Successful Enrichment Research Articles

Enriching rare variants using family-specific linkage information

Genome-wide association studies have been successful in identifying common variants for common complex traits in recent years. However, common variants have generally failed to explain substantial proportions of the trait heritabilities. Rare variants, structural variations, and gene-gene and gene-environment interactions, among others, have been suggested as potential sources of the so-called missing heritability. With the advent of exome-wide and whole-genome next-generation sequencing technologies, finding rare variants in functionally important sites (e.g., protein-coding regions) becomes feasible. We investigate the role of linkage information to select families enriched for rare variants using the simulated Genetic Analysis Workshop 17 data. In each replicate of simulated phenotypes Q1 and Q2 on 697 subjects in 8 extended pedigrees, we select one pedigree with the largest family-specific LOD score. Across all 200 replications, we compare the probability that rare causal alleles will be carried in the selected pedigree versus a randomly chosen pedigree. One example of successful enrichment was exhibited for gene VEGFC. The causal variant had minor allele frequency of 0.0717% in the simulated unrelated individuals and explained about 0.1% of the phenotypic variance. However, it explained 7.9% of the phenotypic variance in the eight simulated pedigrees and 23.8% in the family that carried the minor allele. The carrier’s family was selected in all 200 replications. Thus our results show that family-specific linkage information is useful for selecting families for sequencing, thus ensuring that rare functional variants are segregating in the sequencing samples.

Successive Hydrogen Isotope Separation/Enrichment by Pressure Swing Adsorption Using SZ-13X Column

Focusing on the behavior of hydrogen isotopes onto synthetic zeolites (SZ) at cryogenic temperatures such as liquefied nitrogen, we have been developing a system of pressure swing adsorption (PSA) process for hydrogen isotope separation applicable to fusion fuel processing, environmental tritium safety confinement or recovery of tritium from heavy water reactors. We have previously reported the experimental results of successive isotope separation and enrichment in a H2-D2 mixture by the PSA process using a single column packed with SZ-5A. In this work, a PSA experiment using SZ-13X was carried out in a similar way to the previous experiment for comparing the functional adsorbents. The result shows that the PSA process operation with SZ-13X can make the performance of heavier isotope enrichment with efficiency at a factor of near twice in comparison to the same operation with SZ-5A.

Systematic Studies on TiO2-Based Phosphopeptide Enrichment Procedures upon in-Solution and in-Gel Digestions of Proteins. Are There Readily Applicable Protocols Suitable for Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry-Based Phosphopeptide Stability Estimations?

There have been many successful efforts to enrich phosphopeptides in complex protein mixtures by the use of immobilized metal affinity chromatography (IMAC) and/or metal oxide affinity chromatography (MOAC) with which mass spectrometric analysis of phosphopeptides has become state of the art in specialized laboratories, mostly applying nanoLC electrospray ionization mass spectrometry-based investigations. However, widespread use of these powerful techniques is still not achieved. In this study, we present a ready-to-use phosphopeptide enrichment procedure using commercially available TiO(2)-loaded pipette tips in combination with matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) analyses. Using α-casein as a model protein and citric acid as additive during sample loading, a similar enrichment success can be achieved as compared to applying 2,5- dihydroxy benzoic acid (DHB) for this task. But the DHB-inherited drawbacks are eliminated. In addition, we show that combining DHB and 2,4,6-trihydroxy acetophenone (THAP) as matrix for MALDI-MS measurements retains the sensitivity of DHB for phosphopeptide analysis but adds the homogenous crystallization properties of THAP, enabling preparation of evenly distributed matrix surfaces on MALDI-MS anchor targets, a prerequisite for automated MALDI- MS analyses. Tripartite motif-containing protein 28 and stathmin are two examples for which successful phosphopeptide enrichment of either sodium dodecyl sulfate polyacrylamide gel electrophoresis or two-dimensional gel electrophoresis-separated proteins is shown. Finally, high resolution MALDI Fourier transform ion cyclotron resonance mass spectrometry after phosphopeptide enrichment suggests that chemical dephosphorylation may occur as a side reaction during basic elution of phosphopeptides bound to MOAC surfaces, suggesting that proteome-wide phosphopeptide analyses ought to be interpreted with caution. In contrast, in-depth analysis of phosphopeptide/non-phosphorylated peptide siblings may be used to estimate stability differences of phosphorylation sites in individual proteins, possibly adding valuable information on biological regulation processes.

