Fewer losses in the cascade of care for latent tuberculosis with solo interferon-gamma release assay screening compared to sequential screening.

BACKGROUND: Refugees are at increased risk of developing tuberculosis (TB) soon after resettlement. Targeting high-risk populations for latent tuberculosis infection (LTBI) screening and treatment is an important measure towards eliminating TB in low incidence countries, however, there are low rates of screening and treatment completion in the LTBI cascade of care. The authors hypothesized that an interferon-gamma release assay (IGRA) screening strategy would lead to a higher proportion of refugees completing LTBI screening and treatment, compared to sequential screening with tuberculin skin test (TST) and confirmatory IGRA. METHODS: This retrospective cohort study included eligible refugees screened with a sequential strategy versus a solo-IGRA strategy at different time periods from a centralized refugee clinic. The primary outcome was the proportion completing LTBI screening in each cohort. RESULTS: A total of 471 subjects were included (240 in sequential screening, 231 in solo-IGRA screening). 54% of refugees completed LTBI screening with sequential testing, compared to 85% of those screened with a solo-IGRA. Time to completing screening was also shorter in the solo-QFT group (difference 16.5 days, p < 0.01, 95% confidence interval 9.3, 23.7). There was a higher incidence of LTBI diagnosis in the solo-IGRA group (41 versus 20, p = 0.002). Screening completion was predicted by solo-IGRA screening (aOR 3.74, 95% confidence interval 2.30, 6.09; p < 0.001) and if refugees were privately-sponsored (aOR 2.81, 95% confidence interval 1.53, 5.15; p = 0.001). Treatment completion rates did not differ between groups. CONCLUSION: This study has identified fewer dropouts in the LTBI cascade of care if a solo-IGRA strategy is used for screening. An IGRA should be strongly considered as the screening method for refugees arriving in low-incidence settings if resources are available.

Acetonitrile as a buffer additive for free zone capillary electrophoresis separation and characterization of maize (Zeamays L. ) and sorghum (Sorghum bicolor L. Moench) storage proteins.

An improved method for separating and characterizing maize (Zea mays L.) and sorghum (Sorghum bicolor L. Moench) storage proteins by free zone capillary electrophoresis (FZCE) was developed. Previous electrophoretic methods for analyzing these proteins required high concentrations of urea to maintain protein solubility during separation. To overcome disadvantages of urea, we developed a FZCE method that mimicked reversed-phase high-performance liquid chromatography (RP-HPLC) in that it used high levels of acetonitrile (ACN) at low pH. The optimized FZCE buffer system consisted of 80 mM phosphate-glycine buffer, nominal pH 2.5, containing 60% ACN and a cellulose derivative to dynamically coat capillary walls. Resolution was similar to or higher than that previously achieved by FZCE buffers utilizing 8 M urea as a buffer additive. ACN concentrations of at least 50% were necessary to achieve acceptable separations; this ACN concentration is approximately that necessary to extract these storage proteins. ACN was equally effective as traditional ethanol solvents and 8 M urea for solubilizing maize and sorghum proteins. The ACN-based FZCE buffer system gave high repeatability (<0.3% relative standard deviation, measured over 15 consecutive injections) for migration time. Subclasses of maize and sorghum storage proteins were identified, and genotypes of each cereal were successfully differentiated using ACN-containing buffers. This FZCE method may be applicable for the analysis of other hydrophobic proteins without the use of urea.

High-performance capillary electrophoresis of cereal proteins.

Cereal grains are widely used of human foods and animal feed throughout the world. Cereals provide dietary protein, which also often has a functional role, as wheat gluten does in bread. Cereal proteins are unique in many ways: they are highly complex and heterogeneous, are often difficult to extract, and aggregate readily, making them difficult to characterize. Because of the economic importance and widespread use of cereal proteins, however, many techniques have been used for their analysis. High-performance capillary electrophoresis (HPCE) is one of the newest techniques to be so used. This review describes the development of charge- and size-based HPCE methods for analysis of cereal grain proteins, and the use of these methods for cultivar identification, classification, and prediction of quality. HPCE is versatile, rapid, easily automated, readily quantified, and provides high-resolution separations. Clearly, HPCE is a valuable addition to other methods of cereal protein analysis and should, in time, be applicable to all protein classes from all cereals.

Quantitative genetic studies of A/B zeins using a new model to test non-allelic interactions.

