Simultaneous atomic-resolution electron ptychography and Z-contrast imaging of light and heavy elements in complex nanostructures.

The aberration-corrected scanning transmission electron microscope (STEM) has emerged as a key tool for atomic resolution characterization of materials, allowing the use of imaging modes such as Z-contrast and spectroscopic mapping. The STEM has not been regarded as optimal for the phase-contrast imaging necessary for efficient imaging of light materials. Here, recent developments in fast electron detectors and data processing capability is shown to enable electron ptychography, to extend the capability of the STEM by allowing quantitative phase images to be formed simultaneously with incoherent signals. We demonstrate this capability as a practical tool for imaging complex structures containing light and heavy elements, and use it to solve the structure of a beam-sensitive carbon nanostructure. The contrast of the phase image contrast is maximized through the post-acquisition correction of lens aberrations. The compensation of defocus aberrations is also used for the measurement of three-dimensional sample information through post-acquisition optical sectioning.

Evaluation of the non-toxic mutant of the diphtheria toxin K51E/E148K as carrier protein for meningococcal vaccines.

Diphtheria toxin mutant CRM197 is a common carrier protein for glycoconjugate vaccines, which has been proven an effective protein vector for, among others, meningococcal carbohydrates. The wide-range use of this protein in massive vaccine production requires constant increase of production yields and adaptability to an ever-growing market. Here we compare CRM197 with the alternative diphtheria non-toxic variant DT-K51E/E148K, an inactive mutant that can be produced in the periplasm of Escherichia coli. Biophysical characterization of DT-K51E/E148K suggested high similarity with CRM197, with main differences in their alpha-helical content, and a suitable purity for conjugation and vaccine preparation. Meningococcal serogroup A (MenA) glycoconjugates were synthesized using CRM197 and DT-K51E/E148K as carrier proteins, obtaining the same conjugation yields and comparable biophysical profiles. Mice were then immunized with these CRM197 and DT-K51E/E148K conjugates, and essentially identical immunogenic and protective effects were observed. Overall, our data indicate that DT-K51E/E148K is a readily produced protein that now allows the added flexibility of E. coli production in vaccine development and that can be effectively used as protein carrier for a meningococcal conjugate vaccine.

Rewritable glycochips.

We describe microarraying of carbohydrates for protein screening using either disulfide bridge or Schiff base imine immobilization chemistries on plasmachemical deposited functional nanolayers. The commonly observed issue of nonspecific background binding of proteins is overcome by spotting carbohydrates through a protein-resistant overlayer yielding spatially localized interaction with a reactive functional underlayer.

Tailoring the substrate specificity of the beta-glycosidase from the thermophilic archaeon Sulfolobus solfataricus.

The substrate specificity of the thermophilic beta-glycosidase (lacS) from the archaeon Sulfolobus solfataricus (SSbetaG), a member of the glycohydrolase family 1, has been analysed at a molecular level using predictions from known protein sequences and structures and through site-directed mutagenesis. Three critical residues were identified and mutated to create catalysts with altered and broadened specificities for use in glycoside synthesis. The wild-type (WT) and mutated sequences were expressed as recombinant fusion proteins in Escherichia coli, with an added His(6)-tag to allow one-step chromatographic purification. Consistent with side-chain orientation towards OH-6, the single Met439-->Cys mutation enhances D-xylosidase specificity 4.7-fold and decreases D-fucosidase activity 2-fold without greatly altering its activity towards other D-glycoside substrates. Glu432-->Cys and Trp433-->Cys mutations directed towards OH-4 and -3, respectively, more dramatically impair glucose (Glc), galactose (Gal), fucose specificity than for other glycosides, resulting in two glycosidases with greatly broadened substrate specificities. These include the first examples of stereospecificity tailoring in glycosidases (e.g. WT-->W433C, k(cat)/K(M) (Gal):k(cat)/K(M) (mannose (Man))=29.4:1-->1.2:1). The robustness and high utility of these broad specificity SSbetaG mutants in parallel synthesis were demonstrated by the formation of libraries of beta-glycosides of Glc, Gal, xylose, Man in one-pot preparations at 50 degrees C in the presence of organic solvents, that could not be performed by SSbetaG-WT.

Ready protease-catalyzed synthesis of carbohydrate-amino acid conjugates.

The protease-catalyzed synthesis of amino acid est-carbohydrate conjugates as glycopeptide analogues has been achieved in a highly regioselective and carbohydrate-specific manner using amino acid vinyl ester acyl donors and minimally or completely unprotected carbohydrate acyl acceptors, which together probed active sites of proteases to reveal yield efficiencies that are modulated by the carbohydrate C-2 substitutent, and that may be exploited to allow selective one-pot syntheses.

Controlled site-selective protein glycosylation for precise glycan structure-catalytic activity relationships.

