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     

Parthenogenetic development of dwarf surf dam,Mulinia lateralis,oocytes treated with polar body suppressing agents

Summary

Parthenogenesis following oocyte activation has been observed in a number of marine invertebrates, but the fate of parthenogenesis in bivalve mollusc embryos is unclear. We used the dwarf surf clam, Mulinia lateralis, to examine parthenogenetic development of KC1-activated oocytes using the polar body suppressing agents caffeine and heat or cytochalasin B. Development was followed by epifluorescence microscopy and flow-cytometric analysis using the DNA-specific fluorochrome DAPI. All agents suppressed polar body formation to some degree, putatively increasing the ploidy level and retaining a meiotic centrosome in the zygote; but the zygotes failed to develop normally. Failure of the zygotes to develop suggests that the meiotic centrosome is incapable of participating in mitosis in bivalves.     

Selection of Diazotrophic Bacterial Communities in Biological Sand Filter Mesocosms Used for the Treatment of Phenolic-Laden Wastewater

Agri effluents such as winery or olive mill wastewaters are characterized by high phenolic concentrations. These compounds are highly toxic and generally refractory to biodegradation. Biological sand filters (BSFs) represent inexpensive, environmentally friendly, and sustainable wastewater treatment systems which rely vastly on microbial catabolic processes. Using denaturing gradient gel electrophoresis and terminal-restriction fragment length polymorphism, this study aimed to assess the impact of increasing concentrations of synthetic phenolic-rich wastewater, ranging from 96 mg L^?1 gallic acid and 138 mg L^?1 vanillin (i.e., a total chemical oxygen demand (COD) of 234 mg L^?1) to 2,400 mg L^?1 gallic acid and 3,442 mg L^?1 vanillin (5,842 mg COD L^?1), on bacterial communities and the specific functional diazotrophic community from BSF mesocosms. This amendment procedure instigated efficient BSF phenolic removal, significant modifications of the bacterial communities, and notably led to the selection of a phenolic-resistant and less diverse diazotrophic community. This suggests that bioavailable N is crucial in the functioning of biological treatment processes involving microbial communities, and thus that functional alterations in the bacterial communities in BSFs ensure provision of sufficient bioavailable nitrogen for the degradation of wastewater with a high C/N ratio.     

Gravel dredging alters diversity and structure of riverine fish assemblages

1. Human activities affect fish assemblages in a variety of ways. Large‐scale and long‐term disturbances such as in‐stream dredging and mining alter habitat and hydrodynamic characteristics within rivers which can, in turn, alter fish distribution. Habitat heterogeneity is decreased as the natural riffle–pool–run sequences are lost to continuous pools and, as a consequence, lotic species are displaced by lentic species, while generalist and invasive species displace native habitat specialists. Sediment and organic detritus accumulate in deep, dredged reaches and behind dams, disrupting nutrient flow and destroying critical habitat for habitat specialist species. 2. We used standard ecological metrics such as species richness and diversity, as well as stable isotope analysis of δ¹³C and δ¹⁵N, to quantify the differences in fish assemblages sampled by benthic trawls among dredged and undredged sites in the Allegheny River, Pennsylvania, U.S.A. 3. Using mixed‐effects models, we found that total catch, species richness and diversity were negatively correlated with depth (P < 0.05), while species richness, diversity and proportion of species in lithophilic (‘rock‐loving’) reproductive guilds were lower at dredged than at undredged sites (P < 0.05). 4. Principal components analysis and manova revealed that taxa such as darters in brood hider and substratum chooser reproductive guilds were predominantly associated with undredged sites along principal component axis 1 (PC1 and manova P < 0.05), while nest spawners such as catfish and open substratum spawners including suckers were more associated with dredged sites along PC2 (P < 0.05). 5. Stable isotope analysis of δ¹³C and δ¹⁵N revealed shifts from reliance on shallow water and benthic‐derived nutrients at undredged sites to reliance on phytoplankton and terrestrial detritus at deep‐water dredged sites. Relative trophic positions were also lower at dredged sites for many species; loss of benthic nutrient pathways associated with depth and dredging history is hypothesised. 6. The combination of ecological metrics and stable isotope analysis thus shows how anthropogenic habitat loss caused by gravel dredging can decrease benthic fish abundance and diversity, and that species in substratum‐specific reproductive guilds are at particular risk. The effects of dredging also manifest by altering resource use and nutrient pathways within food webs. Management and conservation decisions should therefore consider the protection of relatively shallow areas with suitable substratum for spawning for the protection of native fishes.     