Development of an off-line capillary column IMAC phosphopeptide enrichment method for label-free phosphorylation relative quantification

Immobilized metal affinity chromatography (IMAC) and metal oxide type affinity chromatography (MOAC) techniques have been widely used for mass spectrometry-based phosphorylation analysis. Unlike MOAC techniques, IMAC requires rather complete removals of buffering reagents, salts and high concentrations of denaturant prior to sample loading in order for the successful enrichment of phosphopeptides. In this study, a simple off-line capillary column-based IMAC phosphopeptide enrichment method can shorten sample preparation time by eliminating the speed-vac step from the desalting process. Tryptic digest peptide samples containing 2 M urea can be directly processed and the entire IMAC procedure can be completed within 6 h. When tryptic digest peptide samples prepared from mouse whole brain tissues were analyzed using our method, an average of 249 phosphoproteins and 463 unique phosphopeptides were identified from single 2-h RPLC–MS/MS analysis (∼88% specificity). An additional advantage of this method is the significantly improved reproducibility of the phosphopeptide enrichment results. When four independent phosphopeptide enrichment experiments were carried out, the peak areas of phosphopeptides identified among four enrichment experiments were relatively similar (less than 16.2% relative standard dev.). Because of this increased reproducibility, relative phosphorylation quantification analysis of major phosphoproteins appears to be feasible without the need for stable isotope labeling techniques.

Removal of sample background buffering ions and myoglobin enrichment via a pH junction created by discontinuous buffers in capillary electrophoresis

Traditional CE sample stacking is ineffective for samples containing a high concentration of salt and/or buffer. We recently reported the use of a discontinuous buffer system for protein enrichment that was applicable to samples containing millimolar concentrations of salt. In this paper, the technique was investigated for samples containing unwanted buffering ions, including TRIS, MES, and phosphate, which are commonly used in biological sample preparation. Using myoglobin as a model protein, the results demonstrated that background buffering ions can be effectively removed or separated from the enriched protein. The key is to use either the acid or the base of the discontinuous buffers to adjust the pH of the sample, such that the net charge of the unwanted buffering ions is near-zero. The successful isolation and enrichment of myoglobin from up to 100 mM TRIS and 50 mM MES was demonstrated. The enrichment factors remained at approximately 200. Removal of phosphate was more challenging because its net charge was anionic in both the acid and the base of the discontinuous buffers. The enrichment was only achievable up to 30 mM of sodium phosphate, the enrichment factors observed were significantly lower, below 50, and the process was delayed due to the higher ionic strength resulted from phosphate. The migration of phosphate during enrichment was studied using a UV-absorbing analogue, phenyl phosphate. In addition, Simul 5.0 was used to simulate the discontinuous buffers in the absence and presence of TRIS and phosphate. The stimulated TRIS and phosphate concentration profiles were generally in agreement with the experimental results. The simulation also provided a better understanding on the effect of phosphate on the formation of the pH junction.

Stoichiometric and molecular evidence for the enrichment of anaerobic ammonium oxidizing bacteria from wastewater treatment plant sludge samples

Anammox enrichments were readily developed from seven municipal wastewater treatment plants (WWTPs) sludge, but not with methanogenic granular sludge from two agro-industrial WWTPs. Only 50 d was required for the first evidence of anammox activity from a return activated sludge obtained from a WWTP operated for nutrient removal. The molar ratios of nitrite and ammonium consumption of approximately 1.32 as well as nitrate and dinitrogen gas product ratios of approximately 0.095 provided evidence of the anammox reaction. The presence of anammox was confirmed by polymerase chain reaction (PCR) using primer sets (PLA46F and AMX820R) specific for anammox bacteria. The 16S rRNA gene fragment of anammox bacteria was detected in seven enrichment cultures (ECs) with demonstrated anammox activity but not in the original inocula from which the ECs were derived and also not in the two methanogenic sludge samples, which indicates the PCR predicted the anammox activity. Two genera, Brocadia and Kuenenia, were successfully identified as the Planctomycetes occurring in the clone libraries of successful anammox enrichments. Brocadia dominated in cultures that were respiked extensively; whereas Kuenenia predominated in cultures that were less aggressively respiked. These findings indicate that respiking management may play an important role on selecting the genus of anammox bacteria. The batch enrichment results clearly illustrate that anammox can be readily enriched from municipal sludge from a wide variety of process operations at WWTPs.

