filtration, and the supernatant was analysed with a high performance anion exchange system  with an AS50 autosampler, GS50 gradient pump, and ED50 PAD system equipped with a CarboPacTotal nitrogen was determined on finely ground barley pre-germinated dry grain and seedlings using an ANCA-SL/GSL elemental analyser , the technique that uses the Dumas procedure for the preparation system, and a stable isotope mass spectrometer. Nitrogen values were multiplied by a factor of 5.45 (g and the supernatant collected. The samples were then re-extracted in 80% (v/v) ethanol for 30min, centrifuged, and the supernatant was collected and pooled with the one from the previous extraction, and dried in a spin-vac. The samples were then reconstituted in water to the correct dilutions for analysis. Soluble sugars were identified and quantified using a gradient high performance anion exchange system (Thermo Fisher Scientific Inc.) with an AS50 autosampler, GS50 gradient pump, and ED50 PAD system equipped with a CarboPac® PA1 column.Barley pre-germinated dry grain and seedlings germinated for 1, 2, 3, 4, 8, and 12 d were used for analysis. Coleoptiles and radicles (if present) were cut off and residual grains were freeze-dried. Freeze-dried residual grain samples were ground in a tissue lyser  operating at 30 Hz for 15 s and passed through a 0.5mm screen. A 10mg aliquot of each flour sample was extracted in 80% (v/v) ethanol at 80 °C for 2h in a thermomixer. The samples were centrifuged for 5min at 13 000 g (Heraeus biofuge pico) for 5min and the pellet was dried at 40 °C. Complete hydrolysis of BGs was conducted using lichenase and β-glucosidase from the mixed-linkage BG kit  following the method provided by the manufacturer . Samples treated in that way were centrifuged, filtered, and the quantification of the resulting glucose units was performed by a high performance anion exchange system (Thermo Fisher Scientific Inc.) with an AS50 autosampler, GS50 gradient pump, and ED50 PAD system equipped with a CarboPac® PA1 column using the run program described before  ethanol, in a boiling water bath for 30min and once for 10min, and 96% ethanol at room temperature for a final wash. The supernatant was removed at the end of every step after centrifugation at 16 000 The α- and β-amylase activities were measured using enzymatic assay kits for these individual enzymes purchased from Megazyme International Ltd. The enzyme assays were performed on 5mg of flour samples and the reaction volumes were scaled down according to the smaller sample weight. For the β-amylase activity, cysteine was added in the extraction buffer to extract the insoluble β-amylase to provide ‘total’ β-amylase activity (as per Megazyme’s protocol).g . The precipitate was suspended in 200 μl of water, layered over 1ml of 80% (w/v) caesium chloride solution, and centrifuged at 13 000 g  for 15min. The starch pellet was washed twice with water, followed by acetone washing and overnight air drying.Starch was extracted and purified from 0.5g of malt flour using a modified version of the protocol described by The amylose content was determined by iodine colorimetry (Granule morphology during germination was studied by scanning electron microscopy (SEM) as described  glutaraldehyde, 4% (w/v) paraformaldehyde, 0.1M sodium cacodylate buffer] including a 1h vacuum treatment, washed in cacodylate buffer at pH 7.3, and post-fixed in 1% (w/v) osmium tetroxide (with 0.1M cacodylate buffer) for 8h at 4 °C. After washing in buffer and water, the samples were dehydrated in a graded acetone series, infiltrated with three different ratios of Spurr resin to acetone, and embedded in Spurr resin within flat moulds. The resin was polymerized in an oven at 60 °C for 8h. Semi-thin section of 2 μm were cut with a histo-diamond knife on a Reichert–Jung supernova ultramicrotome and stained with periodic acid–Schiff’s (PAS) for insoluble carbohydrates containing 1,2-glycol groups and with 1% (w/v) amido black in 7% (w/v) acetic acid for proteins. Sections were viewed in immersion oil in a Nikon Eclipse 80i light and fluorescence microscope. Contrast adjustments were carried out to improve clarity of images but did not alter the overall appearance. Final image processing, cropping, and mounting of the images were done with Adobe Photoshop CS2 and Illustrator CS2.P<0.05/0.01/0.001) compared with the WT.The statistical significance tests were performed using the one-way analysis of variance (ANOVA) statistical tool embedded in SigmaPlot 12.0. The term significant is used in the text only when the change has been confirmed to be significant , where the grain is the only carbon source. During a period of 12 d after imbibition, which was divided into four stages, germination (day 1), early stage of seedling establishment (day 2 to day 4), mid stage of seedling establishment (day 4 to day 