Affinity-free enrichment and mass spectrometry analysis of the ovarian cancer biomarker CA125 (MUC16) from patient-derived ascites.

Developing a mass spectrometry-based assay for the ovarian cancer biomarker CA125 (MUC16) is a desirable goal, because it may enable detection of molecular regions that are not recognized by antibodies and are therefore analytically silent in the current immunoassay. Additionally, the ability to characterize the CA125 proteoforms expressed by individuals may offer clinical insight. Enrichment of CA125 from malignant ascites may provide a high-quality source of this important ovarian cancer biomarker, but a reliable strategy for such enrichment is currently lacking. Beginning with crude ascites isolated from three individual patients with high grade serous ovarian cancer, we enriched for MUC16 using filtration, ion exchange, and size exclusion chromatography and then performed bottom-up proteomics on the isolated proteins. This approach of enrichment and analysis reveals that the peptides detected via mass spectrometry map to the SEA domain and C-loop regions within the tandem repeat domains of CA125 and that peptide abundance correlates with clinical CA125 counts.

Adsorbed Layer Thickness Determination for Convective-Based Media from Pressure Drop Data.

In the present work, we derive a theoretical framework to determine the adsorbed layer thickness from pressure drop measurements for convective-based media without any assumptions about the geometry of the pore structure of the stationary phase matrix. Equations are presented to calculate accuracy of the estimated adsorbed layer thickness as a consequence of measurement error and approximations of the mathematical model. We discovered that there is a minimum in the error for certain pressure drops that results in optimal experimental conditions for determining the adsorbed layer thickness. We demonstrate that the adsorbed layer thickness can be determined with less than 10% error using a wide range of experimental conditions simply from pressure drop data. By careful selection of porous bed dimensions and flow rates, the adsorbed layer thicknesses from subnanometer dimensions up to several hundred nanometers can be determined by measurement of the pressure drop in a range of several bars. The method was experimentally tested on methacrylate monolithic columns using monodisperse latex nanoparticles as a reference standard and two different proteins as unknowns demonstrating close agreement with calculations.

Male circumcision and HIV prevention: looking to the future.

Now that male circumcision has been shown to have a protective effect for men against HIV infection when engaging in vaginal intercourse with HIV-infected women, the research focus needs to shift towards the operational studies that can pave the way for effective implementation of circumcision programs. Behavioral research is needed to find out how people perceive the procedure and the barriers to and facilitators of uptake. It should also assess the risk of an increase in unsafe sex after circumcision. Social research must examine cultural perceptions of the practice, in Africa and beyond, including how likely uncircumcised communities are to access surgery and what messages are needed to persuade them. Advocates of male circumcision would benefit from research on how to influence health policy-makers, how best to communicate the benefits to the public, and how to design effective delivery models.

Design of salt-tolerant membrane adsorbers for viral clearance.

Strong anion exchange chromatography has frequently been employed as a viral clearance step during downstream processing of biological therapeutics. When challenged with viruses having only slightly acidic isoelectric points, the performance of the anion exchange operation becomes highly dependent on the buffer salt concentration, with the virus log reduction value (LRV) dropping dramatically in buffers with 50-150 mM salt. In this work, a series of anion exchange membrane adsorbers utilizing alternative ligand chemistries instead of the traditional quaternary amine (Q) ligand have been developed that overcome this limitation. Four different ligands (agmatine, tris-2-aminoethyl amine, polyhexamethylene biguanide, and polyethyleneimine) achieved >5 LRV of bacteriophage PhiX174 (pI approximately 6.7) at pH 7.5 and up to 150 mM salt, compared to 0 LRV for the Q ligand. By evaluating structural derivatives of the successful ligands, three factors were identified that contributed to ligand salt tolerance: ligand net charge, ligand immobilization density on the membrane, and molecular structure of the ligand-binding group. Based on the results of this study, membrane adsorbers that incorporate alternative ligands provide a more robust and salt tolerant viral clearance-processing step compared to traditional strong anion exchange membrane adsorbers.

Hydrolysis of Whey Protein-Dextran Glycates Made Using the Maillard Reaction.