Bioenergy feedstock‐specific enrichment of microbial populations during high‐solids thermophilic deconstruction

Thermophilic microbial communities that are active in a high-solids environment offer great potential for the discovery of industrially relevant enzymes that efficiently deconstruct bioenergy feedstocks. In this study, finished green waste compost was used as an inoculum source to enrich microbial communities and associated enzymes that hydrolyze cellulose and hemicellulose during thermophilic high-solids fermentation of the bioenergy feedstocks switchgrass and corn stover. Methods involving the disruption of enzyme and plant cell wall polysaccharide interactions were developed to recover xylanase and endoglucanase activity from deconstructed solids. Xylanase and endoglucanase activity increased by more than a factor of 5, upon four successive enrichments on switchgrass. Overall, the changes for switchgrass were more pronounced than for corn stover; solids reduction between the first and second enrichments increased by a factor of four for switchgrass while solids reduction remained relatively constant for corn stover. Amplicon pyrosequencing analysis of small-subunit ribosomal RNA genes recovered from enriched samples indicated rapid changes in the microbial communities between the first and second enrichment with the simplified communities achieved by the third enrichment. The results demonstrate a successful approach for enrichment of unique microbial communities and enzymes active in a thermophilic high-solids environment.

In Vitro Selection of Cathepsin E-Activity-Enhancing Peptide Aptamers at Neutral pH.

The aspartic protease cathepsin E has been shown to induce apoptosis in cancer cells under physiological conditions. Therefore, cathepsin E-activity-enhancing peptides functioning in the physiological pH range are valuable potential cancer therapeutic candidates. Here, we have used a general in vitro selection method (evolutionary rapid panning analysis system (eRAPANSY)), based on inverse substrate-function link (SF-link) selection to successfully identify cathepsin E-activity-enhancing peptide aptamers at neutral pH. A successive enrichment of peptide activators was attained in the course of selection. One such peptide activated cathepsin E up to 260%, had a high affinity (KD; ∼300 nM), and had physiological activity as demonstrated by its apoptosis-inducing reaction in cancerous cells. This method is expected to be widely applicable for the identification of protease-activity-enhancing peptide aptamers.

An overview of the proper generalized decomposition with applications in computational rheology

Abstract We review the foundations and applications of the proper generalized decomposition (PGD), a powerful model reduction technique that computes a priori by means of successive enrichment a separated representation of the unknown field. The computational complexity of the PGD scales linearly with the dimension of the space wherein the model is defined, which is in marked contrast with the exponential scaling of standard grid-based methods. First introduced in the context of computational rheology by Ammar et al. [3,4], the PGD has since been further developed and applied in a variety of applications ranging from the solution of the Schrödinger equation of quantum mechanics to the analysis of laminate composites. In this paper, we illustrate the use of the PGD in four problem categories related to computational rheology: (i) the direct solution of the Fokker-Planck equation for complex fluids in configuration spaces of high dimension, (ii) the development of very efficient non-incremental algorithms for transient problems, (iii) the fully three-dimensional solution of problems defined in degenerate plate or shell-like domains often encountered in polymer processing or composites manufacturing, and finally (iv) the solution of multidimensional parametric models obtained by introducing various sources of problem variability as additional coordinates.