8), and late stage of seedling establishment (day 8 to day 12), the dry mass of the remaining grain and the mass of the emerging parts—coleoptile and radicle—to which some of the original biomass had been remobilized, was measured. Less dry mass was remobilized from the grains containing AO starch, which represents a more resistant starch type . During germination on day 1 and the early stage of seedling establishment, starch content in the WT and the HP line showed almost no reduction, whereas the AO line showed a slow but considerable decrease in starch content. Starch content started to decline markedly at the end of the early stage of seedling establishment in the WT and HP line, but the AO line showed a slower, linear reduction in starch content, demonstrating loss of normal dynamics of starch mobilization. Starch mobilization from HP grain was slower than from the WT, but followed a similar sigmoidal pattern in the time course, ending with almost 30% of residual starch, while the AO line showed persistent reduction, with almost 57% of residual starch. Thus, the AO line efficiently degraded starch initially but both the transgenic lines showed restricted starch degradation, occurring at two different phases: in the HP line during the early stage of seedling establishment and in the AO line at mid and late stages of seedling establishment.The initial contents of starch, protein, and BG in the dry grain are given in Supplementary Table S1 available at JXB online). Sugars resulting from cell wall dissolution contribute energy for the young seedling . The AO line showed a linear and gradual decrease in protein levels, with a higher reduction of 52% by the end of the late seedling establishment stage. Hence, the AO and HP lines both showed compensation of restricted carbon availability with protein mobilization.To investigate further if the differential rates of starch mobilization in the lines re-directed the metabolism to be compensated by another carbon source, the third possible source of carbon, namely, protein, was next analysed. Protein degradation provides the major direct source of amino acids to be directed to translation of vital hydrolytic enzymes for the growing embryo . ProteinSupplementary Table S2 available at JXB online). Total soluble sugar was determined as the sum of the soluble sugar values. In order to obtain information on early metabolism during germination and throughout seedling establishment, the sugars that are either present in the pre-germinated dry grain or resulting from starch and cell wall breakdown were measured in dry grains at 1, 2, 3, 4, 8, and 12 d of germination and seedling establishment. The most striking differential effect in sugar dynamics for the three lines was the general decline in total sugars measured in the AO line as compared with the WT and HP line at mid and late stages of seedling establishment. During germination and the early stage of seedling establishment up to day 3, significant differences were observed for sucrose, fructose, and ribose, where the AO grain had higher levels of sucrose and fructose compared with the WT and HP grain; however, their levels then declined in the AO line in the mid and late seedling establishment stages. Substantial differences were also observed in glucose levels in mid and late stages of seedling establishment, where HP grain showed higher levels than the WT at day 8 (Supplementary Table S2 available at JXB online). There were some differences in the levels of ribose especially in the seedling establishment stage among the WT, HP, and AO lines, indicating differences in nucleotide metabolism. There was no difference in arabinose levels between the WT, HP, and AO lines, indicating no differences in cell wall dissolution and also supporting the observation with the BG levels. Trehalose, which helps to stabilize proteins activated during early germination, showed no differences during germination and the early stages of seedling establishment. In general, all the sugars decreased rapidly at the late stage of seedling establishment, except fructose, ribose, and trehalose. During the late stage of seedling establishment, fructose, ribose, and trehalose showed a gradual increase in the WT and the HP line but remained constant in the AO line.Given the changes in the contents of starch and protein as carbon sources, the levels of soluble sugars representing glucose, fructose, sucrose, arabinose, trehalose, ribose, and maltose were next evaluated  which was carried out as described in the Supplementary protocol for Supplementary Fig. S3 (available at JXB online). A distinct tailing was observed on the TLC plate in the day 8 and day 12 samples of AO, which was not present in the WT and the HP lines, supporting the presence of short chain glucans in the residual AO grain generated by multiple attacks on the rough surfaces