miPIE: NGS-based Prediction of miRNA Using Integrated Evidence.

Methods for the de novo identification of microRNA (miRNA) have been developed using a range of sequence-based features. With the increasing availability of next generation sequencing (NGS) transcriptome data, there is a need for miRNA identification that integrates both NGS transcript expression-based patterns as well as advanced genomic sequence-based methods. While miRDeep2 does examine the predicted secondary structure of putative miRNA sequences, it does not leverage many of the sequence-based features used in state-of-the-art de novo methods. Meanwhile, other NGS-based methods, such as miRanalyzer, place an emphasis on sequence-based features without leveraging advanced expression-based features reflecting miRNA biosynthesis. This represents an opportunity to combine the strengths of NGS-based analysis with recent advances in de novo sequence-based miRNA prediction. We here develop a method, microRNA Prediction using Integrated Evidence (miPIE), which integrates both expression-based and sequence-based features to achieve significantly improved miRNA prediction performance. Feature selection identifies the 20 most discriminative features, 3 of which reflect strictly expression-based information. Evaluation using precision-recall curves, for six NGS data sets representing six diverse species, demonstrates substantial improvements in prediction performance compared to three methods: miRDeep2, miRanalyzer, and mirnovo. The individual contributions of expression-based and sequence-based features are also examined and we demonstrate that their combination is more effective than either alone.

The introduction of radioactive seed localisation improves the oncological outcome of image guided breast conservation surgery.

INTRODUCTION: Radioactive seed localisation (RSL) has become increasingly popular for localisation of non-palpable breast tumours. This is largely due to advantages it offers in terms of practicality and convenience when compared to guide wire localisation (WL). This institute switched from using WL to RSL in September 2014. The primary aim was to assess whether this change improved the accuracy of excision with regards to inadequate margin rates and weight of excision specimens. The secondary aim was to establish whether there is a "learning curve" associated with RSL technique. METHODS: Retrospective data collection was performed for 333 consecutive cases of unifocal non-palpable invasive breast cancers undergoing excision with WL or RSL. An inadequate margin was defined as tumour <1 mm from an inked radial margin. Patient demographics, tumour characteristics and clinical outcomes were compared between WL and RSL cases. RESULTS: 100 WL and 233 RSL cases were included. Patient demographics and tumour characteristics were similar for both groups. Inadequate margin rates were 18% with WL and 8.6% with RSL (p = 0.013). Median specimen weights were 33.3 g with WL and 28.7 g with RSL (p = 0.014). Subdividing the RSL group into the first 100 cases performed (RSL1) and the subsequent 133 cases (RSL2), inadequate margin rates were 13.0% and 5.3% respectively (p = 0.037). Mean specimen weights were similar. CONCLUSION: Switching from WL to RSL results in a significant reduction in both inadequate margin rates and specimen weights. A procedure-specific learning curve is present on first implementation of RSL and following this, inadequate margin rates are further reduced.

Morphological Characterization of the Polyflux 210H Hemodialysis Filter Pores.

Background. Morphological characterization of hemodialysis membranes is necessary to improve pore design. Aim. To delineate membrane pore structure of a high flux filter, Polyflux 210H. Methods. We used a Joel JSM-6010LV scanning electron microscope (SEM) and a SU6600 Hitachi field emission scanning electron microscope (FESEM) to characterize the pore and fiber morphology. The maximal diameters of selected uremic toxins were calculated using the macromolecular modeling Crystallographic Object-Oriented Toolkit (COOT) software. Results. The mean pore densities on the outermost and innermost surfaces of the membrane were 36.81% and 5.45%, respectively. The membrane exhibited a tortuous structure with poor connection between the inner and outer pores. The aperture's width in the inner surface ranged between 34 and 45 nm, which is 8.76-11.60 times larger than the estimated maximum diameter of ß2-microglobulin (3.88 nm). Conclusion. The results suggest that the diameter size of inner pore apertures is not a limiting factor to middle molecules clearance, the extremely diminished density is. Increasing inner pore density and improving channel structure are strategies to improve clearance of middle molecules.

Microbiological assay-trienzyme procedure for total folates in cereals and cereal foods: collaborative study.