Balancing redox cofactor generation and ATP synthesis: Key microaerobic responses in thermophilic fermentations

Geobacillus thermoglucosidasius is a Gram‐positive, thermophilic bacterium capable of ethanologenic fermentation of both C5 and C6 sugars and may have possible use for commercial bioethanol production [Tang et al., 2009; Taylor et al. (2009) Trends Biotechnol 27(7): 398–405]. Little is known about the physiological changes that accompany a switch from aerobic (high redox) to microaerobic/fermentative (low redox) conditions in thermophilic organisms. The changes in the central metabolic pathways in response to a switch in redox potential were analyzed using quantitative real‐time PCR and proteomics. During low redox (fermentative) states, results indicated that glycolysis was uniformly up‐regulated, the Krebs (tricarboxylic acid or TCA) cycle non‐uniformly down‐regulated and that there was little to no change in the pentose phosphate pathway. Acetate accumulation was accounted for by strong down‐regulation of the acetate CoA ligase gene (acs) in addition to up‐regulation of the pta and ackA genes (involved in acetate production), thus conserving ATP while reducing flux through the TCA cycle. Substitution of an NADH dehydrogenase (down‐regulated) by an up‐regulated NADH:FAD oxidoreductase and up‐regulation of an ATP synthase subunit, alongside the observed shifts in the TCA cycle, suggested that an oxygen‐scavenging electron transport chain likely remained active during low redox conditions. Together with the observed up‐regulation of a glyoxalase and down‐regulation of superoxide dismutase, thought to provide protection against the accumulation of toxic phosphorylated glycolytic intermediates and reactive oxygen species, respectively, the changes observed in G. thermoglucosidasius NCIMB 11955 under conditions of aerobic‐to‐microaerobic switching were consistent with responses to low pO₂ stress. Biotechnol. Bioeng. 2013; 110: 1057–1065. © 2012 Wiley Periodicals, Inc.     

Liver in a dish

There exists a worldwide shortage of donor livers for transplant. This may not pose a problem in the future, as Takebe et al. have recently grown functional “liver buds” from stem cells in a dish.Since the discovery of human induced pluripotent stem cells (hiPSCs), the promise of generating organs from patients'' iPSCs has received considerable attention as an alternative to donor organ transplantation. Over the past few years, much progress has been made in the differentiation of various somatic cell types from human pluripotent stem cells (hPSCs). However, only a limited number of reports have described the generation of three-dimensional organoids from human stem cells in vitro, including the optic cup¹, the pituitary epithelium², and from adult stem cells — the gut epithelium³. These experimental systems share several common features: 1) they all begin with ES cells or adult stem cells, 2) the cells grow as floating aggregates, and 3) all three organoids (optic cup, pituitary epithelium, and gut crypt) are epithelial structures⁴. In addition, one particularly unexpected finding has emerged from each of these experiments, namely that a high level of self-organization seems to play a substantial role in establishing local tissue architecture and assembly of the resulting organoid.Despite these remarkable examples of organogenesis in vitro, the likelihood of growing a complex vascularized organ in dish, such as liver, has seemed less plausible. Takebe et al.⁵ have made the implausible possible by focusing on the first steps of organogenesis, namely the cellular interactions that occur during liver bud development. The earliest stage of liver organogenesis involves the outgrowth of a group of endodermal and mesenchymal cells from the posterior foregut that soon thereafter become vascularized to form a liver bud. During these morphogenetic changes, a key element to the formation of a liver bud is the orchestration of signals between three types of cells: liver, mesenchymal and endothelial progenitors. Takebe et al. posited that they might be able to recapitulate liver bud formation in vitro by mixing hepatic endoderm cells together with endothelial and mesenchymal cells. To test this idea, they prepared hepatic endoderm cells (hiPSC-HEs) from hiPSCs by directed differentiation, and then co-cultured them with human umbilical vein endothelial cells (HUVECs) and human mesenchymal stem cells (MSCs). Two days later, the cells had self-assembled into a 5-mm-long, three-dimensional tissue that was reminiscent of “liver bud” structures in vivo. To further mature these hiPSC-derived “liver buds” (hiPSC-LBs), they transplanted them into immune-compromised mice where the hiPSC-LBs connected with the host vasculature within 48 h and formed functional vascular networks similar in density and morphology to those of human adult livers. Transplanted hiPSC-LBs started functioning about 10 days later, producing human albumin and metabolizing drugs in a similar fashion to human liver. Perhaps most remarkably, Takebe et al. demonstrated that these hiPSC-LBs could rescue liver function when transplanted to mice with liver failure.The differences between Takebe and his colleagues'' study and other studies designed to reproduce organogenesis in vitro are that they started with several different cell types; the cells were grown initially in a two-dimensional petri dish; and the result was a solid liver organoid that can be vascularized and function after transplantation. For many, the most striking finding is the high level of self-organization in this experimental differentiation system. By analogy, it is equivalent to delivering all of the materials necessary to build a house to a construction site and returning several days later to find a fully assembled home. Clearly the principles of self-organization and self-assembly are playing much more profound roles during differentiation than we previously thought and it is likely what has been reported by Takebe et al. represents only the tip of the iceberg. One takeaway from the way that Takebe and his colleagues'' tackled the problem of in vitro organogenesis may be their focus on the earliest processes in organ development, as it is likely to identify the right combination of cell types for organogenesis to proceed. Nonetheless, this study has raised several new questions. How does self-organization and self-assembly occur in vitro? What is the molecular logic of this process? How can we manipulate a self-organizing system so that we might guide it in the direction we want it to go? And ultimately, could we use a similar strategy to produce other complex solid organs in vitro, e.g., lung, kidney, and pancreas?As summarized by Takebe et al., this study demonstrates a “proof-of-concept” that “organ-bud transplantation provides a promising new approach to study regenerative medicine”. However, a significant amount of work will be required before these findings can be translated into a therapy. First, these little liver buds do not form a complete adult liver. They are missing a number of critical cell types, chief among them biliary epithelial cells and thus bile ducts. How to produce a fully functional liver remains a challenge. Second, in order to translate these findings into human therapies, a key step will be to scale this process so that one can produce a liver bud large enough for transplantation into humans. Of course, there is always the question about safety when it comes to stem cell-based therapies. Undifferentiated stem cells left in transplants tend to form tumors and the use of oncogenes for iPS reprogramming needs to be resolved before iPS cells can be considered for human therapy. Despite the reality that clinical therapies based on this report remain a distant promise, it is inspirational to consider how quickly the field is moving and exciting to speculate about what might come next. If one considers that a drug has been identified to specifically eliminate pluripotent but not differentiated hPSCs⁶ and that a recent report showed that pluripotent stem cells could be induced from mouse somatic cells by using only small molecules⁷, we may have good reason to believe that one day in the not too distant future we could grow patient-customized organs for transplantation (Figure 1).Open in a separate windowFigure 1This figure outlines the strategy of generating organs from patients'' iPSCs as an alternative to transplantation. Patient-derived pluripotent stem cells (iPSCs) can be differentiated in vitro to desired cell types. As demonstrated by Takebe et al.⁵, different cell types can be co-cultured in dish to recapitulate the earliest process of organogenesis and form three-dimensional organ buds. These in vitro produced organ buds could be used for transplantation in the future.     

