Protein‐energy malnutrition is the most serious nutritional body depletion disorder among infants and young children in developing countries, attributable to inadequate energy and nutrient intake, partly due to high dietary bulk of weaning and infant foods. The gruels fed to children are typically of low nutrient and energy density due to the low flour incorporation rate required for drinking viscosity. The aim of this study was to develop a nutritious product, based on common dry beans and other grains, suitable for supplementary feeding. The optimal processing conditions for desired nutritional and sensory attributes were determined using Response Surface Methodology. For bean processing, soaking for 6, 15, or 24 h, germination for 24 or 48 h, and cooking under pressure for either 10 or 20 min were the independent variables. The processed bean flour's total polyphenol, phytic acid and protein content, the sensory acceptability of the bean‐based composite porridge and its protein and starch digestibility were dependent variables. Based on product acceptability, antinutrients and protein content, as well as on protein and starch digestibility, the optimum processing conditions for the bean flour for infant and young child feeding were 24 h of soaking, 48 h of malting, and 19 min of steaming under pressure. These conditions resulted in a product with the highest desirability. The model equations developed can be used for predicting the quality of the bean flour and the bean‐based composite porridge. Bean optimally processed and incorporated with grain amaranth and rice flours of a ratio of 40: 30: 30, respectively, resulted into flour with high energy, mineral, and nutrient density of the final porridge. The composite is well adaptable to preparation at rural community level. The use of these locally available grains and feasible processes could make a great contribution to nutrition security in sub‐Saharan Africa and other developing countries. In Uganda, infant and childhood malnutrition due to inadequate energy and nutrient density has been associated with the high viscosity of gruels fed to children , improve protein and starch digestibility of porridge from the developed composite flour, evaluate the pasting properties of the composite flour and the acceptability of its porridge, and compare the energy and nutrient  density of porridge from the bean‐based composite flour to those of widely consumed maize and millet porridges.Amaranthus spp.) was procured from farmers in Kamuli district, cleaned by manual sorting and pan‐roasted at 250°C for 5 min, to produce a distinct roasted flavor  was obtained from National Crops Resources Research Institute (NaCCRI) in Uganda, cleaned by manual sorting, batch washed three times with potable water and soaked for 6, 15, and 24 h in water (200% volume for volume). The soaked beans were drained and spread on a plastic tray lined with a thick wet cloth then covered with another wet piece of cloth and left to germinate. The germinated beans were rinsed and steamed under pressure (15 psi at 121°C for 10 or 20 min) using a domestic pressure cooker. The steamed beans were then spread on a metallic tray and dried in a fan oven  at 75°C for 6 h to a final moisture content of 6%. The dried beans were then milled using a Wondermill grinder . To process rice flour, white rice (super brand) was procured from a retail shop in Kampala city, cleaned by manual sorting, and milled using the above mill into fine flour. To process grain amaranth flour, golden colored grain amaranth  was used to determine the optimum level of incorporation of beans, grain amaranth, and rice flours into the composite to significantly contribute to the protein and energy requirements of children aged 2–5 years  (Whitney and Rolfes The desirability function approach (DFA) was used to simultaneously optimize the composite porridge's acceptability, protein and starch digestibility, and the bean flour's total polyphenol, phytate, and protein content. These are very important characteristics of meals fed to infants and young children. The desirability function approach method incorporates desires and priorities for each of the variables and the maximum desirability is 1  on the nutritional quality and consumer acceptability of bean flour and bean/rice/amaranths composite porridge.Each independent variable was varied as shown in Table The variables were expressed individually as a function of the independent variables. The data were fitted to the following second‐order approximating model eq. : (1)Y=B0ε is the random error, Bo the center point of the system, Bi, Bii, and Bij represent the coefficients of the linear, quadratic, and interactive effects, respectively, and Xi, Xi2 and XiXj represent the linear, quadratic, and interactive effects of the independent variables , respectively.Where Y is the response function, Porridges were prepared by dissolving 150 g of the bean‐based composite flour in 400 mL of cold water, adding the resulting paste to 450 mL of boiling water, and boiling for 8 min with constant stirring. Sensory attributes  of the bean‐based composite flour porridges, from each run Table  were evaIn vitro protein digestibility was determined using pepsin‐pancreatin enzyme system  was done at the School of Food Technology, Nutrition and Bio‐engineering sensory laboratory. The panel consisted of 75 males and females aged 18–50 years, familiar with porridge characteristics. The porridges were rated on a 9‐point Hedonic scale where 9 is like extremely and 1 is dislike extremely. The panelists who had consented to participate in the study also gave additional comments on the porridges.Pasting properties of bean‐based composite , finely milled millet, and maize flours  flours were determined using Rapid Visco Analyzer . Peak viscosity, breakdown, final viscosity, set back, peak time, and pasting temperature were read from the pasting profile with the aid of thermocline for windows software  Porridges of the bean‐based composite, millet, and maize flours were prepared at different concentrations in water and boiled for 10 min. The porridges were then placed in a water bath maintained between 54 and 56°C, the recommended consumption temperature for porridges by young children  and pasting properties  of the bean‐based composite, millet, and maize porridges and flours, respectively, were compared using the Statistical Package for Social Scientists 16.0 software program . Means, standard deviations were determined, analysis of variance was performed to calculate significant differences in treatment means and the TUKEY technique (P < 0.05) was used for separation of means using SPSS.To optimize the bean flour processing protocol, data were analyzed by Response Surface Methodology (RSM) procedures using Design‐expert statistical software . Statistical parameters used to relate input variables to responses were Different combinations of common dry bean, grain amaranth, and rice flour exhibited varying protein and energy contents Table . The forThe responses of the preprocessed bean flour and the bean‐based composite porridge varied between different combinations of soaking, germination and steaming time Fig. , Table 5Equation P < 0.0001) increased the acceptability of the composite porridge. Acceptability of the porridges made from cooked beans was low and did not significantly change with soaking time with a mean score of 4.40 on a 9‐point hedonic scale.Soaking and steaming time had a negative and positive linear effect, respectively, on acceptability. Soaking and steaming time had positive interactive effects on acceptability while steaming time had a significant negative quadratic effect on acceptability. Steaming time significantly TotaP = 0.0003).Soaking, germination, and steaming time had a negative linear effect on the bean flour's total polyphenol content. Steaming and germination time had a significant negative interactive effect while only soaking had a positive significant quadratic effect on the total polyphenol content of the bean flour. Increased soaking and germination time significantly reduced polyphenol content of the bean flour and the model was significant (P < 0.0001)Equation Soaking and germination time were the only significant model terms, showing negative linear and interactive effects and positive quadratic effect on phytate content of the bean flour.Starch digestibility of the bean‐based composite porridge was affected by germination and steaming time of the bean flour eq. (5)StarcSteaming and germination time of the beans had positive linear effects while germination and steaming time had negative interactive effect on starch digestibility of the bean‐based composite flour porridge. Only steaming time had a quadratic effect on starch digestibility of the bean‐based composite flour porridge which was negative.P < 0.0001)The relationship between soaking, germination, and steaming time of beans and the bean‐based composite porridge's protein digestibility is represented by equation All the three variables  had positive linear effects, only soaking and germination time had negative interactive and quadratic effects on the bean‐based composite porridge's protein digestibility. Positive interactive effects existed between soaking and steaming time and between germination and steaming time. Steaming time also had a negative quadratic effect on protein digestibility.P = 0.0001) and showed both linear and quadratic effects of soaking and germination time of beans on its flour's protein content.In equation Both soaking and germination time had positive linear effects on protein content while soaking time had a positive quadratic effect and germination had a negative quadratic effect.The processing conditions chosen as optimum were those that resulted in a product with the highest desirability (0.94) Table . These wProximate compositions ) of the bean‐based composite flour were determined and it was found to have substantially good quantities of energy and nutrients Table .P > 0.05) between bean‐based composite and maize porridges, which in turn were significantly preferred to millet porridges. Scores for flavor were not significantly different (P > 0.05) between the bean‐based composite porridge and maize porridge yet it varied significantly between millet and bean‐based composite porridge, with the bean‐based porridge being superior. Overall acceptability scores for the different porridges were not different (P > 0.05) between the