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     

On Glycolysis in Trichomonas vaginalis

SYNOPSIS. The mechanism of carbohydrate dissimilation was studied in cell-free extracts prepared from mass cultures of the trichomonads. Evidence for the presence of all the enzymes associated with the Embden-Meyerhof glycolytic scheme was obtained. Several enzyme systems directly associated with the glycolytic pathway were examined. Two of these, alcohol dehydrogenase and phosphorylase, were not demonstrated in the T. vaginalis extract. The absence of phosphorylase in the presence of a very high glycogen concentration in the cell (20.8%) suggests the possibility of an alternate route. A very active TPN-linked "malic enzyme" was also demonstrated, although no functional citric acid cycle is known for this trichomonad. Based on the experimental evidence and collateral data, a functional Embden-Meyerhof system was suggested for T. vaginalis.     

Fluorometric, Chromatographic, and Spectronic Evidence for the Non-indolic Nature of Citrus Auxin

Evidence for the non-indolic nature of the new citrus auxinis presented on the basis of fluorometric properties, thin-layerchromatography, Ehrlich's colour reaction, paper electrophoresis,and the infra-red spectra determinations. Citrus auxin had alower Rf in TLC than IAA, did not give the typical indole reactionwith Ehrlich's reagent, and behaved differently in electrophoresis.The infra-red spectra also provided preliminary informationconcerning chemical structure. The hypothesis that indolic compoundsconstitute the only natural auxins in higher plants should berevised in view of this evidence that a non-indole auxin existsin higher plants.     

Cold shock and heat shock: a comparison of the protection generated by brief pretreatment at less severe temperatures

Abstract Brief exposure to low (0^oC) or high (40^oC) temperature elicits a protective response that prevents injury when the flesh fly, Sarcophaga crassipalpis Macquart, is subjected to more severe cold (-10^oC) or heat (45^oC). Both the low and high temperature responses were found in all developmental stages of the fly, but were most pronounced in the pupal and pharate adult stages. The protective responses generated by brief exposure to 0 or 40^oC appear similar in that both result in a rapid acquisition of cold or heat tolerance and a loss of protection after the flies are returned to 25^oC. The protection generated by chilling is obvious within 10 min of exposure to 0^oC while a 30 min exposure to 40^oC is required to induce the high temperature protection. High temperature protects against cold shock injury within a narrow range (around 36^oC) but we have no evidence that low temperature can protect against heat injury. We previously demonstrated that the rapid increase in cold tolerance correlates with concomitant increases in glycerol concentration, but in this study we found no significant elevation in glycerol in heat-shocked flies. Thus the physiological and biochemical bases for the rapid responses to cold and heat appear to be different.     

Survival of boll weevil （Coleoptera： Curculionidae） adults after feeding on pollens from various sources

The survival of overwintering boll weevil, Anthonomus grandis grandis （Boheman）, adults on non-cotton hosts in the Lower Rio Grande Valley （LRGV） of Texas was examined from 2001 to 2006. The success of the Boll Weevil Eradication Program, which was reintroduced into the LRGV in 2005, depends on controlling overwintering boll weevil populations. Laboratory studies were conducted using boll weevil adults that were captured in pheromone traps from September through March. The number of adults captured per trap declined significantly in the field from fall to the beginning of spring （3.5-7.0-fold）. The proportion of trapped males and females did not differ significantly. The mean weight of boll weevil adults captured in September was 13.3 mg, while those of captured adults from November to February were significantly lower and ranged from 6.7 to 7.8 mg. Our results show that boll weevil adults can feed on different plant pollens. The highest longevity occurred when adults were fed almond pollen or mixed pollens （72.6 days and 69.2 days, respectively） and the lowest when they fed on citrus pollen or a non-food source （9.7 days or 7.4 days, respectively）. The highest adult survival occurred on almond and mixed pollens [88.0%-97. 6% after 1st feeding period （10 days）, 78.0%-90.8% after 3rd feeding period （10 days）, 55. 0%-83.6% after 5th feeding period （10 days）, and 15.2%-32.4% after lOth feeding period （10 days）]. The lowest adult survival occurred on citrus pollen [52.0%-56.0% after 1st feeding period （10 days）, 13.3% after 3rd and 5th feeding periods （10 days）, and 0 after 6th feeding period （10 days）]. Pollen feeding is not a behavior restricted to adult boll weevils of a specific sex or physiological state. Understanding how boll weevil adults survive in the absence of cotton is important to ensure ultimate success of eradicating this pest in the subtropics.     

A new workerless inquiline in the Lower Attini (Hymenoptera: Formicidae), with a discussion of social parasitism in fungus‐growing ants

Ant inquilines are obligate social parasites, usually lacking a sterile worker caste, which are dependent on their hosts for survival and reproduction. Social parasites are rare among the fungus‐gardening ants (Myrmicinae: tribe Attini) and only four species are known until now, all being inquilines from the Higher Attini. We describe Mycocepurus castrator sp.n. , the first inquiline social parasite to be discovered in the Lower Attini. Our study of the parasite's behaviour and life history supports the conclusion drawn from external morphology: Mycocepurus castrator is an evolutionarily derived inquiline parasite of Mycocepurus goeldii. Inquilines are of great interest to evolutionary biology because it is debated if they originated via sympatric or allopatric speciation. We discuss the life history evolution, behaviour and morphology of socially parasitic, fungus‐growing ants.