In 1996, U.S. Food and Drug Administration regulations mandated the fortification of enriched cereal-grain products with folic acid, thereby emphasizing the need for validated methods for total folates in foods, particularly cereal products. The AOAC Official Methods (944.12, 960.46) currently used for the analysis of folate in foods for compliance purposes are microbiological methods. When the fortification regulations were finalized, no Official AOAC or Approved AACC methods for folate in cereal-grain products were in place. The AOAC Official Method (992.05) for folic acid in infant formula does not incorporate important improvements in the extraction procedure and was not considered suitable for the analysis of folates in foods in general. A microbiological assay protocol using a trienzyme extraction procedure was prepared and submitted for comments to 40 laboratories with recognized experience in folate analysis. On the basis of comments, the method was revised to have the conjugase (gamma-glutamyl-carboxy-peptidase) treatment follow a protease treatment, to include the use of cryoprotected inoculum, and to include the spectroscopic standardization of the standard and optional use of microtiter plates. Thirteen laboratories participated in a collaborative study of 10 required and 10 optional cereal-grain products, including flour, bread, cookies, baking mixes, and ready-to-eat breakfast cereals. The majority of the participating laboratories performed the assay by the standard test tube method; others used the microtiter plate modification for endpoint quantitation with equal success. For the required products, the relative standard deviation between laboratories (RSD(R)) ranged from 7.4 to 21.6% for 8 fortified (or enriched) products compared with expected (Horwitz equation-based) values of 11-20%. RSD(R) values were higher (22.7-52.9%) for 2 unfortified cereal-grain products. For the optional products, the RSD(R) ranged from 1.8 to 11.2% for 8 fortified products. RSD(R) values were higher (27.9-28.7%) for 2 unfortified cereal-grain products. Based on the results of the collaborative study, the microbiological assay with trienzyme extraction is recommended for adoption as Official First Action.

Folate derived from cecal bacterial fermentation does not increase liver folate stores in 28-d folate-depleted male Sprague-Dawley rats.

This study assessed the ability of rats to absorb and store the folate synthesized by cecal bacteria. Male weanling Sprague-Dawley rats were folate depleted by feeding a low folacin AIN93G formulated basal diet for 28 d; they were then fed repletion diets containing folate (0.25-1.0 mg/kg diet), dietary fiber (DF; wheat bran, oat bran, ground corn, wheat germ) or undigested and fermented dietary material (UFDM; polydextrose, inulin) in the presence and absence of an antibiotic (succinylsulfathiazole). Fermentation was stimulated by DF and UFDM and reduced by the antibiotic. In the absence of succinylsulfathiazole, the increase in liver folate (during the repletion phase) was proportional only to the folate content of the diet and did not vary with added DF or UFDM. Adding succinylsulfathiazole lowered total folate excretion from 13.8 +/- 8.2 to 4.8 +/- 2.9 nmol/d (pooled diets, P < 0.00001) in agreement with its role in inhibiting bacterial folate synthesis. In addition, succinylsulfathiazole lowered liver folate in rats fed control and test diets approximately equally with a mean decrease from 11.6 +/- 2.5 to 7.5 +/- 2.5 nmol/g wet liver (pooled diets, P < 0.00001), suggesting that the antibiotic also affected rat folate absorption and/or metabolism. Increased bacterial fermentation and excretion as well as increased bacterial folate production in the presence of added DF and UFDM were demonstrated by increased volatile fatty acid content in cecal and fecal samples (P < 0.000001) and increased diaminopimelic acid, muramic acid and folate in feces (P < 0.00001). The magnitude of these changes depended on the type of DF and UFDM. These results show that bacterially synthesized folate is not substantially absorbed and stored in the liver of Sprague-Dawley male rats.

Automatic detection of coronary artery disease in myocardial perfusion SPECT using image registration and voxel to voxel statistical comparisons.