Glycoproteins occur naturally as complex mixtures of differently glycosylated forms which are difficult to separate. To explore their individual properties, there is a need for homogeneous sources of carbohydrate-protein conjugates and this has recently prompted us to develop a novel method for the site-selective glycosylation of proteins. The potential of the method was illustrated by site-selective glycosylations of subtilisin Bacillus lentus (SBL) as a model protein. A representative library of mono- and disaccharide MTS reagents were synthesized from their parent carbohydrates and used to modify cysteine mutants of SBL at positions 62 in the S2 site, 156 and 166 in the S1 site and 217 in the S1' site. These were the first examples of preparations of homogeneous neoglycoproteins in which both the site of glycosylation and structure of the introduced glycan were predetermined. The scope of this versatile method was expanded further through the combined use of peracetylated MTS reagents and careful pH adjustment to introduce glycans containing different numbers of acetate groups. This method provides a highly controlled and versatile route that is virtually unlimited in the scope of the sites and glycans that may be conjugated, and opens up hitherto inaccessible opportunities for the systematic determination of the properties of glycosylated proteins. This potential has been clearly demonstrated by the determination of detailed glycan structure-hydrolytic activity relationships for SBL. The 48 glycosylated CMMs formed display kcat/KM values that range from 1.1-fold higher than WT to 7-fold lower than WT. The anomeric stereochemistry of the glycans introduced modulates changes in kcat/KM upon acetylation. At positions 62 and 217 acetylation enhances the activity of alpha-glycosylated CMMs but decreases that of beta-glycosylated. This trend is reversed at position 166 where, in contrast, acetylation enhances the kcat/KMs of beta-glycosylated CMMs but decreases those of alpha-glycosylated. Consistent with its surface exposed nature changes at position 156 are more modest, but still allow control of activity, particularly through glycosylation with disaccharide lactose.

Site-selective glycosylation of subtilisin Bacillus lentus causes dramatic increases in esterase activity.

Using site directed mutagenesis combined with chemical modification, we have developed a general and versatile method for the glycosylation of proteins which is virtually unlimited in the scope of proteins and glycans that may be conjugated and in which the site of glycosylation and the nature of the introduced glycan can be carefully controlled. We have demonstrated the applicability of this method through the synthesis of a library of 48 glycosylated forms of the serine protease subtilisin Bacillus lentus (SBL) as single, pure species. As part of our ongoing program to tailor the activity of SBL for use in peptide synthesis, we have screened these enzymes for activity against the esterase substrate succinyl-Ala-Ala-Pro-Phe-S-benzyl. Gratifyingly, 22 enzymes displayed greater than wild type (WT) activity. Glycosylation at positions 62, in the S2 pocket, resulted in five glycosylated forms of SBL that were 1.3- to 1.9-fold more active than WT. At position 217, in the S1' pocket, all glycosylations increased kcat/KM up to a remarkable 8.4-fold greater than WT for the glucosylated enzyme L217C-S-beta-Glc(Ac)3. Furthermore, the ratio of amidase to esterase activity, (kcat/KM)esterase/(kcat/KM)amidase (E/A), is increased relative to wild type for all 48 glycosylated forms of SBL. Again, the most dramatic changes are observed at positions 62 and 217 and L217C-S-beta-Glc(Ac)3 has an E/A that is 17.2-fold greater than WT. The tailored specificity and high activity of this glycoform can be rationalized by molecular modeling analysis, which suggests that the carbohydrate moiety occupies the S1' leaving group pocket and enhances the rate of deacylation of the acyl-enzyme intermediate. These glycosylated enzymes are ideal candidates for use as catalysts in peptide synthesis as they have greatly increased (kcat,KM)esterase and severely reduced (kcat/KM)amidase and will favor the formation of the amide bond over hydrolysis.

The controlled introduction of multiple negative charge at single amino acid sites in subtilisin Bacillus lentus.

The use of methanethiosulfonates as thiol-specific modifying reagents in the strategy of combined site-directed mutagenesis and chemical modification allows virtually unlimited opportunities for creating new protein surface environments. As a consequence of our interest in electrostatic manipulation as a means of tailoring enzyme activity and specificity, we have adopted this approach for the controlled incorporation of multiple negative charges at single sites in the representative serine protease, subtilisin Bacillus lentus (SBL). A series of mono-, di- and triacidic acid methanethiosulfonates were synthesized and used to modify cysteine mutants of SBL at positions 62 in the S2 site, 156 and 166 in the S1 site and 217 in the S1' site. Kinetic parameters for these chemically modified mutant (CMM) enzymes were determined at pH 8.6 under conditions which ensured complete ionization of the unnatural amino acid side-chains introduced. The presence of up to three negative charges in the S1, S1' and S2 subsites of SBL resulted in up to 11-fold lowered activity, possibly due to interference with oxyanion stabilization of the transition state of the hydrolytic reactions catalyzed. Each unit increase in negative charge resulted in a raising of K(M) and a reduction of k(cat). However, no upper limit was observed for increases in K(M), whereas decreases in k(cat) reached a limiting value. Comparison with sterically similar but uncharged CMMs revealed that electrostatic effects of negative charges at positions 62, 156 and 217 are detrimental, but are beneficial at position 166. These results indicate that the ground-state binding of SBL to the standard substrate, Suc-AAPF-pNA, to SBL is reduced, but without drastic attenuation of catalytic efficiency, and show that SBL tolerates high levels of charge at single sites.