Autotrophic biological nitrogen removal from saline wastewater under low DO

AbstractBACKGROUND: This study was conducted to investigate the feasibility and performance of nitrogen removal through the complete autotrophic nitrogen removal over nitrite (CANON) process for saline wastewater in a continuous reactor, and to characterize microorganisms in the sludge from the reactor using DNA‐based techniques.RESULTS: The nitrogen removal experiment in the reactor was operated over five phases for 286 days treating a synthetic sewage of 1.2% salinity at 21–25 °C. At dissolved oxygen (DO) concentrations of 0.5–1.0 mg L−1 and in the presence of glucose, NO2− was accumulated, indicating the activity of ammonia‐oxidizing bacteria (AOB). At DO concentration of 0.5 mg L−1 without organic substrate, the anaerobic ammonium oxidation (Anammox) process was the major pathway responsible for nitrogen removal, with a total nitrogen removal of 70% and an ammonium conversion efficiency of 96%. A maximum ammonium removal rate of 0.57 kg‐N m−3 d−1 was achieved during the experimental period. The concentrations of AOB and Anammox bacteria were monitored over the operation of reactor using quantitative real‐time polymerase chain reaction (qRT‐PCR).CONCLUSION: In this study, autotrophic nitrogen removal process was achieved under salinity condition in a one‐reactor system. An over 100 fold increase of AOB was found due to the increased supply of ammonium at the beginning, then AOB concentration decreased temporarily in correspondence with the decreased DO, and the AOB resumed their concentration at the last phase. The Anammox bacteria abundance was about 150 fold higher than that at the beginning, indicating the successful enrichment of Anammox bacteria in the reactor. Copyright © 2010 Society of Chemical Industry

Verification of hydrogen isotope separation/enrichment by pressure swing adsorption process: Successive enrichment of deuterium using SZ-5A column

We have been developing a system of hydrogen isotope separation using a pressure swing adsorption (PSA) method, aiming at applying it in the fusion fuel cycle and/or the convenient deuterium production for fusion or/and fission reactors. Particularly, for the issue that tritium inventory in the DT fusion system must be as low as possible, the PSA method will be advantageous because its process inventory is reasonably less than such as in a cryogenic distillation system. The PSA system is generally composed of several adsorption columns cooperating in sequential procedure and alternative combination among adsorption, desorption and another preparative process. A variety of operational modes are made suitable for producing a material needed to be enriched or isolated. In this work, supposing the production of deuterium from natural hydrogen or the recovery of tritium from such as a spent nuclear heavy-water moderator, we carried out an experimental series of PSA process system operating successive separation and enrichment of tracer D 2 from a H 2–D 2 mixture, using a single column packed with SZ-5A at 77.4 K. Availability of the PSA hydrogen isotope separation process is endorsed in experimental results.

A multipathogen selective enrichment broth for simultaneous growth ofSalmonella entericaserovar Enteritidis,Staphylococcus aureus, andListeria monocytogenes

A selective enrichment broth (SSL) was formulated to allow concurrent growth of 3 prominent food-borne pathogens: Salmonella enterica serovar Enteritidis, Staphylococcus aureus, and Listeria monocytogenes. Nalidixic acid, lithium chloride, and potassium tellurite were added as the selective agents, while sodium pyruvate and mannitol were employed as the supplemented elements. In the individual growth trial, the target pathogens were capable of growing in SSL to as high as 7-8 log(10) colony-forming units (CFU)/mL after 24 h incubation at 37 degrees C when being inoculated at 50-100 CFU/mL. In the simultaneous growth trial, the 3 combined target pathogens showed similar growth rates. The results show that SSL could support the successful simultaneous enrichment of 3 pathogens; however, SSL inhibited the growth of nontarget bacteria. In the artificial contaminated raw beef and ready-to-eat chicken, a high recovery of these 3 target pathogens was obtained in SSL. Finally, Salmonella Enteritidis, Staphylococcus aureus, and L. monocytogenes were detected from 710 suspicious food samples by SSL with real-time PCR, and no false-positive or -negative results were reported. In summary, SSL has been shown to be a suitable broth for the simultaneous detection of the 3 prominent food-borne pathogens by multipathogen detection on a single-assay platform.

Comparison of Metal and Metal Oxide Media for Phosphopeptide Enrichment Prior to Mass Spectrometric Analyses