A genetic model developed by Bogyo et al. (1988) for quantitatively inherited triploid endosperm characters (an extension of the well-known Mather-Jinks model) is not well-suited for estimating epistatic interaction effects because it requires the assumption that, in segregating loci, all alleles positively affecting a particular character are in one of the inbred parental lines. To better explain zein inheritance in maize, a new model was developed not relying on this assumption. This model was tested by quantitative analysis of A/B zeins, the predominant prolamin storage proteins of maize, using reversed phase high-performance liquid chromatography of two inbred lines, their reciprocal F1 crosses, the F2 generation, backcrosses, and reciprocal backcrosses to both parent lines. The model required epistatic components to be included for an excellent fit for most protein peaks.

Electrophoresis and chromatography of wheat proteins: available methods, and procedures for statistical evaluation of the data.

Analysis of gluten proteins from the wheat grain endosperm has long challenged the analytical chemist. Several hundred unique polypeptides are present, many in large polymers. This complexity, plus useful relationships of composition to genotype and quality, encouraged development and application of electrophoresis and chromatography for gluten analysis. We review the methods of polyacrylamide gel electrophoresis, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, isoelectric focusing and high-performance liquid chromatography available for study of wheat proteins. Singly and in combination, they provide rapid, reproducible, high-resolution separations based on size, charge, or surface hydrophobicity. As challenging and important as the analyses themselves, however, is interpretation of data. Subjective evaluation is sometimes possible, but statistical methods such as similarity scores, clustering, principal components, multiple linear regression, and partial least squares now are increasingly used for data analysis. We review the use of these procedures, and precautions necessary to avoid misinterpretation of data. Optimal evaluation of protein analytical data will enhance the value of such analyses in wheat breeding, marketing, and processing.

Expression of A/B zeins in single and double maize endosperm mutants.

Zeins, the major endosperm proteins in maize (Zea mays L.), are deficient in the essential amino acids lysine and tryptophan. Some mutant genes, like opaque-2 (o2) and floury-2 (fl2), reduce the levels of A- and B-zeins, thereby improving maize's nutritional value. Other mutants, such as amylose-extender (ae), floury-1 (fl1), soft starch (h), dull-1 (du), shrunken-1 (sh1), sugary-1 (su1), sugary-2 (su2), and waxy (wx), primarily affect starch composition, but also alter zein composition. We undertook this study to examine the effects of some of these mutant genes on A/B-zein composition and to study the interactions of these genes in double-mutant combinations. Endosperm prolamins were extracted from inbred B37, ten near-isogenic single mutants (ae, du, fl1, fl2, h, o2, sh1, su1, su2, and wx), and most double-mutant combinations. Zeins in these extracts were fractionated by reversed-phase highperformance liquid chromatography (RP-HPLC) into 22-24 peaks. Of the resulting 22 major peaks the areas of 16 (per milligram endosperm) were significantly affected by individual mutant genes relative to the zein composition of the normal inbred. In combination these genes exhibited significant epistatic interactions in regulating the expression of individual A/B zeins. Epistatic interactions were judged to be significant when the amount of a peak in a double mutant differed from the averages for the peak in the two respective single mutants. The o2 gene, alone and in combination with other mutant genes, significantly decreased the amounts of many individual zeins. The effect of the o2 gene was the greatest of all the genes examined. Various clustering techniques were used to see if mutant effects could be grouped; among these was principal component analysis, a multivariate statistical technique that analyzes all peak sizes simultaneously. Three-dimensional scatter graphs were constructed based on the first three principal components. For the single mutants, these showed no relationships to gene actions; for the double mutants, however, this technique showed that four single mutants, o2, sh1, su1 and su2, had the greatest effects on zein composition when combined with each other and with the remaining six single mutants.

Expression of alcohol-soluble endosperm proteins in maize single and double mutants.