Altering the specificity of subtilisin Bacillus lentus through the introduction of positive charge at single amino acid sites.

The use of methanethiosulfonates as thiol-specific modifying reagents in the strategy of combined site-directed mutagenesis and chemical modification allows virtually unlimited opportunities for creating new protein surface environments. As a consequence of our interest in electrostatic manipulation as a means of tailoring enzyme activity and specificity, we have recently adopted this approach for the controlled incorporation of multiple negative charges at single sites in the representative serine protease, subtilisin Bacillus lentus (SBL). We now describe the use of this strategy to introduce multiple positive charges. A series of mono-, di- and triammonium methanethiosulfonates were synthesized and used to modify cysteine mutants of SBL at positions 62 in the S2 site, 156 and 166 in the S1 site and 217 in the S1' site. Kinetic parameters for these chemically modified mutants (CMM) enzymes were determined at pH 8.6. The presence of up to three positive charges in the S1, S1' and S2 subsites of SBL resulted in up to 77-fold lowered activity, possibly due to interference with the histidinium ion formed in the transition state of the hydrolytic reactions catalyzed.

The impact of elevated serum IgA and race on primary recipient renal allograft survival.

This study correlated overall serum IgA levels in pretransplant (preTx) sera with graft survival. IgA levels, determined by nephelometry, were normally distributed, with a mean level of 255 +/- 139 mg/dl and a median of 234 mg/dl in 631 adult primary kidney allograft recipients and a mean level of 213 +/- 123 mg/dl with a median of 196 mg/dl for 100 retransplant recipients. Improved 3-year survival was associated with a high preTx IgA serum level in primary recipients (Kaplan-Meier analysis, P = 0.01), but not in retransplant patients. After stratifying by race, IgA correlated with graft survival in Caucasian, Hispanic, and "other" (Middle Eastern, Indian subcontinent, and Asian) primary recipients (P < or = 0.04), but not in African Americans. Higher survival rates were not associated with IgA in primary recipients stratified for rejection episodes, blood transfusions, or HLA-DR mismatches. Graft survival was improved in patients with > 2 HLA-AB mismatches and serum IgA above the median. PreTx IgA level and IgA alpha-HLA activity were significantly associated in preTx sera of primary renal allograft recipients (chi 2 = 7.145, P = 0.01), although only 9% (12/133) of sera tested displayed IgA anti-HLA class I reactivity. Thus, enhanced graft survival mediated by elevated serum IgA levels may due in part to competition for allograft HLA class I binding with deleterious Ig subclasses or immune effector cells. Elevated serum IgA may also reflect an altered immunoregulatory state. The results suggest that, depending on the racial group, preTx serum IgA levels are a prognostic indicator of graft survival in primary renal allograft recipients.

Reactivity of serologic tests for the detection of antibody specific to cytomegalovirus. II. Latex agglutination and enzyme-linked immunosorbent assay.

Two hundred seven sera were assayed for antibody-specific for cytomegalovirus (CMV) by two enzyme immunoassays (EIAs; Bioenzabody, Litton Bionetics, and Abbott CMV Total Antibody EIA, Abbott Laboratories) and latex agglutination (LA; CMVScan, Hynson, Westcott and Dunning). The overall accuracy of the LA, Litton EIA, and Abbott EIA was 95.8%, 86.2%, and 88.6%, respectively. Although the Abbott EIA had a sensitivity of 98.8%, the specificity was only 35.5%. The positive predictive values of the LA, Litton EIA, and Abbott EIA were 99.4%, 95.9%, and 88.9%, respectively, while the negative predictive values of each of these tests were 81.1%, 56.2%, and 84.6%, respectively.

Reactivity of serologic tests for the detection of antibody specific to cytomegalovirus.

A total of 212 sera were assayed for antibody specific for cytomegalovirus by complement fixation (CF), indirect immunofluorescence (IFA Electro-Nucleonics Laboratory, Inc., Bethesda, MD), ELISA (Cordis Laboratories, Inc., Miami, FL) and the FIAX system (International Diagnostic Technology, Santa Clara, CA). Correlation of CF with IFA, ELISA, and FIAX was 61%, 78%, and 71%, respectively. Quantitative correlation between IFA and FIAX and ELISA was not possible because of the broad range of reaction intensity of the latter two tests in sera with a particular IFA titer.

Development of competency-based, career-entry examination for clinical laboratory personnel.

The process of developing a competency-based credentialing examination for career-entry practitioners is described, including a review of pertinent literature. A modified Delphi technique was used to achieve consensus among a panel of experts with respect to Career-Entry Statements of Competence. Items were written which were referenced specifically to one or more of the statements. The resulting genralist examination for the technologist and technician personnel levels is the first such examination to be reported in the field of clinical laboratory sciences which utilized a formal process involving such large numbers of practicing professionals.