The purpose of this study was to compare the performance of automatic detection of coronary artery disease (CAD) with that of expert observers. A male and female normal image template was constructed from normal stress technetium-99m single photon emission computed tomography (SPECT) studies. Mean and standard deviation images for each sex were created by registering normal studies to a standard shape and position. The test group consisted of 104 patients who had been routinely referred for SPECT and angiography. The gold standard for CAD was defined by angiography. The test group studies were registered to the respective templates and the Z-score was calculated for each voxel. Voxels with a Z-score greater than 5 indicated the presence of CAD. The performance of this method and that of three observers were compared by continuous receiver operating characteristic (CROC) analysis. The overall sensitivity and specificity for automatic detection were 73% and 92%, respectively. The area (Az) under the CROC curve (+/-1 SE) for automatic detection of CAD was 0.88+/-0.06. There was no statistically significant difference between the performances of the three observers in terms of Az and that of automatic detection (P> or =0.25, univariate Z-score test). The use of this automated statistical mapping approach shows a performance comparable with experienced observers, but avoids inter-observer and intra-observer variability.

Comparison of methods for quantification of transient ischaemic dilation in myocardial perfusion SPET.

The purpose of this study was to compare six methods of measuring the left ventricular (LV) transient ischaemic dilation (TID) ratio during stress-rest myocardial perfusion single-photon emission tomography (SPET). The TID ratio was defined as the mean LV short-axis area at stress divided by the mean LV area of similar slices at rest. The centre of the LV wall was defined as either the maximum, mean or median of the radial short-axis count profiles. The area within the endocardial wall was also calculated for each definition of the LV wall centre. We identified 50 consecutive patients undergoing dipyridamole technetium-99m-tetrofosmin SPET imaging and angiography. Continuous receiver operating characteristic (CROC) analysis showed no significant difference between the six methods in terms of identifying severe coronary artery disease (P >0.47). Algorithms using the mean or the median value in the profile were significantly more robust than those using the maximum (P <0.0005). TID measured by all the algorithms is an indicator of severe coronary disease (P < 0.05). The algorithms compared provide a repeatable, quantitative and specific measure of the TID ratio.

Influence of high dietary threonine on growth and amino acids in blood and tissues of rats.

Diets containing 8 or 15% protein from casein plus limiting amino acids, 25% fat and adequate levels of other nutrients for rat growth were supplemented with 0, 0.5, 1.0, 2.0 or 4% of excess L-threonine. Addition of up to 1% excess threonine had little effect on weight gains or food intakes of weanling rats, but addition of 2 and 4% threonine caused a drastic reduction in weight gains or food intakes (up to 41%); the adverse effect being more severe in rats fed lower protein diets. Addition of graded levels of excess threonine resulted in (5 to 47-fold and 4 to 20-fold) increase in concentration of free threonine in rat plasma and brain, respectively. Addition of excess threonine also caused up to 5-fold increase in plasma level of 3-methylhistidine, suggesting increased muscle protein breakdown.

The protein quality of some enteral products is inferior to that of casein as assessed by rat growth methods and digestibility-corrected amino acid scores.

Protein digestibility and quality of six enteral nutrition products sold in Canada were studied by rat balance and growth methods. Casein+L-methionine, 0.2 g/100 g diet (control) and six enteral products (freeze-dried) were fed as the sole source of protein in diets containing 8.61-9.12 g/100 g protein (N x 6.25) to weanling and 18-mo-old rats for a period of 2 and 1 wk, respectively. A protein-free diet was also included in the feeding studies to permit calculations of true protein digestibility and net protein ratio values. Values for true digestibility of protein as determined in old rats for the control diet and the test products were 95 and 89-93%, respectively. Compared with old rats, protein digestibility values were 5-7 percentage units higher in young rats. The 2-wk relative protein efficiency ratio values (42-56%) or the relative net protein ratio values (61-74%) of the enteral products were considerably lower compared to those of the control (100). Supplementation of an enteral product with cysteine, cysteine + tryptophan, cysteine + threonine or cysteine + tryptophan + threonine caused significant improvement in protein quality; suggesting that the product was limiting in these three amino acids. The protein digestibility-corrected amino acid scores for the enteral products were 43-46, 69-75 and 86-93% by using whole egg, casein and the FAO-WHO (1991) pattern as reference proteins, respectively. The results indicate that these enteral products are inferior to casein in protein quality.

Effect of amino acid supplementation on protein quality of soy-based infant formulas fed to rats.

