Epigenetic profile of the euchromatic region of human Y chromosome.

The genome of a multi-cellular organism acquires various functional capabilities in different cell types by means of distinct chromatin modifications and packaging states. Acquired during early development, the cell type-specific epigenotype is maintained by cellular memory mechanisms that involve epigenetic modifications. Here we present the epigenetic status of the euchromatic region of the human Y chromosome that has mostly been ignored in earlier whole genome epigenetic mapping studies. Using ChIP-on-chip approach, we mapped H3K9ac, H3K9me3, H3K27me3 modifications and CTCF binding sites while DNA methylation analysis of selected CpG islands was done using bisulfite sequencing. The global pattern of histone modifications observed on the Y chromosome reflects the functional state and evolutionary history of the sequences that constitute it. The combination of histone and DNA modifications, along with CTCF association in some cases, reveals the transcriptional potential of all protein coding genes including the sex-determining gene SRY and the oncogene TSPY. We also observe preferential association of histone marks with different tandem repeats, suggesting their importance in genome organization and gene regulation. Our results present the first large scale epigenetic analysis of the human Y chromosome and link a number of cis-elements to epigenetic regulatory mechanisms, enabling an understanding of such mechanisms in Y chromosome linked disorders.

An innovative strategy to recycle permeate in biologics continuous manufacturing process to improve material efficiency and sustainability.

Intensified perfusion processes are an integral part of continuous manufacturing for biopharmaceuticals which enable agile operations and significant reduction in cost of goods. However, they require large volumes of media to support robust cell growth and maintain high productivity, posing substantial challenges to operations, logistics, and process sustainability. This study explores a novel strategy for reprocessing and reusing permeate from perfusion cultures for mAb production. The concept was initially evaluated by recycling permeate, Protein A flow-through (ProA FT) and CEX processed ProA FT in deep-well plate mock perfusion and ambr® 250 perfusion formats. Further processing of ProA FT through a cation exchange depth filter before recycling reduced process impurities such as host cell proteins (HCPs) and DNA. However, a direct replacement of fresh media with spent media reduces nutrient depth which results in a concomitant reduction in productivity. In ambr® 250 bioreactors, recycling of ProA FT at 25%-50% replacement rates (defined as the fraction of recycled material in media) resulted in a 13%-30% reduction in cumulative productivity while maintaining product quality. To mitigate this, we used media concentrates which allowed independent modulation of media depth by replacing a portion of diluent WFI with recycled material. Results from deep-well mock perfusion studies demonstrated that comparable or higher productivities relative to control can be achieved with this approach. Taken together, our study demonstrates the feasibility of recycling permeate in perfusion cultures. Process mass intensity (PMI) calculations reveal that this approach can meaningfully improve material efficiency by reducing water consumption, thereby enhancing overall bioprocess sustainability.

Disulfide trapping of protein complexes on the yeast surface.

Protein complexes are common in nature and play important roles in biology, but studying the quaternary structure formation in vitro is challenging since it involves lengthy and expensive biochemical steps. There are frequent technical difficulties as well with the sensitivity and resolution of the assays. In this regard, a technique that can analyze protein-protein interactions in high throughput would be a useful experimental tool. Here, we introduce a combination of yeast display and disulfide trapping that we refer to as stabilization of transient and unstable complexes by engineered disulfide (STUCKED) that can be used to detect the formation of a broad spectrum of protein complexes on the yeast surface using fluorescence labeling. The technique uses an engineered intersubunit disulfide to covalently crosslink the subunits of a complex, so that the disulfide-trapped complex can be displayed on the yeast surface for detection and analysis. Transient protein complexes are difficult to display on the yeast surface, since they may dissociate before they can be detected due to a long induction period in yeast. To this end, we show that three different quaternary structures with the subunit dissociation constant K(d) approximately 0.5-20 microM, the antibody variable domain (Fv), the IL-8 dimer, and the p53-MDM2 complex, cannot be displayed on the yeast surface as a noncovalent complex. However, when we introduce an interchain disulfide between the subunits, all three systems are efficiently displayed on the yeast surface, showing that disulfide trapping can help display protein complexes that cannot be displayed otherwise. We also demonstrate that a disulfide forms only between the subunits that interact specifically, the displayed complexes exhibit functional characteristics that are expected of wt proteins, the mutations that decrease the affinity of subunit interaction also reduce the display efficiency, and most of the disulfide stabilized complexes are formed within the secretory pathway during export to the surface. Disulfide crosslinking is therefore a convenient way to study weak protein association in the context of yeast display.