In-depth qualitative and quantitative analysis of composite glycosylation profiles and other micro-heterogeneity on intact monoclonal antibodies by high-resolution native mass spectrometry using a modified Orbitrap

Here, we describe a fast, easy-to-use, and sensitive method to profile in-depth structural micro-heterogeneity, including intricate N-glycosylation profiles, of monoclonal antibodies at the native intact protein level by means of mass spectrometry using a recently introduced modified Orbitrap Exactive Plus mass spectrometer. We demonstrate the versatility of our method to probe structural micro-heterogeneity by describing the analysis of three types of molecules: (1) a non-covalently bound IgG4 hinge deleted full-antibody in equilibrium with its half-antibody, (2) IgG4 mutants exhibiting highly complex glycosylation profiles, and (3) antibody-drug conjugates. Using the modified instrument, we obtain baseline separation and accurate mass determination of all different proteoforms that may be induced, for example, by glycosylation, drug loading and partial peptide backbone-truncation. We show that our method can handle highly complex glycosylation profiles, identifying more than 20 different glycoforms per monoclonal antibody preparation and more than 30 proteoforms on a single highly purified antibody. In analyzing antibody-drug conjugates, our method also easily identifies and quantifies more than 15 structurally different proteoforms that may result from the collective differences in drug loading and glycosylation. The method presented here will aid in the comprehensive analytical and functional characterization of protein micro-heterogeneity, which is crucial for successful development and manufacturing of therapeutic antibodies     

THE JUST-ABOUT-RIGHT INTENSITY SCALE: FUNCTIONAL ANALYSES AND RELATION TO HEDONICS

This study assessed the meaning of just‐about‐right (JAR) categories when used with a 5‐point intensity scale. The panelists comprised both consumer and in‐house respondents. The JAR categories have various meanings as perceived by the respondents: “prefer product,”“very good,”“I like the product,”“like it very much,”“highly favorable,”“high acceptability,”“desirable like the product,”“best for the situation” and “correct.” Thus, the JAR meanings invoke preference and acceptability. This article presents two additional analyses to help the product tested. One method of analysis divides below‐JAR deviation from the above‐JAR deviation, with standard statistical procedures applied in each data set. The second method introduces the signal‐to‐noise ratio statistic for analyzing the relation of JAR to overall liking. Both analytic methods provide new ways to look at the JAR data, especially with respect to hedonics and product improvement.     

Human health risks of metals and metalloids in muscle tissue of Synodontis zambezensis Peters, 1852 from Flag Boshielo Dam,South Africa

Muscle tissue from 63 Synodontis zambezensis collected bimonthly in 2013 at Flag Boshielo Dam were analysed for metals and metalloids in a desktop human health risk assessment. The Hazard Quotient, based on a weekly meal of 67 g of fish muscle, exceeded the maximum acceptable level of one for lead, cobalt, cadmium, mercury, arsenic and selenium. The concentrations of these elements were higher in 2013 than those recorded in 2009 and 2012 in other fish species from Flag Boshielo Dam and these may pose a long-term health risk if consumed regularly by impoverished rural communities reliant on fish as a source of protein.     

On the Trail of Atmospheric Mutagens and Carcinogens: A Combined Chemical/Microbiological Approach

Major ecological problems of our polluted troposphere includeairborne toxic chemicals, acid rain and photochemical smog,all three of which are now recognized as being closely relatedchemical phenomena. We also recognize that inorder to developcost-effective strategies for their control, which protect publichealth and the environment, there must be close scientific interactionsbetween chemists and biological scientists. For example, ofrapidly emerging importance is the development of risk assessmentevaluations for specific aspects of each of these problem areas.In preparing such assessments, chemists must define the "exposure,"and biological scientists the "effects." In this paper, I discuss an example of how such close interactionsproved indispensible in our search for atmospheric mutagensand carcinogens. Thus, an integrated chemical/ microbiologicalprocedure for the isolation and identificationof particulatechemical mutagens in respirable diesel soot and ambient particlesis described. Emphasis is placed on our use of the short-term,Ames Salmonella typhimurium bacterial mutagenicity test as arapid, and relatively inexpensive, means of following the biologicalactivities of these environmental mutagens through the chemicalsteps of their separation, isolation and identification fromhighly complex environmental samples. Possible mechanisms offormation of these particulate mutagens are discussed. Theyinclude the reactions of polycyclic aromatic hydrocarbons presenton the surfaces of combustion-generated particles with gaseousco-pollutants such as nitrogen dioxide plus nitric acid, andozone. In discussing this research on a societally "relevant" problem,we illustrate the importance of "Science as a Way of Knowing."We further suggest that this integrated approach to scientificproblem solving by chemical and biological scientists mightserve as an example of a discussion topic on human ecology forundergraduate courses in the natural sciences.     