bean‐based composite and maize porridges. The millet porridge's overall acceptability was significantly different from and inferior to that of both millet and bean‐based composite porridges. Overall acceptability for the bean‐based composite porridge was superior to those of both maize and millet porridges. Mean acceptance scores for color, smell, taste, texture, after taste and mouth feel did not differ significantly (P > 0.05) between bean‐based composite and maize porridge yet were significantly different (P < 0.05) and were superior to those of millet porridge.The mean scores of porridge attributes  on the 9‐point Hedonic scales were compared Table . OverallUse of spindle size 63 of the Brookfield viscometer at 1.5 rpm resulted in higher torque values and was therefore used in the study. The bean‐based composite flour attained viscosity in the range of 2500–3000 at 15%, millet flour at 8% while maize flour at 7% flour (w/v), , its break down, final, and set back viscosities were higher and significantly different from those of millet flour (P < 0.05). The results showed that there were significant differences (P < 0.05) in the pasting temperature of the different flours. The bean‐based composite flour exhibited a significantly higher pasting temperature than both millet and maize flour. The bean‐based composite flour also exhibited the highest peak time that was not significantly different from that of millet (P > 0.05). Maize porridge exhibited a significantly lower peak time (P < 0.05) than both millet and the bean‐based composite flours. The mean final viscosity values were lowest for the bean‐composite and highest for millet flours.On comparison of the bean‐based composite, maize and millet flours' RVA parameters: peak viscosity, breakdown viscosity, final viscosity, setback viscosity, peak time, and pasting temperature, the bean‐based composite had the lowest peak, breakdown, final and set back viscosity  densities (as served) of the bean‐based composite porridge were superior to densities of maize and millet porridges except for sodium that was more in maize porridge than millet and the bean‐based composite porridges Table . Energy Sensory characteristics of a food are key factors in determination of energy and nutrient intake of infants and young children as they consume less of foods with inferior sensory characteristics than those of superior sensory characteristics. Desirable sensory attributes are key factors for foods as it determines acceptability and consumption. In this study, acceptability of the porridge at different levels of bean flour processing increased with increasing steaming time. Cooking improves palatability of foods , Table . Since mEnergy and nutrient density of infants and young children's feeds is a vital factor in their nutritional status as it determines energy and nutrient intake. The bean‐based composite porridge's higher nutrient density as compared to maize and millet Table  is attriThe relatively high viscosity exhibited by maize and millet starch is indicative that the flours are less suitable for infant and young child feeding as infant and young children require less viscous porridges. On the other hand, the low viscosity exhibited by the bean‐based composite starch is indicative that the flour is suitable for infant and young child feeding that requires less viscous porridges. The relatively low energy and nutrient densities exhibited by maize and millet porridges signifies that the porridges may not be suitable for infant and young child feeding that requires high energy and nutrient density. The high energy and nutrient density exhibited by the bean‐based composite porridge shows that the flour is suitable for infant and young child feeding that requires high energy and nutrient density.Soaking, germination, and steaming of bean flour result in significant improvements in the sensory and nutritional quality of the flour and increase its suitability for feeding infants and young children. The optimal processing conditions for dry beans are soaking for 24 h followed by germination for 48 h and steaming under pressure for 19 min, as they result in high acceptability and protein and starch digestibility as well as reduction in antinutrients. Surface Response Methodology (SRM) is a suitable approach to optimizing formulation for complementary foods and the model equations developed closely predicted the quality of the bean flour and bean‐based composite porridge incorporating the processed flour. Dry beans, when optimally processed and composited with other common grains result in a nutrient‐dense complementary food with the potential to contribute to reduction in macro and micronutrient malnutrition among infants and young children.Catherine T. Ndagire, John H. Muyonga, Reddy Manju, and Dorothy Nakimbugwe do not have any conflicting interests.


In this study we identified that a plant specific domain of unknown function, DUF581 is a zf-FCS type zinc finger. Based on HMM-HMM comparison and signature motif similarity we named this domain as FCS-Like Zinc finger (FLZ) domain. A genome wide survey identified that FLZ domain containing genes are bryophytic in origin and this gene family is expanded in spermatophytes. Expression analysis of selected FLZ gene family members of A. thaliana identified an overlapping expression pattern suggesting a possible redundancy in their function. Unlike the zf-FCS domain, the FLZ domain found to be highly conserved in sequence and structure. Using a combination of bioinformatic and protein-protein interaction tools, we identified that FLZ domain is involved in protein-protein interaction.Zinc fingers are a ubiquitous class of protein domain with considerable variation in structure and function. Zf-FCS is a highly diverged group of C Arabidopsis thaliana genome sequencing was completed in late 2000, the functions of a large number of genes are still unknown A. thaliana, functions of about 37% genes are unidentified Identifying gene function and their interaction with other genes with respect to the regulation of growth and development is major task post genome sequencing. Although A. thaliana DUF581 containing protein, MEDIATOR OF ABA-REGULATED DORMANCY 1 (MARD1) was identified from senescence related enhancer-trapping and found to be involved in ABA-mediated seed dormancy and induced during senescence A. thaliana identified many interacting proteins of DUF581 family proteins; however, the biological significance of these interactions remains to be explored DUF581 is a plant specific domain found in all taxa except algae. They are highly conserved across plant kingdom and least explored. An Ectocarpus siliculosus. Zf-FCS is named after the conserved phenyl alanine and serine residues associated with the third cysteine. In metazoans, zf-FCS is largely present in Polycomb-group (PcG) of proteins. PcG proteins are developmental-regulator proteins which silence the expression of downstream proteins through chromatin-remodeling and epigenetic silencing. They form a multi-protein Polycomb Repressive Complex (PRC) which bind to the target gene and alter the epigenetic status of the gene Drosophila melanogaster for silencing the expression of HOX genes which is important in proper embryonic-development D. melanogaster Polyhomeotic protein, interacts with RNA and DNA in non-sequence-specific manner D. melanogaster PcG proteins, Scm-related protein containing four mbt domains (dSfmbt) and Sex comb on midleg (Scm) is mediated by the zf-FCS domains present in both proteins. Both these proteins interact and cooperate synergistically for mediating target gene repression DUF581 show high signature motif similarity with MYM-type Zinc finger with FCS sequence motif (zf-FCS). Zf-FCS is first identified in MYM family proteins which are related to myeloproliferative syndrome and mental retardation A. thaliana FLZ domain proteins and expression analysis of selected FLZ genes are done. Sequence and structure conservation studies identified that unlike the zf-FCS domain, FLZ domain is highly conserved. FLZ domain predicted to form a novel alpha-beta-alpha secondary structure pattern. A combination of bioinformatics and protein-protein interaction tools identified that FLZ acts as a protein-protein interaction module.This study aims to characterize the function of DUF581 protein domain which is exclusive to plants. Using sensitive bioinformatic approaches, we confirmed that DUF581 is a zf-FCS like zinc finger domain. We named this plant specific domain as FCS-Like Zinc finger (FLZ). A genome wide survey identified that FLZ domain has a bryophytic origin and this gene family is expanded in higher plants. Phylogenetic analysis of Blastocystis hominis; however, in our analysis we found that this domain lackedthe conserved alpha-beta-alpha structural pattern specific to the plant DUF581 domain. A total of 757 non-redundant DUF581 genes were identified from 41 plant genomes  domain. The proteins which possess this domain are named as FCS-like zinc finger (FLZ) proteins.DUF581 and zf-FCS domain are members of TRASH clan of PFAM database and show very high similarity in sequence conservation . TRASH sce motif . The aboA. thaliana genome possesses 18 FLZ domain genes  domains while F.ve mrna01033.1 contains an ion-transport protein domain (PF00520), a cyclic nucleotide-binding domain and DUF3354 (PF11834) along with a C-terminal FLZ domain. A FLZ protein in apple, MDP0000136760, shows tandem pentatricopeptide repeats along with an N-terminal FLZ domain.Domain distribution and organization of FLZ family proteins were analyzed by InterProScan Threading/fold recognition is helpful in identifying structural and functional aspects of novel folds even if they possess remote homology with characterized domains A. thaliana FLZ domain containing protein, AT5G47060. We named this protein as FCS-like Zinc Finger 1 (FLZ1). 