Many maize (Zea mays L.) mutant genes exist. Some affect protein content or composition, while others modify carbohydrates or kernel phenotype. In doublemutant lines, two mutant genes are present. We know little about interactions of such genes, however. We therefore examined a normal maize inbred, B37, 10 near-isogenic single mutants and 46 double mutants to analyze quantitative effects on alcohol-soluble endosperm proteins. Proteins were extracted with 70% ethanol0.5% sodium acetate-5% mercaptoethanol, and fractionated by reversed-phase high-performance liquid chromatography (RP-HPLC). Early peaks were alcohol-soluble glutelin (ASG) subunits, while late peaks contained zein. Results were quantified and statistically analyzed. In many double mutants, protein compositions differed significantly from averages of compositions of corresponding single mutants. For example, a high-methionine, water-insoluble ASG is absent when the opaque-2 (o2) gene combines with shrunken-1 (sh1) or surgary-1 (su1). Another water-insoluble ASG nearly doubled when floury-2 (fl2) andsu1 combined. A high-proline, high-histidine, water-soluble ASG nearly doubled in combinations offl2 witho2,su1 and sugary-2 (su2). Zein was about half its expected value wheno2 combined with amylose-extender (ae), floury-1 (fl1), soft-starch (h),sh1 andsu1. Thus, rapid protein extraction and quantitative RP-HPLC showed major new epistatic and synergistic effects of several mutant genes on protein composition. Unexpectedly, these effects often involve genes that primarily affect starch composition or kernel phenotype. Alcohol-soluble proteins often vary in amount, as ino2 lines. They also differ in nutritional value. Thus, RP-HPLC analysis of these proteins can identify nutritionally superior genotypes, and may help explain the basis of such quality.

A 23.8-kD alpha-zein with N-terminal sequence and immunological properties similar to 26.7-kD alpha-zeins.

A 23.8-kD alpha-zein polypeptide, K55PC7, has been shown to be a truncated member of the 26.7-kD alpha-zein class based on its amino acid composition, N-terminal sequence, and immunological properties. This unusual polypeptide was isolated by chromatographing whole alpha-zein from inbred K55. The N-terminal sequence of K55PC7 is highly homologous to those of 4 putative 26.7-kD alpha-zeins but shows no homology to those of 10 putative alpha-zeins that belong to the 23.8-kD class. Its higher valine and lower phenylalanine contents also suggest that K55PC7 is a member of the 26.7-kD class. In addition, studies with antibodies raised to peptides corresponding to regions unique to each of the two alpha-zein classes indicate that K55PC7 has immunological similarity to 26.7-kD alpha-zeins. Peptide mapping data suggest that K55PC7 is not the putative product of the truncated 26.7-kD alpha-zein gene zA1 isolated from inbred W64A and described by Spena et al. [26]. It appears that K55PC7 occurs as a major component in inbred K55 and is a truncated version of a 26.7-kD alpha-zein, arisen either by an internal deletion or premature termination due to a nonsense mutation.

Chromosomal control of wheat gliadin: analysis by reversed-phase high-performance liquid chromatography.

Gliadin proteins of the hexaploid wheat variety 'Chinese Spring', and of its nullisomic-tetrasomic and ditelocentric aneuploid lines, were analyzed by reversed-phase high-performance liquid chromatography (RP-HPLC). Reversed-phase separations were carried out at 70°C on C8 and C18 columns using a gradient of increasing acetonitrile concentration in the presence of 0.1% trifluoroacetic acid. Thirty-five components were separated and all were found to be controlled by genes on the short arms of group 1 and group 6 chromosomes (the complex Gli-1 and Gli-2 loci). Results indicated that gluten polypeptides elute as groups in order of increasing hydrophobicity in the following approximate order: (1) albumins plus globulins, (2) ω-gliadins, (3) high molecular weight (MW) glutenin subunits, (4) α-type gliadins, (5) low MW glutenin subunits, and (6) γ-gliadins. The three distinct protein types coded by genes at the complex Gli-I loci (ω-gliadins, γ-gliadins, and low MW glutenin subunits) thus have uniquely different surface hydrophobicities. Similarly, gene locations for hexaploid 'Cheyenne' gliadins and durum gliadin proteins in the varieties 'Langdon', 'Edmore', and 'Kharkovskaya-5' were determined through RP-HPLC analysis of aneuploid lines. All results confirm known locations of genes for gliadin proteins, and demonstrate that RP-HPLC is a powerful new tool for analysis of gliadins in breeding and genetic studies.

Chromosomal control of glutenin subunits in aneuploid lines of wheat: analysis by reversed-phase high-performance liquid chromatography.

