Using Molecular Markers to Characterize Productivity in Chinese Hamster Ovary Cell Lines

Selection of high producing cell lines to produce maximum product concentration is a challenging and time consuming task for the biopharmaceutical industry. The identification of early markers to predict high productivity will significantly reduce the time required for new cell line development. This study identifies candidate determinants of high productivity by profiling the molecular and morphological characteristics of a panel of six Chinese Hamster Ovary (CHO) stable cell lines with varying recombinant monoclonal antibody productivity levels ranging between 2 and 50 pg/cell/day. We examined the correlation between molecular parameters and specific productivity (q_p) throughout the growth phase of batch cultures. Results were statistically analyzed using Pearson correlation coefficient. Our study revealed that, overall, heavy chain (HC) mRNA had the strongest association with q_p followed by light chain (LC) mRNA, HC intracellular polypeptides, and intracellular antibodies. A significant correlation was also obtained between q_p and the following molecular markers: growth rate, biomass, endoplasmic reticulum, and LC polypeptides. However, in these cases, the correlation was not observed at all-time points throughout the growth phase. The repeated sampling throughout culture duration had enabled more accurate predictions of productivity in comparison to performing a single-point measurement. Since the correlation varied from day to day during batch cultivation, single-point measurement was of limited use in making a reliable prediction.     

An experimental and computational investigation of the post-yield behaviour of trabecular bone during vertebral device subsidence

Interbody fusion device subsidence has been reported clinically. An enhanced understanding of the mechanical behaviour of the surrounding bone would allow for accurate predictions of vertebral subsidence. The multiaxial inelastic behaviour of trabecular bone is investigated at a microscale and macroscale level. The post-yield behaviour of trabecular bone under hydrostatic and confined compression is investigated using microcomputed tomography-derived microstructural models, elucidating a mechanism of pressure-dependent yielding at the macroscopic level. Specifically, microstructural trabecular simulations predict a distinctive yield point in the apparent stress–strain curve under uniaxial, confined and hydrostatic compression. Such distinctive apparent stress–strain behaviour results from localised stress concentrations and material yielding in the trabecular microstructure. This phenomenon is shown to be independent of the plasticity formulation employed at a trabecular level. The distinctive response can be accurately captured by a continuum model using a crushable foam plasticity formulation in which pressure-dependent yielding occurs. Vertebral device subsidence experiments are also performed, providing measurements of the trabecular plastic zone. It is demonstrated that a pressure-dependent plasticity formulation must be used for continuum level macroscale models of trabecular bone in order to replicate the experimental observations, further supporting the microscale investigations. Using a crushable foam plasticity formulation in the simulation of vertebral subsidence, it is shown that the predicted subsidence force and plastic zone size correspond closely with the experimental measurements. In contrast, the use of von Mises, Drucker–Prager and Hill plasticity formulations for continuum trabecular bone models lead to over prediction of the subsidence force and plastic zone.     

The E1B19K oncoprotein complexes with Beclin 1 to regulate autophagy in adenovirus-infected cells

The mechanisms underlying adenovirus-mediated autophagy are currently unknown. Recently, members of the Bcl-2 protein family have been associated with autophagy. It was also reported that the Bcl-2 homology-3 (BH3) domain encompassed by both Beclin 1 and Bcl-2-like proteins is essential for their pro-autophagy or anti-autophagy functions. Here, we report for the first time that E1B19K, the adenovirus BH3 domain protein, interacts with Beclin 1 to initiate autophagy. Using immunoprecipitation assays we showed that expression of E1B19K in the host cell disrupted the physical interactions between Beclin 1 and Bcl-2 proteins. The displacement of Bcl-2 was coincident with the recruitment of PI3KC3 to the Beclin 1/E1B19K complexes. As a result of the changes in the components of the Beclin 1 interactome, there was activation of PI3KC3, as showed by the identification of PI3K-mediated lipid phosphorylation, and subsequent formation of autophagosomes. Importantly, the BH3 functional domain of E1B19K protein was required for the heterodimerization with Beclin 1. We also showed that transfer of E1B19K was sufficient to trigger autophagy in cancer cells. Consistent with these data, mutant adenoviruses encompassing a deletion of the E1B19K gene produced a marked deficiency in the capability of the virus to induce autophagy as showed by examining the lipidation and cleavage of LC3-I as well as the subcellular localization of LC3-II, the decrease in the levels of p62, and the formation of autophagosomes. Our work offers new information on the mechanisms of action of the adenoviral E1B19K protein as partner of Beclin 1 and positive regulator of autophagy.     