50 colonies screened to identify the interacting proteins and 4 genuine interacting proteins are identified. A list of all interacting proteins identified in this study is given in FLZ1 gene were generated  was conducted with an enerated . The N tenerated . In Y2H enerated . In betaenerated . HoweverA. thaliana FLZ domain containing protein, AT5G49120 and checked whether it can interact with PFA-DSP3. It was found that AT5G49120 cannot interact with PFA-DSP3 suggesting that the interaction is very specific to FLZ1 , which is a homologue of flowering time gene CONSTANS (CO). FLZ1 also interacts with STH2 which is mainly involved in light regulated development and shade avoidance MARD1, all three genes analyzed in this study showed transcript accumulation in senescing leaves compared to rosette leaves suggesting the function of FLZ gene family in senescence.Analysis of evolutionary relationship between FLZ genes are a poorly studied class of gene family which is specific to plants. Early efforts in understanding the role of these genes identified that they are related to senescence and ABA mediated seed dormancy A. thaliana FLZ1 protein is indispensable for its interaction with PFA-DSP3 and STH2. However, the strength of the interaction is reduced to half when FLZ domain alone interacted with PFA-DSP3 and STH2 which suggests that the other portions of the protein might be having a helping role in ensuring a tight interaction. Notably, the FLZ is not structurally similar to the protein-protein interaction zf-FCS domains of dSfmbt and Scm (Data not shown). All these results suggest that FLZ domain is a highly diverged group of plant specific zf-FCS which functions as a protein-protein interaction module.In case of identifying the function of DUF, structure based approach is found to be more effective than sequence based search. The function of a protein domain is defined by the fold it forms, so during the course of evolution the structure is likely to be more conserved than the sequence The analysis of secondary structure pattern identified that FLZ form an alpha-beta-alpha secondary structure pattern. Interestingly, this kind of secondary structure pattern is not reported in any classified zinc finger groups so far A. thaliana FLZ gene family members showed an overlap in the expression domain.In short, using a combination of bioinformatics and protein-protein interaction studies, we found that DUF581 is FCS-like zinc-finger which acts as module for protein-protein interaction. They possess a highly conserved and novel secondary structure pattern. FLZ domain containing proteins are plant specific and bryophytic in origin. Local and whole genome duplication resulted in the expansion of this gene family in higher plants. Expression analysis of selected FLZ family genes from 41 species of viridiplantae. Using the key word ‘DUF581’, a search was performed in PFAM, PLAZA v 2.5 and Interpro FLZ genes from Solanaceae were identified from Solanaceae Genomic Resource using InterPro id IPR007650. Members from barley and Cicer arietinum were identified from NCBI BLASTp Picea abies FLZ genes were identified from ConGenIE using BLASTp In this study, we identified Arabidopsis FLZ gene family was generated using MEGA 5 FLZ genes were obtained from Arabidopsis eFP browser For multiple sequence alignment, FLZ and zf-FCS domain sequences were retrieved from PFAM. They were aligned with Clustal X 2.0 and visualized using Mview FLZ1 was cloned in pGBKT7 and used as a bait to screen normalized Mate & Plate Universal Arabidopsis Yeast two-hybrid cDNA library . The interaction of PFA-DSP3 and STH2 was confirmed by cloning them in pGDAT7 and one-to-one interaction check with FLZ1. pGBKT7-53 and pGADT7-T were used as positive control and pGBKT7-Lam and pGADT7-T were used as negative control for the experiments. Deletion constructs of FLZ1 was made in pGBKT7 and interaction was checked with pGDAT7-PFA-DSP3 and pGDAT7-STH2. The primers used for cloning are shown in Yeast two-hybrid assay was conducted using Matchmaker Gold Yeast two-hybrid System  according to manufacturer’s protocol. Bait and prey proteins were co transformed in Y187 yeast strain and β-Galactosidase assay was conducted according to the protocol of Yeast Protocols Handbook . The result was the average of three independent experiments.FLZ1 CDS and deletion fragments and PFA-DSP3 were cloned in pCR8/GW/TOPO vector and transferred to pSAT4-DEST-N (1–174) EYFP-C1 and pSAT5-DEST-C (175-END) EYFP-C1 vectors respectively using Gateway cloning technology . The primers used for cloning are shown in pSAT4-DEST-N (1–174) EYFP-C1 and pSAT5-DEST-C (175-END) EYFP-C1 vectors were used for BiFC Onion peels were subjected to DAPI staining before visualization in confocal scanning microscope. Onion peels were washed with PBS, pH 7.5 and stained with 15 µg/mL DAPI solution for 30 minutes in dark. Peels were again washed with PBS, pH 7.5 and visualized under confocal scanning microscope.Subcellular localization studies were done in onion epidermal cells. FLZ1 and PFA-DSP3 were cloned in pEG104 vector Figure S1Relationship between the members of TRASH clan (CL0175).(PPT)Click here for additional data file.Figure S2Phylogenetic tree of A. thaliana FLZ domain containing proteins.(PPT)Click here for additional data file.Figure S3Expression profile of 3 selected FLZ domain containing genes of Arabidopsis.(PPT)Click here for additional data file.Figure S4Sample result of threading of FLZ domain using Phyre.(PPT)Click here for additional data file.Table S1List of FLZ domain containing proteins in sequenced plant genomes.(XLSX)Click here for additional data file.Table S2List of FLZ1 interacting proteins obtained in yeast two hybrid assay.(DOCX)Click here for additional data file.Table S3Primers used in this study.(DOC)Click here for additional data file.


The neural mechanisms underlying the restorative effects of cognitive training on aging brains remain unclear. To address this issue, we examined the relationship between changes in spontaneous brain activity and cognitive performance that occur after cognitive training.Participants were older adults who were part of a randomized control trial within a larger longitudinal cognitive training study. We conducted single-domain and multi-domain cognitive training in two respective intervention groups. Participants were trained for 1 h, twice a week, for 12 weeks. Cognition was assessed in all participants and magnetic resonance images were obtained at baseline and 1 year after training. To assess spontaneous fluctuations in brain activity, we acquired resting-state fMRI data. Two indices—functional entropy and time-domain entropy—were used to measure the effects of training. Functional entropy increases with aging, and indicates disruptions in functional conectivity. Time-domain entropy decreases with aging, and indicates structural alterations in the brain and blood-flow reduction.p = 0.047) and single-domain groups (p = 9.51 × 10−4) compared with the control group. In the multi-domain group, this was true in the paracentral lobule . Time-domain entropy also improved with training. Compared with controls, time-domain entropy in the multi-domain group decreased less in the inferior frontal gyrus pars opercularis (p = 3.59 × 10−4), the medial part of superior frontal gyrus (p = 1.17 × 10−5), and the thalamus (p = 4.72 × 10−5), while that in the single-domain group decreased less in the cuneus . Additionally, changes in regional entropy for some regions such as hippocampus significantly correlated with improvements in cognitive performance.Seventy participants completed the study: 26 in the multi-domain cognitive training group (70.38 ± 3.30 yrs), 27 in single-domain group (70.48 ± 3.93 yrs), and 17 in a control group (68.59 ± 3.24 yrs). Functional entropy increased significantly less in the multi-domain .The online version of this article (doi:10.1186/s12877-016-0194-5) contains supplementary material, which is available to authorized users. Normal aging of the brain is associated with memory loss and cognitive impairments that can interfere with daily routines  and affeHowever, the neural mechanisms underlying the restorative effects of CogTr on aging brains remains unclear and are subject to debate , 13. TheRecently, using resting-state fMRI to complement traditional task-state fMRI is becoming more common. With resting-state fMRI, brain regions co-activated while not engaged in any specific task can be identified using spontaneous fluctuations in brain activity. Evidence increasingly suggests that temporal fluctuations in resting-state fMRI, in particular low frequency components (<0.1Hz), arise primarily from spontaneous fluctuations in brain metabolism and intrinsic neuronal activity , 19. ResExploring the effects of cognitive training on spontaneous brain activity in older adults may provide fundamental proof for the neural mechanism underlying the effects of cognitive training. Therefore, we conducted a neuroimaging investigation as a sub-study of the longitudinal CogTr program . In the Time-domain entropy was used to characterize the uncertainty of individual time series of spontaneous brain activity that may indirectly reflect the complexity of brain structure. Aging has been found to be associated with a reduction in the time-domain entropy of spontaneous brain activity –26. AddiReduced lateralization of brain activity is another important feature observed in aging populations. This is known as “hemispheric asymmetry reduction in older adults” (HAROLD) . AccordiOur hypothesis can be divided into three parts: 1) Cognitive training leads to changes in the entropy of spontaneous neural activity in healthy older adults, including reductions in the extent to which time-domain entropy decreases and in how much functional entropy increases. 2) Cognitive training reduces HAROLD. 