Protein-polysaccharide glycates are food ingredients that use the Maillard reaction to form a Schiff base linkage between the carbonyl of a polysaccharide and the free amino moiety of a protein. Glycates are excellent emulsification, foaming, and gelling agents in foods and improve protein solubility and heat stability. The present work examined if glycates dissociate by hydrolysis, returning to free un-glycated protein and dextran due to the reversibility of the Schiff base linkage. Hydrolysis of glycates made from whey protein isolate and dextran was measured versus time and temperature, allowing determination of the rate constants and equilibrium constants for glycate hydrolysis. Glycates underwent hydrolysis when placed into aqueous solutions at common food processing temperatures. For example, during hot food storage (60 °C), equilibrium fractional hydrolysis was 44%, whereas at ambient temperature (22 °C), it was 8%. The present work aims to increase the successful use of glycates in new foods by knowing what foods and conditions avoid glycate hydrolysis.

Kinetics of Whey Protein Glycation Using Dextran and the Dry-Heating Method.

Glycation of proteins by polysaccharides via the Maillard reaction improves the functional properties of proteins in foods, such as solubility, heat stability, emulsification, foaming, and gelation. Glycation is achieved by either the dry heating or the wet heating method, and considerable research has been reported on the functionality of the reaction mixture as tested in foods. While the characteristics of the glycates in foods have been well studied, the kinetics and equilibrium yield of the protein-polysaccharide glycation reaction has received little attention. Industrial manufacture of the glycates will require understanding the kinetics and yield of the glycation reaction. This work examined the glycation of whey protein isolate (WPI) and glycomacropeptide (GMP) by using dextran and the dry-heating method at 70 °C and 80% relative humidity. The disappearance of un-glycated protein and the creation of glycated protein were observed using chromatographic analysis and fluorescence laser densitometry of sodium dodecyl sulfate-polyacrylamide gels. Data were fit using a first-order reversible kinetic model. The rate constants measured for the disappearance of un-glycated protein by sodium dodecyl sulfate-polyacrylamide (SDS-PAGE) (k = 0.33 h-1) and by chromatographic analysis (k = 0.38 h-1) were not statistically different from each other for WPI-dextran glycation. Dextran glycation of GMP was slower than for WPI (k = 0.13 h-1). The slower rate of glycation of GMP was attributed to the 50% lower Lys content of GMP compared to WPI. Yield for the dry-heating dextran glycation method was 89% for WPI and 87% for GMP. The present work is useful to the food industry to expand the use of glycated proteins in creating new food products.

Dietary glycomacropeptide supports growth and reduces the concentrations of phenylalanine in plasma and brain in a murine model of phenylketonuria.

Phenylketonuria (PKU) is a genetic disorder caused by deficiency of phenylalanine hydroxylase (PAH) that requires life-long adherence to a low-phenylalanine (Phe) diet. Glycomacropeptide (GMP) is uniquely suited to the nutritional management of PKU, because pure GMP contains no Phe. Our aim was to assess how ingestion of diets containing GMP support growth and affect the concentrations of amino acids in plasma and brains of mice with a deficiency of PAH, the Pah(enu2) mouse (PKU mouse). Experiments were conducted in 4- to 6-wk-old wild-type (WT) (C57Bl/6) and PKU mice fed diets containing 20% protein from casein, amino acids, or GMP supplemented with limiting indispensable amino acids (IAA). PKU mice fed the GMP diet showed gains in body weight, feed efficiency, and a protein efficiency ratio that did not differ from the amino acid diet. The concentrations of isoleucine and threonine in plasma showed a significant 2- to 3-fold increase for WT and PKU mice fed GMP compared with casein or amino acid diets, respectively. PKU mice fed the GMP diet had decreased concentrations of Phe in plasma (11% decrease) and in 5 regions of the brain (20% decrease) compared with the amino acid diet. The concentration of Phe in the brain was inversely correlated with the concentrations of isoleucine, threonine, and valine in plasma (R2 = 0.74; P < 0.0001), suggesting competitive inhibition of Phe transport into the brain. In summary, PKU mice fed GMP showed comparable growth and reduced concentrations of Phe in plasma and the brain compared with an amino acid diet. These data support the use of GMP supplemented with IAA as an alternative source of dietary protein for individuals with PKU.

A consensus rating method for small virus-retentive filters. I. Method development.