The powder forms of soy-based infant formulas obtained from four manufacturers were fed to weanling rats for two weeks, as the sole source of protein in diets containing 8% protein, 20% fat, and adequate amounts of minerals and vitamins. The relative protein efficiency ratio (RPER) and the relative net protein ratio (RNPR) values (casein + methionine = 100) of diets containing unsupplemented formulas were 71-81 and 78-85, respectively. Supplementation of the formula diets with lysine (0.2%), methionine (0.2%), threonine (0.1%) or tryptophan (0.05%) increased the level of the supplemental amino acid in rat serum but generally failed to improve the RPER or RNPR values. Addition of all four essential amino acids to the formula diets, however, caused a marked improvement in their protein quality (RPER or RNPR values = 100). The data suggested that proteins in soy-based formulas could be marginally co-limited in several indispensable amino acids.

Influence of dietary soybean trypsin inhibitors and DL-ethionine on sulfur amino acid adequacy of diets for young rats.

Weanling male Wistar rats were fed 20% protein diets based on casein or either of two combinations of soy protein isolate and ground raw soy providing three levels of soybean trypsin inhibitors (SBTI; 0, 448 and 808 mg of trypsin inhibited per 100 g of diet respectively). DL-ethionine was included at three levels (0, 0.05% and 0.10%) with each level of SBTI. After 4, 8 and 12 weeks of ad libitum feeding, diets containing SBTI without DL-ethionine were associated with decreases in weight gain, feed efficiency, serum cholesterol and serum urea nitrogen. Higher levels of triglycerides, glutamate pyruvate transaminase (SGPT) and altered serum free amino acid levels were also found. Increased dietary levels of DL-ethionine also resulted in deficits in growth and feed efficiency, decreased serum cholesterol, increased SGPT and similar alterations in serum free amino acids. Combination of dietary SBTI with DL-ethionine resulted in even greater growth deficits and serum cholesterol decreases as well as increases in SGPT and serum triglycerides and changes in serum free amino acid levels. Methionine deficiency in the young rats fed SBTI and DL-ethionine was indicated by the changes in serum amino acids and growth deficits. Moderation of some effects over the 12 week test period suggested decreased methionine requirements in the older rats.

Amino acid rating method for evaluating protein adequacy of infant formulas.

Amino acid profiles and/or protein digestibility (by the rat balance method) were determined for various forms (powder, ready-to-use, liquid concentrate, etc.) of cow's milk- and soy-based infant formulas obtained from 4 manufacturers. The essential amino acid data of the formulas were compared with that of human milk for the calculation of amino acid scores (based on the single most limiting amino acid). The product of amino acid score and total protein (g/100 kcal) was then termed "amino acid rating." Amino acid scores for the milk- and soy-based formulas ranged from 59 to 90 and from 59 to 81%, respectively, due to deficiencies in sulfur amino acids and/or tryptophan. Because of significantly higher total protein contents (g/100 kcal) of soy- (2.65-3.68) and milk-based (2.20-2.95) infant formulas compared to human milk (1.5), the relative amino acid ratings (human milk = 100) for all infant formulas except 2 liquid concentrates (having values of 87%) were above 100%. Values for true digestibility of protein in milk- and soy-based formulas ranged from 87 to 97 and from 92 to 95%, respectively. When corrected for protein digestibility, the relative amino acid ratings for all the milk-based liquid concentrates were below 100% (77-98%).

Differences in protein digestibility and quality of liquid concentrate and powder forms of milk-based infant formulas fed to rats.

Protein and amino acid digestibility and protein quality of liquid concentrate and/or powder forms of infant formulas were studied by rat balance and growth methods. Casein plus methionine (control) and eight formulas were fed to weanling rats as the sole source of protein in diets containing 8% protein (nitrogen X 6.25). Values for true digestibility of protein, lysine, methionine, or cystine (85-92%) in liquid concentrates were up to 13% lower than those in powders. Similarly, the 2-wk relative protein-efficiency ratio values (64-85%) or the relative net protein ratio values (78-94%) of liquid concentrates were up to 25% lower than those for powders. Lower levels of bioavailable lysine and methionine plus cystine in liquid concentrates compared with powders (prepared by the same manufacturer) would suggest that inferior protein quality of liquid concentrates may be due to more heat treatment involved in their preparation.

Relationship between dietary fiber levels and protein digestibility in selected foods as determined in rats.