3) Brain changes induced by cognitive training have correlations with cognitive performance. Because the effects of different cognitive training methods remain unknown, we investigated the effects of multi-domain and single-domain cognitive training.The study was approved by the Human Research Ethics Board of Tongji Hospital in Shanghai, China. Written informed consent was obtained from all participants before the experiment (LL (H)-09-04).Participants were from a longitudinal CogTr study. The longitudinal CogTr study was a randomized control trial in healthy older adults, using three groups, including two intervention groups (single-domain CogTr and multi-domain CogTr training) and one wait-list control group . DetailsParticipants were healthy older adults with normal functional capacity, living independently in the community. They were recruited between March 2008 and April 2008 from three community centers around Tongji Hospital through a notice/broadcast dispatched by the local community service institute. Eligibility criteria included: age 65–75 years; education ≥ 1 year; no difficulties with hearing, vision, or communication; no severe physical disease or psychotic disorder; and no obvious cognitive decline . ExclusWe used a controlled and assessor-blind design to test the effect of CogTr on spontaneous brain activity and cognitive capacity. Magnetic resonance imaging (MRI) was performed for all participants at baseline and at 1 year after training ending.We conducted single-domain or multi-domain CogTr in the two respective intervention groups. Participants completed 1 h of training, twice a week, for 12 weeks. The training procedure was administered in a classroom at Tongji Hospital. The multi-domain training targeted memory, reasoning, problem-solving strategies, visuospatial map-reading skills, and making handcraft. The single-domain CogTr focused specifically on reasoning, including the “towers of Hanoi” puzzle, numerical reasoning, Raven’s progressive matrices, and verbal reasoning. During the first 15 min of every training session, the trainer gave a lecture about a common disease in aging people such as hypertension. Then, the participants were taught about a certain cognitive strategy or technique via a slide presentation for the next 30 min. The training tasks were administered mainly via pen/paper. The newly learned skill was reinforced by solving a few real-life problems . The coAll participants were, initially, given a cognitive capacity assessment, and this was taken as the baseline for any subsequent changes. One year after intervention another cognitive capacity assessment was conducted. The measures included the Repeatable Battery for the Assessment of Neuropsychological Status  , aligned along the anterior commissure-posterior commissure line were acquired. For each subject, a total of 155 volumes were acquired, resulting in a total scan time of 310 s. Subjects were instructed simply to rest with their eyes closed, not to think of anything in particular, and not to fall asleep. Subsequently, high-resolution T1-weighted anatomical images were acquired in the sagittal orientation using a magnetization-prepared rapid gradient-echo sequence  on each subject.Scanning was performed using a Siemens 3.0 Tesla Allegra scanner  at East China Normal University, Shanghai, China. Foam padding was used to minimize head motion for all subjects. Functional images were acquired using a single-shot, gradient-recalled echo planar imaging sequence . Thirty-two transverse slices (field of view = 240 × 240 mmhttp://www.fil.ion.ucl.ac.uk/spm), Resting-State fMRI Data Analysis Toolkit (REST V1.8) (http://restfmri.net/forum/), and Data Processing Assistant for Resting-State fMRI (DPARSF V2.2) (http://restfmri.net/forum/dparsf_v2_2). The remaining functional scans were first corrected for within-scan acquisition-time differences between slices, and then realigned to the middle volume to correct for inter-scan head motions. Subsequently, the functional scans were spatially normalized to a standard template . We then assumed the brain entropy was the Shannon entropy . Thus, wst usage , we callregional functional entropy.2) The alternative approach was to calculate the entropy for every single brain region. This method considers the functional links between a selected region and the other 89 regions. Thus, we could extract 89 Pearson correlation coefficients for each region by comparing its BOLD time series with those of the other 89 regions. We also separated these into 20 equal-width class intervals, and calculated the Shannon entropy of each region. We called the entropy of each brain region the t-distribution to calculate the p value of the Pearson correlation coefficient, and used linear regression to remove the effects of age, gender, and education. We used the t-tests to compare measurements between each intervention group and control group. Additionally, a Chi-squared test was employed to test the comparability of gender, and an F test was utilized to test the comparability of age and education between the different groups.In this paper, we used several statistical methods. The first and most basic one was the Pearson product-moment correlation coefficient. This was used to describe the relationship between each pair of brain regions and allowed us to quantify the relationship between the two types of entropy and other relevant quantities. Additionally, we used the Approximate Entropy  to extraWe also collected cognitive behavioral data using the RBANS, CWST, visual reasoning tests, and TMT scores, and used before and after (1 year later) differences in these measures to calculate changes in cognitive ability. We used linear regression to determine the relationship between behavioral score changes and age, gender, and education in the control participants. We then took the control scores as a baseline and calculated the theoretical score-change in the multi-domain and single-domain training groups based on their age, gender, and education. We then compared the difference between the predictions and the actual values, and considered any difference to be an effect of the CogTr. We applied the same method when comparing the entropy values.It should be emphasized that our results are primarily based on entropy—an informational concept—at the time and functional connectivity levels. To have a direct comparison with more conventional approaches, we also performed functional connectivity analysis and provide details in Additional file n = 26, mean age 70.38 ± 3.30 yrs), single-domain , and control . Sixty-five participants completed the baseline MRI scanning. No significant differences in demographics were found between the participants who responded to advertisements and included in the imaging subsample. Fifty-four participants completed the 1-year follow-up. The dropout rate during the study was 28.57 %. The flow of participants through the entire study is illustrated in Fig. p > 0.05). Subject characteristics are summarized in Table Eighty-two participants responded and were assessed for baseline MRI scanning eligibility. Among them, 70 were eventually included in the MRI subsample. They were divided into three groups based on type of cognitive training: multi-domain  in the multi-domain CogTr group. Specifically, it was especially improved in language , attention  and delayed memory . In contrast, the single-domain CogTr group showed no significant changes. Moreover, CWST and TMT scores did not show any significant improvement in either group. Lastly, the visual reasoning score improved significantly in the single-domain CogTr group , and trended toward improvement in the multi-domain CogTr group .RBANS test score significantly improved  and single-domain CogTr  groups. Additionally, the mean decrease in time-domain entropy was found to be smaller in both multi-domain  and single-domain CogTr group  when compared with the prediction for people with no training, as shown in Fig. We found a number of differences between groups that were associated with spontaneous brain activity. The mean increase in functional entropy was significantly reduced in both multi-domain CogTr , the medial part of superior frontal gyrus , and the thalamus . Similarly, time-domain entropy decreased significantly less the cuneus of those in the single-domain training group compared with controls . In the Multi-domain training group, time-domain entropy in the amygdala (right hemisphere) correlated significantly with that in the middle orbitofrontal cortex (right hemisphere) . However, time-domain entropy did not correlate between any pairs of regions in the single-domain training group .As shown in Yao et al’s work , regionat = 2.48, p = 0.012). In contrast, it did not decrease less in the single-domain training group   Fig.  and b. At = 1.72, p = 0.050), the orbital part of the superior frontal gyrus , and the putamen  of the multi-domain CogTr group. However, there was no similar significant reduction in the single-domain CogTr group.Time-domain entropy in the middle temporal gyrus significantly increased with age, functional entropy significantly decreased with age in the orbital part of the superior frontal gyrus, and significantly increased with age in the putamen  and positively (but not significant) correlated with delayed memory in the multi-domain group. In the multi-domain group, regional functional entropy of the calcarine fissure and the surrounding cortex was significantly negatively correlated with the visuospatial test . Additionally, entropy trends in the two regions were significantly reduced in the training groups (p = 0.050).We found that the change of regional entropy in some regions correlated significantly with improvements in behavioral data. As shown in Fig. In the results, we analyzed changes in the entropy of time series and functional connectivity from resting-state fMRI data. The trend of the functional entropy to increase, as observed in aging individuals, was significantly reduced in both groups that underwent cognitive training. Moreover, reduced lateralization, assessed by different entropy indices, was significantly suppressed in the multi-domain cognitive training group. Further, differences in gray matter changes were reduced in both training groups. Finally, behavioral improvements in memory and attention were significantly correlated with brain activity changes after training. In conclusion, our results provide explicit evidence that cognitive training can induce plastic changes at the level of intrinsic activity patterns in aging individuals. This is mainly through modifications in functional connectivity and brain structure, which are thus likely to be part of the neural mechanisms underlying the effects of cognitive training.To our knowledge, this study is the first exploration of the effects of CogTr on functional connectivity in the aging brain from different aspects, as measured by entropy. Combining the analysis cognitive behavioral data, we conclude that while both types of CogTr could improve cognitive capacity in aging individuals, multi-domain training has more advantages in the cognitive domains that it affects.We should emphasize that our results are primarily based on the informational concept of entropy, at the time and functional connectivity levels. To have a direct comparison with more conventional approaches, we analyzed the changes in functional connectivity between 4005 links in the different groups. We found one link that survived Bonferroni correction in the multi-domain training group, implying that connectivity was significantly improved with multi-domain training. The link in question was from the amygdala (right hemisphere) to the middle orbitofrontal cortex (right hemisphere). Li et al. explored the effects of multimodal intervention over a 6-week period, comprising cognitive training, Tai Chi exercise, and group counseling in the elderly, and found that intervention selectively affected resting-state functional connectivity between the medial prefrontal cortex and medial temporal lobe, which has some common features with our result. Differences could be because while they assessed short-term effects, we assessed long-term ones . No linkThe results also suggest that the target of CogTr should be the dysfunction of cortical circuits that have been assumed to play a role in cognitive aging , 52. TheAt the regional level, training in the multi-domain group slowed down the decrease of time-domain entropy in the opercula part of the inferior frontal gyrus, the medial part of the superior frontal gyrus, and the thalamus. Similarly, training in the single-domain group significantly reduced the decrease in time-domain entropy in the cuneus. These results suggest that regions involved in speech production , self-awWe also explored the effect of CogTr on the lateralization of time-domain and functional entropy. Lateralization can be viewed as a marker of brain activity efficiency. Consistent with a study having a larger dataset and wider age distribution , we founAt the regional level, the rate at which entropy asymmetry decreased was significantly reduced in the middle temporal gyrus, orbital part of superior frontal gyrus, and putamen of those in the multi-domain group. In fMRI experiments, these regions have been related to self-awareness , movemenTo determine whether the entropy changes were relevant to behavior, we examined the relationship between the two. Changes in regional time-domain entropy of the hippocampus correlated significantly and positively with the change in delayed memory observed in the single-domain group, indicating the role of the hippocampus in the delayed memory process. In contrast, changes of regional functional entropy in the calcarine fissure and the surrounding cortex were significantly and negatively correlated with the change in visuospatial behavior observed in the multi-domain group. These regions function in primary sensory abilities such as vision and audition .Several aspects of the present study should be taken into account when interpreting our results. First, the sample size was modest and may be susceptible to type II errors, obscuring true-positive effects. Bigger sample sizes are needed to verify the effect of CogTr on the aging brain. Second, the addition of a control group of young subjects could help determine whether CogTr training-induced changes are specific to older participants or whether they could also benefit younger adults. It would also help more directly determine whether the brains in the intervention groups get “younger” than those of the aging controls. Finally, it should be again emphasized that most of our results arise from an analysis based on the notion of entropy. We found that in the multi-domain training group, there was one functional link that was significantly modified, but there was no such link in the single-domain training group. In this sense, multi-domain training was better than single-domain training, and was consistent with the results based on entropy. Therefore, the entropy analysis revealed reliable results. We note that our entropy approach revealed more information than a direct comparison using functional connectivity analysis alone, which implies that entropy had more statistical power. We also conducted a functional connectivity analysis and found results similar to the entropy experiments (Additional file In conclusion, CogTr can induce plastic changes at the level of intrinsic activity patterns in older people, mainly by modifying functional connectivity. Thus, changes in functional connectivity are likely to be a key neural mechanism underlying the CogTr effect. Multi-domain CogTr demonstrated more advantages than single-domain CogTr, perhaps because multi-domain CogTr involves more regions related to cognition. Larger sample sizes are urgently needed to study CogTr-induced changes in brain activity, cognition capacity, and their relationship.


Methylation of a CpG dinucleotide at cytosine C5 (mCpG) is a major modification in vertebrate genomes associated with epigenetic gene silencing. CpG methylation recruits proteins which specifically recognise this motif and these methylated DNA binding proteins then recruit enzymes which chemically and physically alter chromatin, inducing transcriptional repression. Although most CpG motifs are methylated, short (500–2000 bp) CG-rich regions, known as CpG islands (CGIs), found within ~60 % of human gene promoters remain non-methylated Bird . How the4/5CXXCXXC and CXXRC motifs that provide the amino acid residues that coordinate the two zinc ions means that the main domain architecture, despite sequence variation between the three types, is similar in all CXXC domains.To investigate the possibility that proteins are targeted to these CGIs, Voo et al.  used a lCXXC domains are found in various chromatin binding proteins: CFP1 is a member of the SETDB1 complex responsible for H3K4 methylation; MLL protein is also a H3K4 methyltrasferase; KDM2A and KDM2B are H3K36 demethylase enzymes. DNMT1 and MBD1 are unique amongst CXXC domain containing proteins in that they can also bind hemi-methylated and methylated CpG respectively as well as non-methylated CpG. As well as the methylated DNA binding domain, MBD1 also contains a transcriptional repressor domain (TRD) and depending on splice variant, 2 or 3 CXXC domains. The TRD is capable of transcriptional repression independently of the MBD domain, and MBD1 induces transcriptional repression by recruiting the histone H3-K9 methyl transferase enzyme SETDB1. After DNA replication, this histone methylation is heritably maintained due to MBD1 remaining at the replication fork through its interaction with chromatin assembly factor 1. Once replication has occurred, DNMT1 activity fully methylates hemimethylated CpG motifs allowing MBD1 to rebind to the methyl-CpG  as an N-terminal glutathione-S transferase (GST) fusion. Protein expression was induced (OD600 0.6–0.8) with 0.3 mM IPTG at 13 °C overnight. Cells were harvested and resuspended in phosphate buffered saline solution (PBS) pH 7.3 (10 ml per 1 l of culture). Bacteria were disrupted using BugBuster™ (Novagen) and DNA digested with Benzonase® nuclease (Novagen). The protein was purified by affinity chromatography using glutathione Sepharose 4 Fast Flow resin and the tag removed on-column by PreScission Protease. To remove EDTA from the PreScission protease it was first dialysed against 50 mM Tris–HCl (pH 8.0), 150 mM NaCl, 1 mM DTT using a Thermo Scientific Slide-A-Lyzer Dialysis Cassette, 7000 molecular weight cut off (MWCO). Protein samples were buffer exchanged into 5 mM deuterated imidazole, 250 mM NaCl, 1 mM deuterated DTT and concentrated to a final volume of 570 µl using a Vivaspin 20 5000 MWCO centrifugal concentrator (Sartorius Stedim). For isotopically labelled protein, the bacteria were cultured using M9 minimal containing 1 g/l 15N-ammonium chloride.MBD1 CXXC1 domain, residues 167–222, was amplified from the EST clone, accession number CF552871 (Geneservice Ltd ID 30529682) using the PCR primers: hMBD1 CXXC1 forward 5′ GGGATCCGAGCAGAGAATGTTTAAG 3′; hMBD1 CXXC1 reverse 5′ CTCGAGTCAGCTCCTTTCCACAATC 3′. The plasmid pGEX-6p1  containing the CXXC domain from hMBD1 was over expressed in 1H TOCSY, 2D 1H NOESY and 2D 1H COSY spectra were recorded on unlabelled protein in samples containing 5 % D2O and 100 % D2O. 2D 15N HSQC, 3D 15N HSQC-TOCSY, 3D 15N HSQC-NOESY, HNHA and HNHB were recorded using 15N labelled protein. All NOESY experiments used for structure calculation were recorded with a mixing time of 100 ms and at a temperature of 293 K. Data was processed with AZARA and analyzed with CCPN analysis software  were assigned by using the 2D 15N T1, T2 and heteronuclear-NOE relaxation data were recorded at 600.13 MHz (1H) and 293 K and the data were analyzed according to the Lipari–Szabo model-free formalism using the program FAST-model free  for each residue was estimated using the r2r1_tm script , which calculates τc from the ratio of R2/R1. Residues with NOE <0.6 or with an R2/R1 ratio more than one standard deviation from the mean were omitted. The molecular rotational time (τm) of 5.67 ± 0.141 ns for the entire molecule was then estimated by taking the mean τc of the remaining residues.15N NOESY-HSQC and 2D 1H NOESY spectra. In addition to the 1327 NOE distance restraints, 38 3JHN-Hα (extracted from a 3D HNHA spectrum), 17 3JHN-Hβ restraints (derived from a 3D HNHB spectrum) and zinc co-ordination restraints for each of the cysteines  were also included and are summarised in Table Structure calculations were carried out using ARIA 2.3 (Ambiguous Restraints for Iterative Assignment)  adopts a crescent shape incorporating two zinc ions Fig. , MBD1 CX2), 8 by model 2 , 18 by model 4  and 20 by model 5 . Five of the eight residues were not fitted to any model , while two were not assessed because their amides could not be assigned  and one is a proline residue (199P).Fast-model free was able to assign internal dynamic models to 47 out of 56 residues Fig. , 1 repre2 values and internal correlation times on the pico- to nano-second time scale. The loop region is further characterised by requiring the S2f function to fit the internal motion to a model. Residues 175–196 and 208–217 have low coordinate RMSD over the structure ensemble and mean S2 values 0.766 and 0.837 respectively, indicating only modest internal motion. The second cysteine in each cluster, C179 and C191, has a lower S2 value, 0.684 and 0.679 respectively, indicating more internal motion compared to other residues in the well-structured region. Both cysteine clusters have rapid internal motion in the low ps time scale, while the region around cluster one also undergoes chemical exchange.The results show the two termini and loop region are dynamic with low S199PHDV202, has a significant effect on the structure of the loop region since P199 does not allow the hydrophobic side chain of H200 to pack up against β protons of C194 to form a helix, resulting in an elongated loop  multi-protein PRC1 complex). Since hMBD1 CXXC3 does not interact with Ring1b (Sakamoto et al.