Virus filters are membrane-based devices that remove large viruses (e.g., retroviruses) and/or small viruses (e.g., parvoviruses) from products by a size exclusion mechanism. In 2002, the Parenteral Drug Association (PDA) organized the PDA Virus Filter Task Force to develop a common nomenclature and a standardized test method for classifying and identifying viral-retentive filters. One goal of the task force was to develop a test method for small virus-retentive filters. Because small virus-retentive filters present unique technical challenges, the test method development process was guided by laboratory studies to determine critical variables such as choice of bacteriophage challenge, choice of model protein, filtration operating parameters, target log10 reduction value, and filtration endpoint definition. Based on filtration, DLS, electrospray differential mobility analysis, and polymerase chain reaction studies, a final rating based on retention of bacteriophage PP7 was chosen by the PDA Virus Filter Task Force. The detailed final consensus filter method was published in the 2008 update of PDA Technical Report 41. Virus Filtration.

Fractionation of Glycomacropeptide from Whey Using Positively Charged Ultrafiltration Membranes.

Fractionation of the bovine glycomacropeptide (GMP) from the other proteins in cheese whey was examined using ultrafiltration membranes surface modified to contain positively charged polymer brushes made of polyhexamethylene biguanide. By placing a strong positive charge on a 1000 kDa ultrafiltration membrane and adjusting the pH of whey close to the isoelectric point of GMP, a 14-fold increase in selectivity was observed compared to unmodified membranes. A one stage membrane system gave 90% pure GMP and a three-stage rectification system gave 97% pure GMP. The charged membrane was salt-tolerant up to 40 mS cm-1 conductivity, allowing fractionation of GMP directly from cheese whey without first lowering the whey conductivity by water dilution. Thus, similarly sized proteins that differed somewhat in isoelectric points and were 50⁻100 fold smaller than the membrane molecular weight cut-off (MWCO), were cleanly fractionated using charged ultrafiltration membranes without water addition. This is the first study to report on the use of salt-tolerant charged ultrafiltration membranes to produce chromatographically pure protein fractions from whey, making ultrafiltration an attractive alternative to chromatography for dairy protein fractionation.

Milk Protein Concentration Using Negatively Charged Ultrafiltration Membranes.

In this work, milk protein concentrate (MPC) was made using wide-pore negatively charged ultrafiltration membranes. The charged membranes were used for a six-fold volume concentration of skim milk and subsequent diafiltration to mimic the industrial MPC process. The charged 100 kDa membranes had at least a four-fold higher permeate flux at the same protein recovery as unmodified 30 kDa membranes, which are currently used in the dairy industry to make MPC. By placing a negative charge on the surface of an ultrafiltration membrane, the negatively charged proteins were rejected by electrostatic repulsion and not simply size-based sieving. Mass balance models of concentration and diafiltration were developed and the calculations matched the experimental observations. This is the first study to use wide-pore charged tangential-flow membranes for MPC manufacturing. Additionally, a unique mass balance model was applied, which accurately predicted experimental results.

Designing medical foods for inherited metabolic disorders: why intact protein is superior to amino acids.

Phenylketonuria and tyrosinemia are inherited metabolic disorders characterized by high blood levels of phenylalanine (Phe) or tyrosine (Tyr), due to mutations in genes affecting Phe and Tyr metabolism, respectively. The primary management is a lifelong diet restricted in protein from natural foods in combination with medical foods comprised mixtures of synthetic amino acids. Compliance is often poor after childhood leading to neuropsychological sequela. Glycomacropeptide, an intact 64 amino acid glycophosphopeptide isolated from cheese whey, provides a new paradigm for the management of phenylketonuria and tyrosinemia because glycomacropeptide contains no Phe and Tyr in its pure form, and is also a prebiotic. Medical foods made from glycomacropeptide have been used successfully for the management of phenylketonuria and tyrosinemia. Preclinical and clinical studies demonstrate that intact protein from glycomacropeptide provides a more acceptable and physiologic source of defined protein compared to amino acids in medical foods. For example, harmful gut bacteria were reduced, beneficial short chain fatty acids increased, renal workload decreased, protein utilization increased, and bone fragility decreased using intact protein versus amino acids. Advances in biotechnology will propel the transition from synthetic amino acids to intact proteins for the management of inherited metabolic disorders.

Inactivation Kinetics of Pathogens during Thermal Processing in Acidified Broth and Tomato Purée (pH 4.5).