Several affinity resins consisting of ionic metals or metal oxides were investigated for their phosphopeptide enrichment capabilities with subsequent mass spectrometric analyses. Commercially-available enrichment metal oxide affinity chromatography (MOAC) resins using manufacturer's and/or published protocols were compared and evaluated for the most efficient and selective method that could be implemented as a standard enrichment procedure. From these comparative analyses, using a tryptic digest of casein proteins, it was determined that in our hands, two of the resins out-performed the others based on a variety of criteria, including the number of phosphorylation sites identified during MS analyses, the lower numbers of nonspecifically bound peptides observed, and the limits of detection. Applicability of these enrichment resins to a complex biological mixture was investigated. For this work, a mixture of avian histones was digested, subjected to titanium dioxide phosphopeptide enrichment, and analyzed by mass spectrometry. Eight phosphorylated tryptic peptides were observed following enrichment and subsequent LC/MS/MS analyses. Of note, seven of the eight phosphopeptides were not observed without titanium dioxide enrichment. From these analyses, four sites of phosphorylation were unequivocally determined, two of which have not been reported previously. Four additional phosphopeptides were observed; however, the site of phosphorylation could not be distinguished but was localized to one of two possible amino acids. These methods should aid in the investigation of proteins post-translationally modified with phosphate, especially those present at low concentrations as was demonstrated by successful enrichment at the femtomole level.

Optimized IMAC−IMAC Protocol for Phosphopeptide Recovery from Complex Biological Samples

Immobilized metal ion affinity chromatography (IMAC) is widely used for phosphopeptide enrichment. However, the robustness, efficiency, and specificity of this technique in large-scale phosphoproteomics studies are still disputed. In this study, we first compared three widely used IMAC materials under three different conditions. Fe(III)-nitrilotriacetic acid (NTA) IMAC resin was chosen due to its superior performance in all tests. We further investigated the solution ionization efficiency change of the phosphoryl group and carboxylic group in different acetonitrile-water solutions and observed that the ionization efficiencies of the phosphoryl group and carboxylic group changed differently when the acetonitrile concentration was increased. A magnified difference was achieved in high acetonitrile content solutions. On the basis of this concept, an optimized phosphopeptide enrichment protocol was established using Fe(III)-NTA IMAC resin and it proved to be highly selective in the phosphopeptide enrichment of a highly diluted standard sample (1:1000) prior to MALDI MS analysis. We also observed that a higher iron purity led to an increased IMAC enrichment efficiency. The optimized method was then adapted to phosphoproteome analyses of cell lysates of high protein complexity. From either 20 microg of mouse sample or 50 microg of Drosophila melanogaster sample, more than 1000 phosphorylation sites were identified in each study using IMAC-IMAC and LC-MS/MS. We demonstrate efficient separation of multiply phosphorylated peptides from singly phosphorylated peptides with successive IMAC enrichments. The rational improvements to the IMAC protocol described in this study provide more insights into the factors that affect IMAC performance for phosphopeptide recovery. The improved IMAC-IMAC method should allow more detailed characterization of phosphoproteins in functional phosphoproteomics research projects.

Sequential Fe3O4/TiO2 enrichment for phosphopeptide analysis by liquid chromatography/tandem mass spectrometry

Protein phosphorylation regulates a wide range of cellular functions and is associated with signaling pathways in cells. Various strategies for enrichment of phosphoproteins or phosphopeptides have been developed. Here, we developed a novel sequential phosphopeptide enrichment method, using magnetic iron oxide (Fe(3)O(4)) and titanium dioxide (TiO(2)) particles, to detect mono- and multi-phosphorylated peptides. In the first step, phosphopeptides were captured on Fe(3)O(4) particles. In a subsequent step, any residual phosphopeptides were captured on TiO(2) particles. The particles were eluted and rinsed to yield phosphopeptide-enriched fractions that were combined and analyzed using liquid chromatography/tandem mass spectrometry (LC/MS/MS). The validity of this sequential Fe(3)O(4)/TiO(2) enrichment strategy was demonstrated by the successful enrichment of bovine alpha-casein phosphopeptides. We then applied the sequential Fe(3)O(4)/TiO(2) enrichment method to the analysis of phosphopeptides in L6 muscle cell lysates and successfully identified mono- and multi-phosphorylated peptides.

Evolution of specific RNA motifs derived from pan-protein interacting precursors

In vitro evolution of nucleic acid aptamers is a powerful tool to investigate the structure–function relationship of natural occurring RNA–protein interaction motifs. Otherwise, it also allows the identification of novel RNA-based ligands that can be used to investigate a target’s function in its native environment. However, artifacts have been described during in vitro selection procedures hampering the successful enrichment of aptamers. Here we describe a novel observation, namely the enrichment of pan-protein binding RNA sequences. We demonstrate that evolution of specific target binding sequences originating from a pan-protein binding RNA precursor is possible in general. Our data demonstrate that the mutual co-variation of an ancestor molecule can be applied for the evolution of specific target binding RNA sequences. These results might have implications in the context of the RNA world theory, exemplifying a possible evolutionary route towards protein-specific RNA molecules from a common ancestor.