B2 ordered NiAl is known for its poor room temperature (RT) ductility; failure occurs in a brittle like manner even in ductile single crystals deforming by single slip. In the present study NiAl was severely deformed at RT using the method of high pressure torsion (HPT) enabling the hitherto impossible investigation of multiple slip deformation. Methods of transmission electron microscopy were used to analyze the dislocations formed by the plastic deformation showing that as expected dislocations with Burgers vector  For theTherefore, it seems important to get a deeper insight into the microstructural evolution of NiAl in order to understand the difficulties in the plastic deformation of NiAl at RT. In the present study, the method of high pressure torsion (HPT) 2From B2 ordered NiAl single crystals (Ni–49.5 at%Al), disk-shaped samples (6 mm in diameter and ∼0.5 mm thick) were prepared by wire sawing and subsequent grinding. They were deformed by HPT for up to 22 turns at RT applying a pressure of 8 GPa. After 22 turns, the deformation corresponds to a nominal shear strain 33.1In k-value. For the present study, it is important that an analysis of fundamental reflections and superlattice reflections in separate Williamson–Hall plots yields CSD sizes that are within the error margins of their combined analysis. This means that the CSD size and the size of the ordered domains do not differ significantly, indicating a chemically highly ordered phase.44.1In NiAl, the occurrence of dislocations other than In addition to the ysis cf. , Fig. 3 The relatively high density of The reactions leading to 4.2The fact that no free ions cf. , Fig. 3 Our observations are therefore in agreement with the sophisticated calculation of the core properties and predictions of the mobility of dislocations in NiAl as found in the literature The 4.3The formation of sessile dislocations by the reaction of glissile ones as encountered in this study of HPT deformed NiAl can be compared with dislocation reactions in fcc metals and alloys. There, the initial situation seems to be similar: The mobile dislocations 4.4A to point B in such a way that The 3D dislocation networks that are frequently formed during HPT deformation cf.  contain sile cf. . The netsile cf. . Taking In contrast to tensile experiments the high quasi-hydrostatic pressure of 8 GPa applied during HPT deformation can give rise to a different scenario. A possibility is the formation of microcracks that do not transform into catastrophic cracks because the high pressure can lead to crack closure. But when the hydrostatic pressure is taken away, the elastic stresses built up in the sample during deformation will lead to the formation of cracks. This is confirmed by the experimental findings: An acoustic emission is heard during pressure release at the end of the HPT deformation and the samples show cracking that can be seen with the bare eye. In this context, it is proposed that the nano- and submicrometer voids and cracks observed in Cu and Cu–1 wt%Pb deformed by equal channel angular pressing 4.53Au, Ni3Al and in FeAl having L12 and B2 long-range order, respectively 3Al where the glide dislocations contain superlattice intrinsic stacking faults In a recent publication the hypothesis was put forward that chemical disordering by deformation is caused by glide dislocations containing distinct antiphase boundary (APB) faults rder cf. , Fig. 5.100〉 cf.  are perfThe preservation of the B2 long-range order in NiAl is of specific importance for the mechanical behavior of NiAl deformed at RT. The absence of chemical disordering (that would lead to a transition from the B2 to the bcc structure) inhibits the activation of 5•HPT deformation of NiAl enables to carry out the up till now impossible investigation of multiple slip deformation at RT.•The TEM study shows that the glissile •3D dislocation networks containing •During HPT deformation the high quasi-hydrostatic pressure of 8 GPa prevents the sample from catastrophic failure. It is therefore proposed that the cracks observed after deformation form in the moment of pressure release.•By HPT deformation the chemical order is not significantly reduced even in nanocrystalline regions of the sample. This is explained by the lack of antiphase boundary dissociated dislocations. Therefore, the slip system does not change with increasing strain. This is crucial for the occurrence of the •Our results on sessile dislocation reactions are able to explain by an orientation dependence the scatter of the results of single crystal ductility of NiAl found in the literature.