Thermal inactivation kinetics for single strains of Shiga toxin-producing Escherichia coli (STEC), Listeria monocytogenes, and Salmonella enterica were measured in acidified tryptic soy broth (TSB; pH 4.5) heated at 54°C. Inactivation curves also were measured for single-pathogen five-strain cocktails of E. coli O157:H7, L. monocytogenes, and S. enterica heated in tomato purée (pH 4.5) at 52, 54, 56, and 58°C. Inactivation curves were fit using log-linear and nonlinear (Weibull) models. The Weibull model yields the time for a 5-log reduction (t*) and a curve shape parameter (ß). Decimal reduction times (D-values) and thermal resistance constants (z-values) from the two models were compared by defining t* = 5D* for the Weibull model. When the log-linear and Weibull models match at the 5-log reduction time, then t* = 5D* = 5D and D = D*. In 18 of 20 strains heated in acidified TSB, D and D* for the two models were not significantly different, although nonlinearity was observed in 35 of 60 trials. Similarly, in 51 of 52 trials for pathogen cocktails heated in tomato purée, D and D* were not significantly different, although nonlinearity was observed in 31% of trials. At a given temperature, D-values for S. enterica << L. monocytogenes < E. coli O157:H7 in tomato purée (pH 4.5). When using the two models, z-values calculated from the D-values were not significantly different for a given pathogen. Across all pathogens, z-values for E. coli O157:H7 and S. enterica were not different but were significantly lower than the z-values for L. monocytogenes. These results are useful for supporting process filings for tomato-based acidified food products with pH 4.5 and below and are relevant to small processors of tomato-based acidified canned foods who do not have the resources to conduct research on and validate pathogen lethality.

Routine, rapid HIV testing.

HIV testing identifies HIV-positive persons, allowing for reduced future HIV transmission while simultaneously providing policy makers with surveillance data to inform policy planning. If current costs of HIV testing were reduced, these funds could be redirected to increase testing rates or to expand treatment. The cost of testing is lowered and impact increased if noninvasive (oral and urine), rapid-testing modalities are utilized, pretest counseling uses cost-efficient counseling methods (e.g., video, pamphlets, small group discussions), and opt-out consent strategies are implemented while posttest counseling is more narrowly targeted to HIV-positive persons. Rather than relying on one international standard, customizing HIV testing procedures to local environments may be more efficient and effective. In the United States, laboratories with substantial HIV testing revenues are likely to be most resistant to altering current practices. However, AIDS researchers, policy makers, and advocates may dramatically influence the epidemic's course by encouraging flexibility and innovation in HIV-testing guidelines.

Preventing HIV among Latino and African American gay and bisexual men in a context of HIV-related stigma, discrimination, and homophobia: perspectives of providers.

HIV-related stigma, discrimination, and homophobia impede community-based efforts to combat HIV disease among Latino and African American gay and bisexual men. This commentary highlights ways to address these social biases in communities of color in Los Angeles, California, from the perspectives of staff from HIV prevention programs. Information was collected from HIV prevention program staff participating in a 2-day symposium. The outcomes from the symposium offer strategies for developing and implementing HIV prevention services for Latino and African American gay and bisexual men, which include: (1) addressing social biases present in a community that can hinder, and even prohibit, utilization of effective HIV prevention programs; (2) recasting HIV prevention messages in a broader social or health context; (3) developing culturally appropriate HIV prevention messages; (4) exploring new modalities and venues for delivering HIV prevention messages that are appropriate for gay and bisexual men of color and the communities in which they live; and (5) broadening the target of HIV prevention services to include service providers, local institutions and agencies, and the community at-large. These strategies underscore the need to consider the social and contextual factors of a community when designing and implementing HIV prevention programs.

Heat penetration and thermocouple location in home canning.

We processed applesauce, tomato juice, and cranberries in pint jars in a boiling water canner to test thermal processing theories against home canning of high-acid foods. For each product, thermocouples were placed at various heights in the jar. Values for f h (heating), f cl (cooling), and F 82.2°C (lethality) were determined for each thermocouple location, and did not depend substantially on thermocouple location in accordance with heat transfer theory. There was a cold spot in the jar, but the cold spot during heating became the hot spot during cooling. During heating, the geometric center was the last to heat, and remained coldest the longest, but during coooling, it was also the last to cool, and remained hottest the longest. The net effect was that calculated lethality in home canning was not affected by thermocouple location. Most of the lethality during home canning occurred during air cooling, making cooling of home canned foods of great importance. Calculated lethality was far greater than the required 5-log reduction of spores in tomato juice and vegetative cells in cranberries, suggesting a wide margin of safety for approved home-canning processes for high-acid foods.

Method for chromatographic analysis of whey protein-dextran glycation products.

