In vitro assessment of some sperm function following exposure to levonorgestrel in human fallopian tubes

Background  

The mechanism of action of levonorgestrel (LNG) as emergency contraception (EC) remains a subject of debate and its effect on sperm function has been only partially explained. The aim of this study was to assess whether LNG at a similar dose to those found in serum following oral intake for EC could affect spermatozoa when exposed to human fallopian tubes in vitro.     

Identification and classification of host cell proteins during biopharmaceutical process development

As significant improvements in volumetric antibody productivity have been achieved by advances in upstream processing over the last decade, and harvest material has become progressively more difficult to recover with these intensified upstream operations, the segregation of upstream and downstream processing has remained largely unchanged. By integrating upstream and downstream process development, product purification issues are given consideration during the optimization of upstream operating conditions, which mitigates the need for extensive and expensive clearance strategies downstream. To investigate the impact of cell culture duration on critical quality attributes, CHO-expressed IgG1 was cultivated in two 2 L bioreactors with samples taken on days 8, 10, 13, 15, and 17. The material was centrifuged, filtered and protein A purified on a 1 ml HiTrap column. Host cell protein (HCP) identification by mass spectrometry (MS) was applied to this system to provide insights into cellular behavior and HCP carryover during protein A purification. It was shown that as cultivation progressed from day 8 to 17 and antibody titer increased, product quality declined due to an increase in post-protein A HCPs (from 72 to 475 peptides detected by MS) and a decrease in product monomer percentage (from 98% to 95.5%). Additionally, the MS data revealed an increase in the abundance of several classes of post-protein A HCPs (e.g., stress response proteins and indicators of cell age), particularly on days 15 and 17 of culture, which were associated with significant increases in total overall HCP levels. This provides new insight into the specific types of HCPs that are retained during mAb purification and may be used to aid process development strategies.     

The miR-17-5p microRNA is a key regulator of the G1/S phase cell cycle transition

Background

MicroRNAs are modifiers of gene expression, acting to reduce translation through either translational repression or mRNA cleavage. Recently, it has been shown that some microRNAs can act to promote or suppress cell transformation, with miR-17-92 described as the first oncogenic microRNA. The association of miR-17-92 encoded microRNAs with a surprisingly broad range of cancers not only underlines the clinical significance of this locus, but also suggests that miR-17-92 may regulate fundamental biological processes, and for these reasons miR-17-92 has been considered as a therapeutic target.

Results

In this study, we show that miR-17-92 is a cell cycle regulated locus, and ectopic expression of a single microRNA (miR-17-5p) is sufficient to drive a proliferative signal in HEK293T cells. For the first time, we reveal the mechanism behind this response - miR-17-5p acts specifically at the G1/S-phase cell cycle boundary, by targeting more than 20 genes involved in the transition between these phases. While both pro- and anti-proliferative genes are targeted by miR-17-5p, pro-proliferative mRNAs are specifically up-regulated by secondary and/or tertiary effects in HEK293T cells.

Conclusion

The miR-17-5p microRNA is able to act as both an oncogene and a tumor suppressor in different cellular contexts; our model of competing positive and negative signals can explain both of these activities. The coordinated suppression of proliferation-inhibitors allows miR-17-5p to efficiently de-couple negative regulators of the MAPK (mitogen activated protein kinase) signaling cascade, promoting growth in HEK293T cells. Additionally, we have demonstrated the utility of a systems biology approach as a unique and rapid approach to uncover microRNA function.     

Analysis of fouling and breakthrough of process related impurities during depth filtration using confocal microscopy

Titer improvement has driven process intensification in mAb manufacture. However, this has come with the drawback of high cell densities and associated process related impurities such as cell debris, host cell protein (HCP), and DNA. This affects the capacity of depth filters and can lead to carryover of impurities to protein A chromatography leading to early resin fouling. New depth filter materials provide the opportunity to remove more process related impurities at this early stage in the process. Hence, there is a need to understand the mechanism of impurity removal within these filters. In this work, the secondary depth filter Millistak+ X0HC (cellulose and diatomaceous earth) is compared with the X0SP (synthetic), by examining the breakthrough of DNA and HCP. Additionally, a novel method was developed to image the location of key impurities within the depth filter structure under a confocal microscope. Flux, tested at 75, 100, and 250 LMH was found to affect the maximal throughput based on the max pressure of 30 psi, but no significant changes were seen in the HCP and DNA breakthrough. However, a drop in cell culture viability, from 87% to 37%, lead to the DNA breakthrough at 10% decreasing from 81 to 55 L/m² for X0HC and from 105 to 47 L/m² for X0SP. The HCP breakthrough was not affected by cell culture viability or filter type. The X0SP filter has a 30%–50% higher max throughput depending on viability, which can be explained by the confocal imaging where the debris and DNA are distributed differently in the layers of the filter pods, with more of the second tighter layer being utilized in the X0SP.     