S. cerevisiae Tho1 have been determined.In this study, high-resolution structures of both the N-terminal DNA-binding SAP domain and the C-terminal RNA-binding domain of  et al.  by coupling transcription with mRNA processing and export. THO is thought to be formed from five subunits, Tho2p, Hpr1p, Tex1p, Mft1p and Thp2p, and recent work has determined a low-resolution structure of the complex [Poulsen  al. 2014, PLoS On The resulting plasmids were transformed into Escherichia coli C41 (DE3) cells. Cells were grown in 2×TY medium at 37°C to mid-log phase and were induced with 1 mM IPTG. The temperature was reduced to 22°C and the cells were grown for a further 16 h. Isotopically labelled domains were prepared by growing cells in K-MOPS . The elution volumes of both domains were consistent with their being monomeric. Double-deionized water was used to make the buffer solutions.DNA encoding the N- and C-terminal domains of Tho1 were amplified from 2.3.M in 90% H2O, 10% D2O containing 20 mM potassium phosphate pH 6.5, 100 mM NaCl, 5 mM β-mercaptoethanol. All spectra were acquired using a Bruker DRX800, DRX600 or DMX500 spectrometer equipped with pulsed field gradient triple resonance at 25°C, and were referenced relative to external sodium 2,2-dimethyl-2-silapentane-5-sulfonate (DSS) for proton and carbon signals or liquid ammonia for those of nitrogen. Assignments were obtained using standard NMR methods using 13C/15N-labelled, 15N-labelled, 10%13C-labelled and unlabelled protein samples NH, HNCACO, HNCO, HBHA(CO)NH and 1H–13C HSQC. Additional assignments were made using two-dimensional TOCSY and DQF–COSY spectra. Distance constraints were derived from two-dimensional NOESY spectra recorded with a mixing time of 120 ms. Torsional angle constraints were obtained from an analysis of C′, N, Cα, Hα and Cβ chemical shifts using TALOS H—O(A) corresponded to 1.5–2.5 Å and (D)N—O(A) to 2.5–3.5 Å. The three-dimensional structures of the domains were calculated using the standard torsion-angle dynamics simulated-annealing protocol in CNS v.1.2 .Protein samples prepared for NMR spectroscopy experiments were typically at 1.5 m15N HSQC spectra of 200 µM Tho1 SAP domain were recorded in the presence of 200 µM self-complementary oligonucleotides corresponding to either a typical histone cluster scaffold attachment region sequence (5′-AGAAAATAATAAAATAAAACTAGCTATTTTATATTTTTTC-3′) or a random dsDNA sequence (5′-TCCTGATCAGGA-3′). The potential interaction with dsRNA was also measured using the 30-mer dsRNA oligonucleotide 5′-GGACAGCUGUCCCUUCGGGGACAGCUGUCC-3′. The potential interaction of the C-terminal domain with RNA was measured using 15N HSQC spectra recorded for 200 µM C-terminal Tho1 domain in the absence and presence of several 200 µM RNA and DNA oligonucleotides including 18-mer polyA, 18-mer polyU, 18-mer polyG, 18-mer polyC, the 30-mer dsRNA 5′-GGACAGCUGUCCCUUCGGGGACAGCUGUCC-3′ and the 20-mer ssRNA 5′-CUUGUACAUAGUUGGCCAUA-3′.To monitor the interaction of the SAP domain with DNA and RNA, 3.3.1.JPred and Phyre both predicted Tho1 to contains two α-helical clusters, with Phyre predicting an additional helical motif at the C-terminus . Careful analysis of the sequence and disorder prediction suggested that the additional α-helix predicted by Phyre would be unlikely to form. A 15N HSQC spectrum of a clone comprising residues 51–218 showed no additional resonances in the regions expected for structured residues . Subsequently, a number of clones were created to investigate the structures of the domains.us Fig. 1a. Caref3.2.S. cerevisiae Tho1 were used for NMR assignment and structural analysis. We could assign 98% of the backbone resonances . All of the observable side-chain proton resonances were assigned using a combination of homonuclear and triple-resonance experiments as described in §1HN and 15N resonance assignments for the proteins are shown by the single-letter code followed by the sequence number in the 1H–15N HSQC  and 3b), respectively. The SAP domain is composed of two α-helices (residues 9–19 and 27–42) connected by a structured loop in the helix–extended-loop–helix (HEH) motif typical of this fold. The N-terminus was structured from residue 2 onwards, whereas the C-terminal tail has few medium-range or long-range NOEs and was disordered. The structures of several SAP domains have been determined previously, with most having a role in DNA binding and chromosomal reorganization  and HNRNPUL1 , a protein that is also involved in the nuclear export of mRNA. DNA-binding experiments have revealed that the SAP domain of S. cerevisiae Tho1 has the potential to bind DNA  constraints. A summary of all conformational constraints and statistics is presented in Table 1c) and 3d), respectively. The domain is composed of two antiparallel α-helices (residues 122–141 and 147–162) connected by a structured loop. Each of the antiparallel helices has a hydrophobic face and these faces pack together. The fold is further stabilized by two leucine residues in the C-terminal helix that interact with hydrophobic residues at the N-terminal end of helix 1 and the C-terminal end of helix 2. The N-terminus was structured from residue 120 onwards, whilst the C-terminal tail contained a structured loop and a small α-helix (residues 170–175). The residues after Ser179 contained no medium-range or long-range NOEs. A structure-comparison search using DALI revealed a similarity (r.m.s.d. of 2.7 Å over 50 residues) between the helix–turn–helix motif formed by the first two helices and the fold of the WHEP RNA-binding domain, which is found in multiple copies in a number of higher eukaryotic aminoacyl-transfer RNA synthetases. The C-terminal region of Tho1 has been shown to bind RNA , the domain expressed (residues 119–183) exhibited no potential to bind RNA . The domain may still have the potential to bind to RNA, but the exact nature and sequence of the RNA required for binding is unknown. Alternatively, the domain may require the contribution of additional residues of Tho1 that were not included in the expression constructs used for this study.3.5.et al., 2006et al., 2004et al., 2004et al., 2007et al., 2010et al., 2001et al., 2002et al., 2004et al., 2004a) reveals that the hydrophilic faces of the C-terminal ends of both of the helices in the helix–turn–helix motif are highly conserved. Each helix ends with a glycine residue, which is preceded by a phenylalanine that projects into solvent . The residue preceding the phenylalanine and the residues one and two helical turns back from it are also highly conserved as either arginine or lysine. This produces two very similar potential RNA-binding sites at opposite ends of the domain that could, for example, interact with two copies of the same RNA sequence separated by a particular number of bases or specifically orientated within a structural motif. Inspection of the sequences of the C-terminal region of CIP29 shows that it contains a second closely spaced copy of this module, which can be readily identified by the presence of the lysine–arginine–phenylalanine–glycine sequence motif . Two copies of this motif are also present in CIP29 homologues from other animal species. The Arabidopsis Tho1 homologue MOS11 (Germain et al., 2010A human protein, CIP29, has been proposed from sequence alignment to be a homologue of yeast Tho1 (Jimeno nt Fig. 4b. The rif Fig. 4c. Two c4.et al., 2006We report here the solution structures of the N-terminal SAP domain and C-terminal domain of yeast Tho1. The structures of the domains provide potential insight into the structure of related domains in the Tho1/CIP29/MOS11 family of proteins. The location of the DNA-binding site of the Tho1 SAP domain was shown to be similar to that observed in other SAP domains. The putative RNA binding of the C-terminal domain was investigated, although none was detected. Further work will be required to determine exactly which region of yeast Tho1 is responsible for RNA binding (Jimeno Saccharomyces cerevisiae Tho1, SAP domain, 4uzwPDB reference: C-terminal domain, 4uzxPDB reference: 10.1107/S2053230X16007597/pq5029sup1.pdfSupporting Information: Supplementary Figures S1-S4.. DOI:


The factor VIII C2 domain is essential for binding to activated platelet surfaces as well as the cofactor activity of factor VIII in blood coagulation. Inhibitory antibodies against the C2 domain commonly develop following factor VIII replacement therapy for hemophilia A patients, or they may spontaneously arise in cases of acquired hemophilia. Porcine factor VIII is an effective therapeutic for hemophilia patients with inhibitor due to its low cross-reactivity; however, the molecular basis for this behavior is poorly understood. In this study, the X-ray crystal structure of the porcine factor VIII C2 domain was determined, and superposition of the human and porcine C2 domains demonstrates that most surface-exposed differences cluster on the face harboring the “non-classical” antibody epitopes. Furthermore, antibody-binding results illustrate that the “classical” 3E6 antibody can bind both the human and porcine C2 domains, although the inhibitory titer to human factor VIII is 41 Bethesda Units (BU)/mg IgG versus 0.8 BU/mg IgG to porcine factor VIII, while the non-classical G99 antibody does not bind to the porcine C2 domain nor inhibit porcine factor VIII activity. Further structural analysis of differences between the electrostatic surface potentials suggest that the C2 domain binds to the negatively charged phospholipid surfaces of activated platelets primarily through the 3E6 epitope region. In contrast, the G99 face, which contains residue 2227, should be distal to the membrane surface. Phospholipid binding assays indicate that both porcine and human factor VIII C2 domains bind with comparable affinities, and the human K2227A and K2227E mutants bind to phospholipid surfaces with similar affinities as well. Lastly, the G99 IgG bound to PS-immobilized factor VIII C2 domain with an apparent dissociation constant of 15.5 nM, whereas 3E6 antibody binding to PS-bound C2 domain was not observed. Hemophilia A is an X-linked bleeding disorder resulting from dysfunctional blood