A chromatographic method for the analysis of whey protein isolate (WPI)-dextran glycates was developed in this work that is useful for quantification of sample purity and concentration, and as a sample-preparation method for subsequent analysis by gel electrophoresis (SDS-PAGE) and laser-light scattering. Glycation was by the Maillard reaction between WPI and dextran of 3 different sizes. Glycate fractions from each dextran were collected and analyzed by fluorescent and glycoprotein staining of gels, bicinchoinic acid protein assay, and static and dynamic laser light scattering. The weight-average molecular mass of the glycates was 27-34 kDa (from 3.5 kDa dextran), 32-39 kDa (from 10 kDa dextran), and 250-270 kDa (from 150 kDa dextran). The new method was used to characterize the kinetics of the glycation reaction, which followed a reversible pseudo first-order model. The kinetics of decomposition of the purified glycate by hydrolysis was also examined. The new method is rapid (25 min) and quantitative, and is the first chromatographic method for direct analysis of WPI-dextran glycation products.

Chromatographic purification and characterization of whey protein-dextran glycation products.

A process for the food-grade preparative-scale production and chromatographic purification of whey protein isolate (WPI)-dextran glycates was developed in this work. The Maillard reaction was used to produce the glycates in aqueous solution. A 5 mL cation exchange column and salt step gradients were utilized to elute the glycated protein at low salt and unreacted protein at high salt. The process was scaled-up 160-fold to an 800 mL column. Glycated products were analyzed by SDS-PAGE, BCA protein assay and glycoprotein carbohydrate estimation kit, MALDI-TOF and static and dynamic light scattering. Glycated protein was relatively pure, containing only traces of beta-lactoglobulin, and it was heterogeneous due to oligo-glycation. It had a molecular mass of 26-35 kDa by static light scattering, and 22-67 kDa by MALDI-TOF. Mono-glycated protein would have been 23.8 kDa from beta-lactoglobulin (18.6 kDa) and dextran (5.2 kDa). This work demonstrated the utility of cation exchange chromatography for the large-scale purification of glycated proteins using food-grade chemicals and procedures.

Monoliths for the purification of whey protein-dextran conjugates.

Proteins conjugated to neutral biopolymers are of keen interest to the food and pharmaceutical industries. Conjugated proteins are larger and more charge shielded than un-reacted proteins, making purification difficult using conventional beaded chromatographic supports because of slow mass transfer rates, weak binding, and viscous solutions. Past methods developed for pharmaceuticals are unsuitable for foods. In this work, a food-grade whey protein-dextran conjugate was purified from a feed solution also containing un-reacted protein and dextran using either a column packed with 800 mL of a beaded support that was specifically designed for purification of conjugated proteins or an 8 mL tube monolith. The monolith gave a similar dynamic binding capacity as the beaded support (4-6 g/L), at a 42-fold greater mass productivity, and 48-fold higher flow rate, albeit at somewhat lower conjugate purity. Performance of the monolith did not depend on flow rate. In conclusion, monoliths were found to be well suited for the purification of whey protein-dextran conjugates.

Ingredients and pH are key to clear beverages that contain whey protein.

A challenge of shelf stable beverages that contain whey protein is that a small portion of protein can be denatured and aggregated during thermal processing, resulting in a turbid solution or white precipitate that consumers perceive as a defect. In this study, 3 approaches were taken to reduce turbidity in heat-treated beverages that contain whey protein: (1) centrifugation to remove insoluble protein aggregates, (2) addition of ingredients, and (3) alteration of pH in the range from 3.0 to 4.0. At pH 3.6 and below, all samples were essentially clear both before and after heating for all ingredients. At a pH of 3.8 and above, ingredient selection was crucial to solution clarity after heat treatment. At a pH of 4.0, addition of salts at both 10 and 50 mM increased the turbidity significantly compared to the control, which contained only whey protein in water. Neither addition of sugars at 25, 50, and 100 g/L, nor addition of sugar alcohols at 25 g/L significantly affected turbidity after heat treatment compared to the control. However, sugar alcohols added at 50 or 100 g/L significantly reduced turbidity after heat treatment compared to the control. Removal of insoluble protein aggregates by centrifugation prior to heat treatment resulted in a statistically significant decrease in turbidity after heat treatment. Understanding these results at the molecular level will assist food scientists in selecting processing treatments, ingredients, and pH in the development of shelf stable clear beverages that contain whey protein.