Abstract LB-221: Differential aberrant DNA methylation predicts for Tarceva response in NSCLC

Abstract Recent phase III clinical trials have shown that some non-small cell lung cancer (NSCLC) patients without epidermal growth factor receptor mutations derive clinical benefit from erlotnib, a small molecule EGFR inhibitor. Currently there is no way to identify this important patient subset. In an effort to explore whether aberrant DNA methylation profiles could be used to better define the subset of patients with NSCLC who might respond to erlotinib, we have undertaken an integrated genomics approach employing both gene expression profiling and methylated DNA immunoprecipitation (meDIP). In the initial phase of this study, we used a series of twelve NSCLC cell lines without EGFR mutations, which we defined in a previous report as either sensitive or resistant to erlotinib, based on an in vitro microtiter assay (cell titer glow). We treated each cell line with the DNA methylation inhibitor 5-aza-2′-deoxycytidine (5-azadC) qod for 6 days and confirmed gene induction using quantitative RT-PCR (QPCR). We then performed gene expression profiling on the mock treated (DMSO) and 5-azadC (1 M) treated cell lines using the Affymetrix U133P 2.0 chip. Consistent with previous reports we found that the expression levels of large numbers of genes are affected by 5-azadC treatment, and in both directions. In addition, since it is known that 3′ biased gene expression arrays can miss expression changes in alternatively spliced (and methylated) variants of genes (such as RASSF1A and p16), we sought to improve our ability to discover differentially methylated regions (DMR) by using meDIP in parallel with gene expression profiling. We performed meDIP on DNA prepared from cell pellets made at the same time as we did the 5-azadC treatments. We confirmed successful immunoprecipitation and enrichment of methylated DNA using QPCR for several targets including the imprinted H19 locus, as well as several others. Analysis of these data suggests that thousands of loci are differentially methylated between the twelve cell lines, and that there are several hundred DMRs which differentiate between Tarceva sensitive and resistant NSCLC lines. While there was overlap between the meDIP and gene expression data, there were many genes that appeared only on one or the other platform. However, we confirmed that both approaches identify loci that are differentially methylated by direct sodium bisulfite sequencing between the sensitive and resistant cell lines. Subsequent validation of the DMRs in a broader panel of NSCLC lines shows that our approach has successfully identified markers which differentiate between Tarceva sensitive and resistant cell lines. Future studies will determine whether these DMRs can distinguish between tumors that respond to erlotnib and those that are resistant. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-221.

Optimization of folate deposition in eggs through dietary supplementation of folic acid over the entire production cycle of Hy-Line W36, Hy-Line W98, and CV20 laying hens

SUMMARY Supplementing laying hen diets with folic acid at 4 mg/kg of diet for 3 wk led to the production of folate-enriched eggs with an approximately 3-fold increase in egg folate concentration relative to a regular commercial egg. However, because long-term consistency in folate deposition is important for successful folate enrichment of natural foods such as eggs, 2 studies were designed to determine the level of folic acid required to maximize egg folate content and consistency throughout the entire cycle of egg production. In study 1, Hy-Line W36 and W98 laying hens (n = 156/treatment) received a barley-based diet containing 0 and 4 mg/kg of folic acid, whereas in study 2, a total of 1,248 Hy-Line CV20 laying hens (n = 416/treatment) received a barley-based ration supplemented with folic acid at 0, 2, or 4 mg/kg of diet. Both studies lasted for eleven 28-d periods. Response criteria included production parameters, egg quality measurements, and measures of egg folate content. Results of both long-term production studies support the use of 4 mg of folic acid/kg of diet as the recommended level of folic acid supplementation in terms of maximizing egg folate concentration over time. This is supported by the higher egg folate content and more consistent egg folate deposition at 4 mg of folic acid/kg of diet as compared with 0 and 2 mg of folic acid/kg of diet. Hy-Line W98, a strain of hens selected for large egg mass, benefited more from increased dietary folic acid in terms of increased egg production than did Hy-Line W36 and CV20 hens, strains selected for better egg efficiency and livability. Sensitivity and responsiveness to increasing dietary folic acid concentration in terms of egg folate content were, however, similar for all strains. In conclusion, eggs of laying hens were consistently enriched with folate by dietary supplementation with 4 mg of folic acid/kg of diet throughout the production cycle.