coagulation factor VIII (fVIII), affecting 1 in 5,000 males worldwide. The most effective treatment for hemophilia A consists of repeated therapeutic infusions of either plasma-derived or recombinant fVIII, commonly referred to as fVIII replacement therapy . There aap-A3-C1-C2  with a ½” titanium horn attached to a Branson Sonifier 450 . The cell lysate was clarified by centrifugation at 16,000 RPM for 30 minutes at 4°C , and the supernatant was sequentially filtered through a 5 and 0.45 micron filter, respectively. The filtered supernatant was allowed to incubate with TALON resin  for 60 minutes at 4°C with rocking. The TALON resin was allowed to settle and was washed with ten column volumes of Wash Buffer I  and ten column volumes of Wash Buffer II . The porcine C2 domain was eluted from the TALON column with five column volumes of Elution Buffer  and then dialyzed against High Salt Buffer  overnight. Affinity-purified porcine C2 domain was further purified by size exclusion chromatography with a Superdex 75 column  at 1 mL/min in High Salt Buffer, and the purified fractions were buffer exchanged into Storage Buffer , concentrated to 2 mg/mL, flash frozen in liquid nitrogen and stored at -80°C for future crystal trials. The human fVIII C2 domain and the 3E6  and G99  antibodies were expressed and purified as previously described . St. St6 aff2 domain , which i2 domain . The lar2 domain .The high resolution X-ray crystal structure of the porcine fVIII C2 domain, along with a comparison of sequence differences with the human fVIII C2 domain and recent X-ray crystallographic evidence of inhibitory antibody epitopes, allows for an atomic description of the low cross-reactivity that is possessed by porcine factor VIII  33]. St. St33]. D) were calculated from each binding curve .To further understand the cross-reactive potentiality of porcine fVIII, both human and porcine fVIII C2 domains were analyzed for inhibitory antibody binding by ELISA. Following the immobilization of 3E6 and G99 IgG to ELISA plates, both human and porcine C2 domains were serially diluted and subsequently incubated in the ELISA plates, as previously described . The appng curve . The claectively . By conts tested . This des tested . HoweverHigh resolution X-ray crystal structures of both the human and porcine fVIII C2 domains allow for comparisons between the electrostatic surface potentials of each protein, which may provide further evidence for the mechanism by which fVIII associates with negatively charged membrane surfaces . Upon deD values for human and porcine C2 domains based on PS-binding ELISA curves were approximately 45 nM and 75 nM, respectively. To further inspect the importance of K2227, the K2227A and K2227E mutants of the human C2 domain were generated and subjected to the PS-binding ELISA, which resulted in comparable or modestly lower affinities as well, with approximate KD values of 60 nM and 120 nM, respectively  is informative for the purpose of understanding the molecular basis of low cross-reactivity by porcine fVIII . As descThe current working hypothesis for how fVIII binds negatively charged surfaces, primarily through the C1 and C2 domains, states that the solvent exposed, hydrophobic residues, which reside on the end of the beta hairpin loops, embed within the nonpolar nature of the membrane interior . The favIn this study, the X-ray crystal structure of the porcine C2 domain, along with calculations of electrostatic surface potential and equilibrium binding data, support a model for PS membrane binding where the face containing the classical epitopes contributes directly Figs. , 5. SpecThe development of an inhibitory immune response to fVIII remains a significant complication to both hemophilia A treatment as well as acquired hemophilia. To further understand the nature of this immune response, X-ray crystallographic structure determination combined with antibody binding studies provide a foothold from which new, more active and less immunogenic fVIII replacement therapeutics may be developed. Recent high-resolution epitope mapping of anti-C2 antibodies has provided a comprehensive study of epitope hotspots throughout the structure of the fVIII C2 domain . Lastly,


K), a central amidase domain and a C-terminal SH3b cell wall-binding domain. CHAPK cleaves bacterial peptidoglycan between the tetra-peptide stem and the penta-glycine bridge.Bacteriophages encode endolysins to lyse their host cell and allow escape of their progeny. Endolysins are also active against Gram-positive bacteria when applied from the outside and are thus attractive anti-bacterial agents. LysK, an endolysin from staphylococcal phage K, contains an N-terminal cysteine-histidine dependent amido-hydrolase/peptidase domain (CHAPK domain of LysK was crystallized and high-resolution diffraction data was collected both from a native protein crystal and a methylmercury chloride derivatized crystal. The anomalous signal contained in the derivative data allowed the location of heavy atom sites and phase determination. The resulting structures were completed, refined and analyzed. The presence of calcium and zinc ions in the structure was confirmed by X-ray fluorescence emission spectroscopy. Zymogram analysis was performed on the enzyme and selected site-directed mutants.The CHAPK revealed a papain-like topology with a hydrophobic cleft, where the catalytic triad is located. Ordered buffer molecules present in this groove may mimic the peptidoglycan substrate. When compared to previously solved CHAP domains, CHAPK contains an additional lobe in its N-terminal domain, with a structural calcium ion, coordinated by residues Asp45, Asp47, Tyr49, His51 and Asp56. The presence of a zinc ion in the active site was also apparent, coordinated by the catalytic residue Cys54 and a possible substrate analogue. Site-directed mutagenesis was used to demonstrate that residues involved in calcium binding and of the proposed active site were important for enzyme activity.The structure of CHAPK domain of LysK was determined, suggesting the location of the active site, the substrate-binding groove and revealing the presence of a structurally important calcium ion. A zinc ion was found more loosely bound. Based on the structure, we propose a possible reaction mechanism. Future studies will be aimed at co-crystallizing CHAPK with substrate analogues and elucidating its role in the complete LysK protein. This, in turn, may lead to the design of site-directed mutants with altered activity or substrate specificity.The high-resolution structure of the CHAP Myoviridae family of the Caudovirales order, with a genome of 148,317 bp-2-(hydroxymethyl)-1,3-propanediol) in between the two monomers of the asymmetric unit of CHAPGH15 and a MES and HEPES molecule in the case of the native and derivative structures of CHAPK, respectively. The calcium ion is in exactly the same position, as are its coordinating residues and the EF-hand-like domain in which it is incorporated. No zinc ion was observed in the CHAPGH15 crystals.The higher resolution of the CHAPGH15 domain. As observed for CHAPK, it was found that mutating the active site residue Cys54 affected bacterial lysis activity strongly. Mutating the calcium ion coordinating residues Asp45, Asp46 and Asp56 also diminished activity about ten-fold, while Tyr49 and His51 seem less important, the same as we observed.Gu et al. also performed site-directed mutagenesis studies, but on K domain of LysK at 1.8 Å resolution (1 Å = 0.1 nm). The structure has the papain-type fold with a long loop between the two amino-terminal alpha-helices. The structure suggests the location of the active site near a hydrophobic groove, with Cys54, His117 and Glu134 forming the catalytic triad. The substrate most likely binds to the hydrophobic groove.We determined the structure of the CHAPK. It likely has a structural role, stabilizing the protein fold. It may also be involved in ensuring the correct location of the peptidoglycan inside the catalytic cleft or in the stabilization of the negative charge of the tetrahedral intermediate during catalysis. A zinc ion was also found and is likely more loosely bound, as it is less buried, has less protein ligands and could be exchanged for a methylmercury ion upon derivatization. Its role, if any, may be regulatory.A calcium ion was found tightly bound to the protein. Its ligands are the side-chains of Asp45, Asp47 and Asp56, plus the backbone oxygens of Tyr49 and His51, all in the amino-terminal domain specific to CHAPK domain in the complete LysK protein. This may allow site-directed mutation to modulate the peptidoglycan specificity and activity of both the CHAPK and LysK enzymes.Based on the structure, we propose a possible reaction mechanism, involving all three residues of the likely catalytic triad. Future studies will include co-crystallization with peptidoglycan analogues and elucidating the role of the CHAPK was expressed, purified, crystallized and crystallographic data was collected as described[CHAPescribed,12. A coescribed. The modescribed and refiescribed and takiescribed. To confescribed. Validatescribed. RefinemCrystal contact analysis was done with PISA; other aK mutants were created using the QuikChange II Site-Directed Mutagenesis Kit from Agilent  as per the manufacturer’s instructions. Crude cell lysate was analyzed for over-expression using sodium dodecyl sulphate gel electrophoresis and for ability to lyse Staphylococcus aureus cells using zymographic gels as described previously[CHAPeviously.The authors declare that they have no competing interests.RK purified the protein and performed site directed mutagenesis and zymogram activity tests; MSG crystallized the protein. MSG and CGD collected X-ray diffraction and fluorescence data. MSG, CGD and MJvR performed structure solution and refinement. MSG, RK, AC and MJvR analyzed the structure. MSG and MJvR drafted the first version of the manuscript. AC initiated the project, while AC and MJvR supervised it. All authors helped to write and improve the manuscript and approved the final version.Sequence aligment underlining the colour coding of Figure Click here for file