Bacterial and archaea community present in the Pine Barrens Forest of Long Island, NY: unusually high percentage of ammonia oxidizing bacteria

Of the few preserved areas in the northeast of United States, the soil in the Pine Barrens Forests presents a harsh environment for the microorganisms to grow and survive. In the current study we report the use of clustering methods to scientifically select the sampling locations that would represent the entire forest and also report the microbial diversity present in various horizons of the soil. Sixty six sampling locations were selected across the forest and soils were collected from three horizons (sampling depths). The three horizons were 0-10 cm (Horizon O); 11-25 cm (Horizon A) and 26-40 cm (Horizon B). Based on the total microbial substrate utilization pattern and K-means clustering analysis, the soil in the Pine Barrens Forest can be classified into four distinct clusters at each of the three horizons. One soil sample from each of the four clusters were selected and archaeal and bacterial populations within the soil studied using pyrosequencing method. The results show the microbial communities present in each of these clusters are different. Within the microbial communities present, microorganisms involved in nitrogen cycle occupy a major fraction of microbial community in the soil. High level of diversity was observed for nitrogen fixing bacteria. In contrast, Nitrosovibrio and Nitrosocaldus spp are the single bacterial and archaeal population respectively carrying out ammonia oxidation in the soil.     

Discovery of novel human breast cancer microRNAs from deep sequencing data by analysis of pri-microRNA secondary structures

MicroRNAs (miRNAs) are key regulators of gene expression and contribute to a variety of biological processes. Abnormal miRNA expression has been reported in various diseases including pathophysiology of breast cancer, where they regulate protumorigenic processes including vascular invasiveness, estrogen receptor status, chemotherapy resistance, invasion and metastasis. The miRBase sequence database, a public repository for newly discovered miRNAs, has grown rapidly with approximately >10,000 entries to date. Despite this rapid growth, many miRNAs have not yet been validated, and several others are yet to be identified. A lack of a full complement of miRNAs has imposed limitations on recognizing their important roles in cancer, including breast cancer. Using deep sequencing technology, we have identified 189 candidate novel microRNAs in human breast cancer cell lines with diverse tumorigenic potential. We further show that analysis of 500-nucleotide pri-microRNA secondary structure constitutes a reliable method to predict bona fide miRNAs as judged by experimental validation. Candidate novel breast cancer miRNAs with stem lengths of greater than 30 bp resulted in the generation of precursor and mature sequences in vivo. On the other hand, candidates with stem length less than 30 bp were less efficient in producing mature miRNA. This approach may be used to predict which candidate novel miRNA would qualify as bona fide miRNAs from deep sequencing data with approximately 90% accuracy.     

An assessment of the effect of harvest time and harvest method on biomass loss for Miscanthus × giganteus

This study examines the amount of biomass loss occurring in Miscanthus × giganteus crop at harvest. The study assesses loss incurred as a direct result of the harvest systems employed to collect the material along with examining how the time of harvest effects the amount of loss occurring over the spring harvest window. Pre harvest losses of 4.8–5.1% were measured prior to harvest. There was no significant difference between pre harvest loss and post harvest loss when a self‐propelled forage harvester fitted with a maize harvesting header was used to harvest the crop. The use of a conditioner mower and baler significantly increased crop losses to 9.4–14.1%. This demonstrates that correct selection of the harvest system can significantly increase biomass recovery. Additional losses were measured at headlands when the mower/baler system was used, but headland losses will not occur when self‐propelled forage harvesters are utilized. Losses were significantly greater in the area beside the swath after the baler pass when compared to prior to baling. This study has shown that correct selection of harvest systems can significantly increase biomass recovery, with no significant difference in pre harvest loss or harvest loss occurring as a result of cutting the M. × giganteus crop at different dates during the harvest window (March 1st, March 25th, April 21st).