Glutenin subunits from nullisomic-tetrasomic and ditelocentric lines of the hexaploid wheat variety 'Chinese Spring' (CS) and from substitution lines of the durum wheat variety 'Langdon' were fractionated by reversed-phase high-performance liquid chromatography (RP-HPLC) at 70 °C using a gradient of acetonitrile in the presence of 0.1% trifluoroacetic acid. Nineteen subunits were detected in CS. The presence and amounts of four early-eluted subunits were found, through aneuploid analysis, to be controlled by the long arms of chromosomes 1D (1DL) (peaks 1-2) and 1B (1BL) (peaks 3-4). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that these four subunits are the high molecular weight subunits of glutenin, which elute in the order 1Dy, 1Dx, 1By, and 1Bx. Similar amounts of 1DL subunits were present (6.3 and 8.8% of total glutenin), but 1BL subunits differed more in abundance (5.4 and 9.5%, respectively). Results indicate that most late-eluting CS glutenin subunits were coded by structural genes on the short arms of homoeologous group 1 chromosomes: 6 by 1DS, 5 by 1AS, and 4 by 1BS. Glutenin of tetraploid 'Langdon' durum wheat separated into nine major subunits: 6 were coded by genes on 1B chromosomes, and 3 on 1A chromosomes. Gene locations for glutenin subunits in the tetraploid durum varieties 'Edmore' and 'Kharkovskaya-5' are also given. These results should make RP-HPLC a powerful tool for qualitative and quantitative genetic studies of wheat glutenin.

Separation of cereal proteins by reversed-phase high-performance liquid chromatography.

Cereal proteins have been extremely difficult to purify and characterize owing to their heterogeneity, poor solubility and tendency to polymerize. High-performance liquid chromatography (HPLC) on a 300 A reversed-phase (RP)(C18) support (Syn Chropak RP-P), using acetonitrile as organic modifier in the presence of trifluoroacetic acid, has been found to be capable of high-resolution separations of these proteins; the resolution is often better than that obtained by any other chromatographic or electrophoretic method. Examples are presented showing separations of low-molecular-weight gliadins, omega-gliadins and ethanol-soluble reduced glutenin subunits from wheat and of zein from corn. In addition, proteins may be directly extracted from ground single kernels and subsequently analyzed by RP-HPLC; applications in genetic studies, in breeding programs and in varietal identification are proposed. In addition to its high resolution, RP-HPLC is superior to most other methods in speed, sensitivity, reproducibility and suitability for quantitation. Polypeptide chains of molecular weight up to 133,000 are recovered in high yields and the column capacity is high, demonstrating that RP-HPLC is suitable for both preparative and analytical separations of proteins. RP-HPLC resolves proteins primarily on the basis of differences in surface hydrophobicity, so it therefore complements, rather than duplicates, other techniques that separate proteins on the basis of size or charge. RP-HPLC promises to become an invaluable technique for the fractionation and characterization of proteins from cereals and other sources.

Cereal prolamin evolution and homology revealed by sequence analysis.

Prolamin mixtures were isolated from oats, rice, normal and high-lysine sorghum, two varieties of pearl millet, two strains of teosinte, and gamma grass and subjected to NH2-terminal amino acid sequence determinations. In each case (except for rice, whose prolamins apparently have blocked or unavailable NH2-terminal residues), primarily a single sequence was observed despite significant heterogeneity, suggesting that prolamin homology in each cereal arose through duplication and mutation of a single ancestral gene. Comparisons were then made to prolamin sequences previously determined for wheat, corn, barley, and rye. Within genera, different varieties or subspecies exhibited few differences, but more distantly related genera, subtribes, and tribes showed increasingly large differences. Within the subfamily Festucoideae, no homology was apparent between prolamins of oats and those of the subtribe Triticinae (including wheat, rye, and barley, for which prolamin homology was previously demonstrated). Within the subfamily Panicoideae, corn was shown to be closely related to teosinte but more distantly to Tripsacum. Sorghum was shown to have diverged less from corn than had millet. These comparisons demonstrate that prolamin sequence analyses can successfully predict and clarify evolutionary relationships of cereals.

Disulfide bonds: key to wheat protein functionality.

Disulfide bonds in wheat proteins are major factors that determine the properties of the proteins and their functionality in wheat flour. The gliadin proteins contain mostly intramolecular disulfide bonds. In contrast, the high-molecular-weight glutenins are formed by disulfide linkages of several diverse polypeptide chains which have been separated and characterized. The linkage of these proteins in a fairly linear array contributes to the unique viscoelastic properties of glutenin. The glutenin has been separated into two fractions differing in molecular weight. The amount of highest molecular weight component is correlated with the rheological behavior of the flours from different wheat varieties. Various oxidizing and reducing agents are widely used to alter the functional behavior of wheat proteins by the action on sulfhydryl and disulfide groups.