Ribulose 1, 5-bisphosphate Carboxylase/ Oxygenase Activities in Leaves of Greenhouse Roses

An enzyme assay was developed to measure the initial and Mg²⁺–CO₂activated forms of Ribulose 1,5-bisphosphate Carboxylase/Oxygenase(Rubisco) in rose leaves. The assay was verified by co-extractionof the leaflets with partially purified spinach Rubisco andthrough correlation with net photosynthetic rates of individualleaflets (r²=0.7324). Changes in activities were measured asa function of depth of leaves in the canopy for two cultivarsof greenhouse hybrid tea roses. Initial Rubisco activity declinedwith increasing canopy depth for both cultivars. The activatedform of the enzyme, however, remained constant with canopy depthfor cv. Red Success; but increased with canopy depth, then declinedafter mid-canopy in the cv. Royalty. Rubisco activities werealso measured in the cv. Red Success grown in CO₂ enriched environments(100 mm³ dm^–3) at three humidity levels. The activitieswere not significantly affected by humidity treatment. However,there was a trend for plants grown at lower humidity to havehigher activated activities. Key words: Humidity, Rubisco, Rosa ? hybrida, Royalty, Red Success     

Tissue MicroRNAs as Predictors of Outcome in Patients with Metastatic Colorectal Cancer Treated with First Line Capecitabine and Oxaliplatin with or without Bevacizumab

Purpose

We tested the hypothesis that expression of microRNAs (miRNAs) in cancer tissue can predict effectiveness of bevacizumab added to capecitabine and oxaliplatin (CAPEOX) in patients with metastatic colorectal cancer (mCRC).

Experimental Design

Patients with mCRC treated with first line CAPEOX and bevacizumab (CAPEOXBEV): screening (n = 212) and validation (n = 121) cohorts, or CAPEOX alone: control cohort (n = 127), were identified retrospectively and archival primary tumor samples were collected. Expression of 754 miRNAs was analyzed in the screening cohort using polymerase chain reaction (PCR) arrays and expression levels were related to time to disease progression (TTP) and overall survival (OS). Significant miRNAs from the screening study were analyzed in all three cohorts using custom PCR arrays. In situ hybridization (ISH) was done for selected miRNAs.

Results

In the screening study, 26 miRNAs were significantly correlated with outcome in multivariate analyses. Twenty-two miRNAs were selected for further study. Higher miR-664-3p expression and lower miR-455-5p expression were predictive of improved outcome in the CAPEOXBEV cohorts and showed a significant interaction with bevacizumab effectiveness. The effects were strongest for OS. Both miRNAs showed high expression in stromal cells. Higher expression of miR-196b-5p and miR-592 predicted improved outcome regardless of bevacizumab treatment, with similar effect estimates in all three cohorts.

Conclusions

We have identified potentially predictive miRNAs for bevacizumab effectiveness and additional miRNAs that could be related to chemotherapy effectiveness or prognosis in patients with mCRC. Our findings need further validation in large cohorts, preferably from completed randomized trials.     

Survival of Wood Duck Ducklings and Broods in Mississippi and Alabama

ABSTRACT Although North American wood ducks (Aix sponsa) are well-studied throughout their range, researchers know little about demographic and environmental factors influencing survival of ducklings and broods, which is necessary information for population management. We studied radiomarked female and duckling wood ducks that used nest boxes and palustrine wetlands at Noxubee National Wildlife Refuge (NNWR) in Mississippi, USA, in 1996–1999, and riverine wetlands of the Tennessee-Tombigbee Rivers and Waterway (TTRW) system in Alabama in 1998–1999. We estimated survival of ducklings and broods and evaluated potentially important predictors of duckling survival, including age and body mass of brood-rearing females, hatch date of ducklings, duckling mass, brood size at nest departure, inter-day travel distance by ducklings, site and habitat use, and daily minimum air temperature and precipitation. At NNWR, survival of 300 radiomarked ducklings ranged from 0.15 (95% CI = 0.04-0.27) to 0.24 (95% CI = 0.13-0.38) and was 0.21 (95% CI = 0.15-0.28) for 1996–1999. Our overall estimate of brood survival was 0.64 (n = 91; 95% CI = 0.54-0.73). At TTRW, survival of 129 radiomarked ducklings was 0.29 in 1998 (95% CI = 0.20-0.41) and 1999 (95% CI = 0.13-0.45) and was 0.29 (95% CI = 0.20-0.40) for 1998–1999. Our overall estimate of brood survival was 0.71 (n = 38; 95% CI = 0.56-0.85). At NNWR, models that included all predictor variables best explained variation in duckling survival. Akaike weight (w_i) for the best model was 0.81, suggesting it was superior to other models (<0.01 < w_i < 0.18). We detected 4 competing models for duckling survival at TTRW. Inter-day distance traveled by ducklings was important as this variable appeared in all 4 models; duckling survival was positively related to this variable. Patterns of habitat-related survival were similar at both study areas. Ducklings in broods that used scrub-shrub habitats disjunct from wetlands containing aggregations of nest boxes had greater survival probabilities than birds remaining in wetlands with such nest structures. Managers may increase local wood duck recruitment by promoting availability of suitable brood habitats (e.g., scrub-shrub wetlands) without aggregations of nest boxes that may attract predators and by dispersing nest boxes amid or adjacent to these habitats. We did not determine an optimal density of nest boxes relative to local or regional population goals, which remains important research and conservation needs.     

Source of an egg kairomone for Trissolcus basalis, a parasitoid of Nezara viridula

Abstract. The eggs of the southern green stink bug, Nezara viridula (L.) (Hemiptera: Pentatomidae), are successfully attacked by Trissolcus basalis (Woll.) (Hymenoptera: Scelionidae) and are recognized as hosts by a secretion applied to the egg chorion. This secretion is produced by the follicular cells in the proximal region of the ovariole of the female pentatomid and functions as an adhesive for attaching the eggs to the oviposition substrate. The adhesive and kairomone activity could be partially removed with water. This water extract elicited host recognition behaviour in T. basalis when applied to glass beads which stuck together as the extract dried. The adhesive and kairomonal activity was removed completely with acetone since acetone-washed host eggs were not recognized by T. basalis. Application of the acetone extract to glass beads stimulated ovipositional probes by T. basalis. The adhesive appeared to be composed of a mucopolysaccharide–protein complex.