of the AO starch granules.Iodine complexation is an useful method for detecting the content of linear α-1,4 glucan chains in a starch-containing sample. These glucan segments can be free or linked by α-1,6 branch linkages such as in amylopectin, and their lengths distinctly determine the spectra of the iodine complexes formed, where long segments stain deep blue and blue-green, and shorter chains stain more weakly, having purple, red, and faint red colours with decreasing lengths . The optFig. S2, available at JXB online ] ]  (MacGregonline)] , whereasonline)] . The AO online)] .The mobilization of starch, proteins, and lipids in the barley endosperm was also investigated by histochemical analyses at different time points . BesidesThere were remarkable differences during germination among the WT and the two lines with regard to starch granule morphology and rate of degradation, and also differences regarding the appearance of insoluble carbohydrates in general, and the distribution of proteins and lipids. All images are from longitudinal sections through the embryo–endosperm junction of the endosperm.Before germination, the starch granules from HP endosperm  resembleAt 4 d of germination, the starch granules from the WT  were higMore dramatic changes in the PAS reaction were seen within the tissues at 8 d of germination, as starch was almost completely degraded within the entire WT endosperm , J. This2O, and therefore the only opportunity for the grain and germinating plantlet system to generate energy is through respiration of storage compounds. However, a major part of the grain storage compounds are also remobilized to support anabolic pathways of the new plantlet. It was observed that total dry weight biomass was removed less efficiently , which differed in their starch structure with respect to amylose content . This was unexpected since phosphorylation is known to stimulate starch degradation  and, to a lesser extent, arabinoxylan are the main cell wall components in a barley grain -β-ey grain . BG decley grain  and playIn summary, low starch carbon availability is compensated by re-directing the metabolism to protein degradation. These results show that perturbations of starch metabolism in a seedling can affect other metabolic fluxes, in this case protein metabolism, providing essential carbon without severely endangering the seedling’s survival.Polysaccharide and protein degradation are directly and metabolically linked to sugar metabolism, and the soluble sugar levels increase dramatically upon germination. In the present study, there was no significant difference in total soluble sugar levels in the initial stages of germination between the WT and HP, while AO showed slightly higher levels of sucrose, fructose, and glucose. There have been extensive studies done to establish the mechanism of regulation by sugars and GA in barley , whereasEndogenous α-amylases are the most abundant hydrolases and they play a central role in the mobilization of starch during germination and seedling establishment . However, there was no significant difference between expression levels of either of these two genes among HP and WT grains, indicating that the storage starch phosphorylation level does not affect the expression of these putative phosphoglucan phosphatases in grains.Direct dephosphorylation of starch by the two chloroplastic phosphoglucan phosphatases SEX4 and LSF2 is important for degradation of transitory starch in the leaves of It was hypothesized that the differences in germination metabolism between the three starch types can be attributed solely to the starch structure. However, the data indicate that the pleiotropic metabolic effects in the modified lines, in particularly indirect effects on amylase activity, also result in different sugar levels in the grain and different amylolytic capacities. Thus, the combined effects of starch granule degradability and amylolytic activity levels were translated into vastly different compensatory carbon flows in the different lines. The differential starch granule degradation patterns observed by microscopy were possibly directly attributed to different starch granule susceptibility towards hydrolytic attack as initially directed by granule topography and at later stages by the inner morphology. These interaction effects are complex and depend on various factors such as surface pores on the starch granule  optical section of purified WT and AO starch granules to investigate multiple hila.Figure S3. Thin-layer chromatography of glucan fragments.Figure S4. qPCR of putative phosphoglucan phosphatase gene expression in barley grains.Table S1. Values and statistical significance of grain reserve components and amylolytic enzyme activities are presented as mean±SE of three replicates.Table S2. Values and statistical significance of soluble sugars are presented as mean±SE of three replicates.Figures S1–S4.Protocols for