Differential expression of specific sulphate transporters underlies seasonal and spatial patterns of sulphate allocation in trees

Sulphate uptake and its distribution within plants depend on the activity of different sulphate transporters (SULTR). In long‐living deciduous plants such as trees, seasonal changes of spatial patterns add another layer of complexity to the question of how the interplay of different transporters adjusts S distribution within the plant to environmental changes. Poplar is an excellent model to address this question because its S metabolism is already well characterized. In the present study, the importance of SULTRs for seasonal sulphate storage and mobilization was examined in the wood of poplar (Populus tremula × P. alba) by analysing their gene expression in relation to sulphate contents in wood and xylem sap. According to these results, possible functions of the respective SULTRs for seasonal sulphate storage and mobilization in the wood are suggested. Together, the present results complement the previously published model for seasonal sulphate circulation between leaves and bark and provide information for future mechanistic modelling of whole tree sulphate fluxes.     

Broadscale Specificity in a Bark Beetle–Fungal Symbiosis: a Spatio-temporal Analysis of the Mycangial Fungi of the Western Pine Beetle

Whether and how mutualisms are maintained through ecological and evolutionary time is a seldom studied aspect of bark beetle–fungal symbioses. All bark beetles are associated with fungi and some species have evolved structures for transporting their symbiotic partners. However, the fungal assemblages and specificity in these symbioses are not well known. To determine the distribution of fungi associated with the mycangia of the western pine beetle (Dendroctonus brevicomis), we collected beetles from across the insect’s geographic range including multiple genetically distinct populations. Two fungi, Entomocorticium sp. B and Ceratocystiopsis brevicomi, were isolated from the mycangia of beetles from all locations. Repeated sampling at two sites in Montana found that Entomocorticium sp. B was the most prevalent fungus throughout the beetle’s flight season, and that females carrying that fungus were on average larger than females carrying C. brevicomi. We present evidence that throughout the flight season, over broad geographic distances, and among genetically distinct populations of beetle, the western pine beetle is associated with the same two species of fungi. In addition, we provide evidence that one fungal species is associated with larger adult beetles and therefore might provide greater benefit during beetle development. The importance and maintenance of this bark beetle–fungus interaction is discussed.     

Fouling of an anion exchange chromatography operation in a monoclonal antibody process: Visualization and kinetic studies

Fouling of chromatographic resins over their operational lifetimes can be a significant problem for commercial bioseparations. In this article, scanning electron microscopy (SEM), batch uptake experiments, confocal laser scanning microscopy (CLSM) and small‐scale column studies were applied to characterize a case study where fouling had been observed during process development. The fouling was found to occur on an anion exchange (AEX) polishing step following a protein A affinity capture step in a process for the purification of a monoclonal antibody. Fouled resin samples analyzed by SEM and batch uptake experiments indicated that after successive batch cycles, significant blockage of the pores at the resin surface occurred, thereby decreasing the protein uptake rate. Further studies were performed using CLSM to allow temporal and spatial measurements of protein adsorption within the resin, for clean, partially fouled and extensively fouled resin samples. These samples were packed within a miniaturized flowcell and challenged with fluorescently labeled albumin that enabled in situ measurements. The results indicated that the foulant has a significant impact on the kinetics of adsorption, severely decreasing the protein uptake rate, but only results in a minimal decrease in saturation capacity. The impact of the foulant on the kinetics of adsorption was further investigated by loading BSA onto fouled resin over an extended range of flow rates. By decreasing the flow rate during BSA loading, the capacity of the resin was recovered. These data support the hypothesis that the foulant is located on the particle surface, only penetrating the particle to a limited degree. The increased understanding into the nature of the fouling can help in the continued process development of this industrial example. Biotechnol. Bioeng. 2013; 110:2425–2435. © 2013 Wiley Periodicals, Inc.     

Latitudinal variation in the competition‐colonisation trade‐off reveals rate‐mediated mechanisms of coexistence

Theories of species coexistence often describe a trade‐off between colonising and competitive abilities. In sessile marine invertebrates, this trade‐off can manifest as trends in species distributions relative to the size of isolated patches of substrate. Based on their abilities to find available substrate and competitively exclude neighbours, good colonisers tend to dominate smaller patches, whereas better competitors tend to monopolise larger patches. In theory, species with equivalent colonising and competitive abilities should display similar distributions across patch sizes. We used patch size to observe this manifestation of the competition‐colonisation trade‐off over 20° of latitude. The trade‐off was more readily observed at lower latitudes and was proportional to the ‘ecological age’ of communities (i.e. the degree of resource acquisition and likelihood of species interactions). Results suggest that ecological age may mediate the prominence of stochastic or deterministic coexistence mechanisms and will depend on the rate of ecological processes.