Enhancing Essential Amino Acids and Health Benefit Components in Grain Crops for Improved Nutritional Values

Improving essential amino acids or protein content, along with other phytonutrients in the food crops, will affect a great portion of the world population, especially in developing countries where rice grain is the main source of protein. Malnutrition, including deficiencies in protein/energy, iron/zinc, vitamin A, and iodine, causes a total 24,000 deaths per day worldwide. The problem is severe where rice is the major staple food. Protein deficiency involves both the quantity (amount) and quality (the content in essential amino acids) of the dietary protein. Various interventions, such as distribution, fortification, dietary diversification, and measures against infectious diseases, have been applied to reduce deficiency disorders. The problem, however, remains unsolved. Developing genetically novel lines with elevated content of essential amino acids together with other health benefit components becomes more feasible for the enhancement of breeding techniques, genomics, molecular manipulations, and genetic engineering. Advancement in basic genetic and genetic engineering has resulted in successful enrichment of some essential amino acids, such as lysine (Lys), tryptophan (Trp), and methionine (Met). Successful genetic enhancement has been largely restricted to the maize crop through enrichment of grain Lys and to some extends Trp. Since rice is the main source of calories and protein intake for billions of people, enhancing essential amino acids in rice represents a tremendous challenge. This paper will discuss and review the current status in basic genetics, molecular genetics, and genetic engineering associated with the enhancement of amino acids and other health benefit components in major grain crop improvement. Patents and future efforts associated with enhancing nutritional quality of the grain will also be reviewed as a concerted effort to solve the malnutrition problem and improve the quality of life worldwide.

Distribution of anammox bacteria in domestic WWTPs and their enrichments evaluated by real-time quantitative PCR

Enrichment of anaerobic ammonium oxidation (anammox) bacteria using five activated sludges in three domestic wastewater treatment plants (WWTPs) were processed in a short term of 70 days and evaluated by real-time quantitative PCR (RTQ-PCR). Before the enrichment, building phylogenetic trees of Planctomycetes phylum in four reactors of sequencing batch reactor (SBR), anoxic and oxic reactors of anaerobic–anoxic–oxic (A2O) process, and rotating biological contactor (RBC) revealed six groups of distantly relative genera of Planctomyces, Pirellula, Gemmata, Isophaera, Candidatus and putative anammox bacteria. All clones of Candidatus sp. were affiliated with anammox bacteria and the majority of anammox clones were related to Planctomycete KSU-1 (AB057453). The discovery of anammox bacteria in raw activated sludges provided a partial rationale for the utilization of activated sludge as a seeding source of the anammox process. To verify the activity of anammox bacteria in the activated sludges, enrichment cultivations were conducted using SBRs. The enrichment of anammox bacteria resulted in the significant anammox activity of three samples. Quantification of 16S rRNA gene of anammox bacteria using RTQ-PCR showed the highest concentration of anammox bacteria of 2.48 ± 0.22 × 109 copies of 16S rRNA gene/mg-volatile suspended solids (VSS), which was the same order of magnitude as that of the referential granular anammox sludge, 6.23 ± 0.59 × 109 copies of 16S rRNA gene/mg-VSS, taken from an anammox upflow anaerobic sludge blanket (UASB) reactor. The doubling time of anammox bacteria enriched in this study was 1.18 days. The growth yield of anammox bacteria enriched in this study was 4.75 ± 0.57 × 106 copies of 16S rRNA gene/mg of ammonium- and nitrite-nitrogen, which was similar to 4.50 ± 0.61 × 106 copies of 16S rRNA gene/mg of ammonium- and nitrite-nitrogen for the referential anammox sludge. Substrate uptake rates of three successful enrichments at the end of the enrichment were comparable to those of granular and suspended anammox sludges. Rapid enrichment of anammox bacteria using activated sludge could offer an alternative method for obtaining a large volume of seeding anammox sludge.