Many scientists attempt to publish their work in a journal with the highest possible journal impact factor (IF). Despite widespread condemnation of the use of journal IFs to assess the significance of published work, these numbers continue to be widely misused in publication, hiring, funding, and promotion decisions , 2.There are a number of problems with this approach. First of all, the journal IF is a journal-level metric, not an article-level metric, and its use to determine the impact of a single article is statistically flawed since citation distribution is skewed for all journals, with a very small number of articles driving the vast majority of citations , 4. FurtASM journals focus on publishing high-quality science that has been rigorously peer reviewed by experts and evaluated by academic editors. The primary mission of ASM is to advance microbial science. At the recent Journals Board meeting that took place during ASM Microbe 2016 in Boston, MA, the editors in chief and the ASM leadership decided to no longer advertise the IFs of ASM journals .Our goal is to avoid contributing further to the inappropriate focus on journal IFs. Although this action by itself may have little effect on a practice that is deeply entrenched in the biological sciences, we hope that removing IFs from ASM journal websites makes a statement of principle that will be emulated by other journals.


Philosophical Transactions A has been produced as an integral component of the Royal Society's celebrations of the 350th anniversary of the publication of the first issue of PhilosophicalTransactions, the world's oldest scientific journal. The issue contains 16 commentaries, each of which is focused on a major scientific advance in the physical, mathematical or engineering sciences since 1665 that was published in Philosophical Transactions. A corresponding theme issue of the journal's biological sciences counterpart, Philosophical Transactions B, has also been produced  A Spot in One of the Belts of Jupiter; An Account of Micrographia, or the Physiological Descriptions of Minute Bodies, Made by Magnifying Glasses; and Of the Mineral of Liege, Yielding Both Brimstone and Vitriol, and the Way of Extracting Them Out of It, Used at Liege. Thus, the wide scientific range of the journal was established in this first issue.Philosophical Transactions Giving Some Account of the Present Understanding, Studies and Labours of the Ingenious in Many Considerable Parts of the World ; this arose due to his scientific correspondence with a Dutch colleague.In its early years, the production of Philosophical Transactions was undertaken by a series of scientists, the majority of whom were the serving Secretary of the Royal Society. This required a considerable devotion to the cause since the Editor-Secretary bore the financial burden of publishing the journal. The Royal Society's reputation became increasingly linked to that of Transactions. During the period 1720–1750, the society and the journal both experienced biting satirical attacks from writers such as Jonathan Swift (e.g. Gulliver's Travels) and the actor, apothecary and naturalist John Hill. This ridicule led to the journal being brought under the direct control of the Royal Society in 1752 and renamed as the Philosophical Transactions of the Royal Society in 1786. Further reform followed in the nineteenth century, when Charles Babbage wrote an open letter, denouncing Fellows who used the Royal Society as a social club and calling for a new category of Fellow: working scientists who published their work in Philosophical Transactions. By the late 1830s, peer review was made more rigorous and  involved two or more referees, one of which could be the Editor, a position then still held by the Secretary of the Society.Following Oldenburg's death in 1677, the responsibility of editing Philosophical Transactions A, covering the physical sciences, and Philosophical Transactions B, covering the biological sciences. In the later twentieth century much of the administrative and marketing responsibilities for the journals were taken on by in-house publishing editors, with a Fellow at the helm of each journal supported by a large, internationally and scientifically diverse editorial board.By the late nineteenth century, the breadth and scope of scientific discovery had increased to such an extent that (in 1886) it was decided to divide the journal into two components, Philosophical Transactions would adopt a unique publishing style. Each issue would focus on a specific topic of current scientific importance and be guest-edited by one (or more) leading scientist(s) in the field. Each year, several issues of both Philosophical TransactionsA and B are based on the presentations given at a Royal Society Discussion or Theo Murphy