Samples of 15 food products and feces obtained by feeding them to rats were analysed for dietary fiber fractions. The food products were added as the sole source of protein in 8% protein diets, making up 8.8-51.6% of the diets. Diets were supplemented with 0.54-5.00% purified cellulose to make them more comparable in total fiber. Fiber analyses of food products revealed that the protein sources provided 0.06-7.27% total dietary fiber. The true protein digestibility in rats was negatively correlated with the total food fiber level (r = -0.69, P less than 0.01) or with the food cellulose level (r = -0.82, P less than 0.01) but it was positively correlated (r = +0.81, P less than 0.01) with the purified cellulose level. No relationship was found between protein digestibility and fiber fermentability. Results indicate that several food fiber fractions and possibly associated substances influenced protein digestibility. Purified cellulose did not have the same physiological behavior as food cellulose from the viewpoint of protein digestibility and fiber fermentability.

Digestibility of protein and amino acids in selected foods as determined by a rat balance method.

Values (%) for true digestibility of crude protein and individual amino acids in 20 selected foods were determined by the rat balance (fecal) method. The products were fed as the sole source of protein in diets containing 8% crude protein (N x 6.25). Lowest true protein digestibility values (79-84) were obtained for pinto beans, kidney beans and lentils; intermediate values (89-92) were obtained for chick peas, beef stew, skim milk (over heated), rolled oats, whole wheat cereal, and pea protein concentrate; and highest values (94-100) were obtained for sausage, macaroni-cheese, rice-wheat gluten cereal, skim milk, tuna, soy isolate, peanut butter, chicken frankfurters, beef salami, casein and casein + methionine. In animal foods, peanut butter and soy isolate, the differences between true digestibility of crude protein and most individual amino acids were less than 5%. However, the values for true digestibility of methionine and cystine were up to 44% lower than those of crude protein in pinto beans, kidney beans, lentils, chick peas and pea concentrate. In these legumes, digestibility of crude protein was not a good predictor of digestibility of the limiting amino acids.

Relationship between amino acid scores and protein quality indices based on rat growth.

Protein efficiency ratio (PER), relative PER (RPER), net protein ratio (NPR) and relative NPR (RNPR) values, and amino acid scores were calculated for 20 food products (casein, casein + Met, beef salami, skim milk, tuna, chicken frankfuters, sausage, heated skim milk, peanut butter, rolled oats, soy isolate, chick peas, pea concentrate, kidney beans, wheat cereal, pinto bean, lentils, rice-wheat gluten cereal, macaroni-cheese, and beef stew). In most cases, PER, RPER, NPR or RNPR ranked the products in the same order and positive correlations among the protein quality methods were highly significant (r = 0.98-0.99). Amino acid scores (based on the first limiting amino acid, Lys-Met-Cys, Lys-Met-Cys-Trp or lys-Met-Cys-Trp-Thr) were positively correlated to the PER, RPER, NPR or RNPR data (r = 0.61-0.75). Inclusion of the correction for true digestibility of protein improved the correlations between amino acid scores and the indices based on rat growth. The correlations were especially high between Lys-Met-Cys scores (corrected for true digestibility of protein) and PER, RPER, NPR or RNPR (r = 0.86-0.91). Inclusion of the correction for true digestibility of individual amino acids did not result in further improvements of the correlations in most cases. It is concluded that adjusting amino acid scores for true digestibility of protein would be sufficient and further correction for digestibility of amino acids would be unnecessary in mixed diets.

Complete amino acid analysis in hydrolysates of foods and feces by liquid chromatography of precolumn phenylisothiocyanate derivatives.

The amino acid analysis method using precolumn phenylisothiocyanate (PITC) derivatization and liquid chromatography was modified for accurate determination of methionine (as methionine sulfone), cysteine/cystine (as cysteic acid), and all other amino acids, except tryptophan, in hydrolyzed samples of foods and feces. A simple liquid chromatographic method (requiring no derivatization) for the determination of tryptophan in alkaline hydrolysates of foods and feces was also developed. Separation of all amino acids by liquid chromatography was completed in 12 min compared with 60-90 min by ion-exchange chromatography. Variation expressed as coefficients of variation (CV) for the determination of most amino acids in the food and feces samples was not more than 4%, which compared favorably with the reproducibility of ion-exchange methods. Data for amino acids and recoveries of amino acid nitrogen obtained by liquid chromatographic methods were also similar to those obtained by conventional ion-exchange procedures.