A phylogeographic study of the stoneplant Conophytum (Aizoaceae; Ruschioideae; Ruschieae) in the Bushmanland Inselberg Region (South Africa) suggests anemochory

The Bushmanland Inselberg Region (BIR) of South Africa provides an ideal system to study population interactions, as these inselbergs function as islands of Succulent Karoo surrounded by Nama Karoo vegetation. The population genetics of four Conophytum taxa endemic to the quartz-associated habitats of inselbergs in the BIR were investigated using amplified fragment length polymorphisms (AFLP), namely C. marginatum subsp. haramoepense, C. marginatum subsp. marginatum, C. maughanii, and C. ratum. Conophytum marginatum colonizes the quartz outcrops on the summits of the inselbergs, while C. maughanii and C. ratum occupy quartz patches at the summit and base of the inselbergs. A total of 24 populations were sampled to assess genetic differentiation between populations of each species, specifically between summit and base populations of C. ratum, eastern and western populations of C. maughanii and populations of the two subspecies of C. marginatum. Moderate levels of genetic differentiation were recovered between the summit and base populations of C. ratum, with an indication of some genetic connectivity between the populations. Slight differentiation between the eastern and western populations of C. maughanii was recovered, however, this was not reflected in the PCoA and STRUCTURE results. In C. marginatum, no significant genetic differentiation was recovered between populations of the subspecies. These results may reflect evidence of different dispersal mechanisms in the species, with the genetic connectivity between populations of C. ratum possibly indicating dispersal through hygrochastic capsules, while genetic connectivity between populations of C. maughanii and C. marginatum may, for the first time, suggest long-distance dispersal, i.e. anemochory. This study provides the first insights into population interactions across the BIR and highlights the importance of conservation in the region, particularly of the Gamsberg, in light of the recent mining activities.     

Characterization and homology modelling of a novel multi-modular and multi-functional Paenibacillus mucilaginosus glycoside hydrolase

Extremophiles - Glycoside hydrolases, particularly cellulases, xylanases and mannanases, are essential for the depolymerisation of lignocellulosic substrates in various industrial bio-processes. In...