meeting. This approach has proved to be successful and, currently, 26 issues of both Philosophical TransactionsA and B are published annually.In 1996, given significant competition from specialist journals, the decision was made that Philosophical Transactions has published a significant number of the most important scientific discoveries and developments accomplished since 1665, the selection of the landmark papers in the physical, mathematical or engineering sciences to be celebrated in this theme issue represented a considerable challenge. The process commenced with the consideration of over 300 possible publications from which a ‘long list’ of ca 70 papers was produced. These papers were then carefully reviewed and subjected to the following considerations:(i) the paper must have had a high scientific impact when published and remain influential today;Philosophical Transactions A currently covers;(ii) the need to ensure a balanced representation of the disciplines that (iii) the desirability of including contributions from all of the various time periods across the journal's 350 year history; and(iv) due to the considerable competition and the limitations of space, no scientist could be represented by more than one publication.Given that The 16 landmark papers selected received the approval of the journal's Editorial Board. The fact that only two of these papers celebrate the achievements of female scientists is a sad reflection of the historical under representation of women in the world of science.Philosophical Transactions A with an insight into the essence of each of the major scientific developments, a perspective of their significance and an appreciation of some of the pertinent human dimensions.Each landmark paper is celebrated by a commentary that places the scientific advance in its historical and scientific context and discusses how the new knowledge obtained has influenced developments within its scientific field, together with considerations of the wider impact of the discovery. I greatly enjoyed reading these accounts and I hope that that they will provide readers of this theme issue of Philosophical Transactions A illustrates the major contributions to the development of scientific knowledge and understanding made by the journal during its lifetime. In addition to celebrating past achievements, we look forward to the journal continuing to play a significant role in providing authoritative and readily accessible accounts of recent notable scientific achievements, together with developments in prospect and challenges beyond our present horizons.This theme issue of


Food & Nutrition Research (FNR) was recently awarded a very high Impact Factor of 3.226, making it one of the top journals serving nutrition scientists and researchers. I hope FNR's high Impact Factor will contribute to an even greater number of high-quality submissions on nutrition from around the world. The increase in the journal's Impact Factor is the result of the combined efforts of the authors who chose to publish their good work in this journal, as well as those of the editors, editorial board members, and reviewers, along with the team managing the journal. I congratulate all of these individuals and thank them for their invaluable contributions.In addition to a new Impact Factor, the journal has undergone several changes related to editorial content and now publishes on a wide variety of nutrition- and food-related topics, with papers from every corner of the globe. Indeed, over the last two years, there has been a surge in the number of submissions from beyond Europe, making it a truly global journal. In this context, I am very pleased to announce that some eminent nutritionists have accepted our invitation to write commentaries on topical issues of the day.FNR editorially. The increase in the Impact Factor along with the worldwide popularity of FNR has certainly leant us a renewed energy as we work to further develop FNR. Among other changes, is a rapid completion of the review process and publication of articles once accepted. As always, FNR papers can be downloaded from any corner of the world, making it stand out among nutrition journals.No doubt we are all very pleased with the increased popularity of the journal among nutritionists worldwide and the recent changes that we have made to further strengthen In addition to the above, an expansion of the editorial team is underway. The rapid increase in the number of manuscripts as well as the diverse nature of submissions has brought forth the need to revitalize and renew the editorial board as well as to recruit new section editors with expertise in diverse nutrition fields. Announcements regarding these items are forthcoming.Food & Nutrition Research. I am truly honored and privileged to serve the International Nutrition Community as Editor-In-Chief of FNR.This is my first Editorial that I write as an Editor-In-Chief of Asim K. DuttaroyEditor-In-ChiefFood & Nutrition Research


This study investigates the asymptotic performance of the Quadratic Discriminant Function (QDF) under correlated and uncorrelated normal training samples. This paper specifically examines the effect of correlation, uncorrelation considering different sample size ratios, number of variables and varying group centroid separators ( Discriminant analysis (DA) as a topic in Multivariate Statistical Analysis has attracted much research interest over the years, with the evaluation of discriminant functions when the covariances matrices are unequal with moderate sizes being well explained by Wahl and Kronmal . Linear Marks and Dunn  approachW and Z formed under the assumption of independent training observation. Asymptotic expected error rate for W under the model  when the covariance matrices are heterogeneous with the data of interest being correlated, sample size ratios being unequal, considering different number of variables and varying values of group centroid separator (To evaluate the performance of QDF for correlated and uncorrelated training samples of distributions, we considered a Monte Carlo study with multivariate normally correlated random data generated for three populations with their mean vector Mean vector separator which is set at Sample sizes which are also specified. Here 14 values of The number of variables for this study is also specified. The number of variables are set at 4, 6 and 8 following Murray  who consThe size of population 1 Factors considered in this study were:Series of subroutines were written in MatLab to perform the simulation and discrimination procedures on QDF. Below are the important ones.Generalization of classification procedure for more than two discriminating groups (ie from 2 to Allocate mentclass2pt{minimSuppose that the ith population is defined asThe constant d in Eq. , since ix to the population mean The quadratic score x to Allocate In practice, the Suppose that the joint densities of mentclass2pt{minimThis function is easily extended to the 3 group classification where 2 cut off points are required for assigning observations to the 3 groups (Johnson and Wichern This section presents the performance of QDF when the training data are correlated and then when they are uncorrelated.Evaluating the effect of sample size on QDF with respect to the correlated normal distribution for mentclass2pt{minimmentclass2pt{minimFrom Table For the uncorrelated distribution from Table entclass1pt{minimaThe coefficients of variation generally increased exponentially and stabilized with increasing total sample size and number of variables in The coefficients of variation in correlated normal distribution in Fig. The effect of number of variables on the QDF under the correlated and uncorrelated normal distribution are discussed under this subsection.The graphs of the results for sample size ratio 1:1:1 of the situations of 4, 6 and 8 variables are shown in Fig. The coefficients of variation in this distribution for ratio 1:1:1 in Fig. From Fig. The coefficients of variation shown in Fig. This section presents the results of our investigation on the effect of the Mahalanobis distance on QDF for correlated normal distribution. Considering the correlated normal distribution in Fig. The coefficients of variation of sample size ratio 1:1:1 with increasing total sample size in Fig. From Fig. mentclasspt{minimaThe study focussed on the asymptotic performance of the QDF under correlated and uncorrelated normally distributed training samples. Under this distribution, the performance of the QDF under varying sampling ratios, selected number of variables and different group centroid separators were extensively studied. The QDF recorded minimum misclassification error rates and high variability as the sample size increased asymptotically under correlated normal distribution, thereby increasing the accuracy of classification of observations with the function. The performance of the QDF deteriorated when the sample size ratio was 1:2:3 as f Lawoko  where th


This retracts the article “The effects of 21 and 23 milimeter aortic valve prosthesis on hemodynamic performance and functional capacity in young adults” in Vol. 30 No. 2 from page 356-360, which was published in final edited form because of ethical misconduct as one of the authors copied some portion from an article published in Turkish language which was detected later. The authors have been notified and agree with the retraction. Retracted on April 15, 2014.Pak J Med Sci. 2014;30(2):356–360.Yener AU, Ozcan S, Budak AB, Genc SB, Ozkan T, Cicek OF. The effects of 21 and 23 milimeter aortic valve prosthesis on hemodynamic performance and functional capacity in young adults. http://dx.doi.org/10.12669/pjms.302.4468doi: