Sortase as a Tool in Biotechnology and Medicine

We have harnessed two reactions catalyzed by the enzyme sortase A and applied them to generate new methods for the purification and site-selective modification of recombinant protein therapeutics.

We utilized native peptide ligation —a well-known function of sortase A— to attach a small molecule drug specifically to the carboxy-terminus of a recombinant protein. By combining this reaction with the unique phase behavior of elastin-like polypeptides, we developed a protocol that produces homogenously-labeled protein-small molecule conjugates using only centrifugation. The same reaction can be used to produce unmodified therapeutic proteins simply by substituting a single reactant. The isolated proteins or protein-small molecule conjugates do not have any exogenous purification tags, eliminating the potential influence of these tags on bioactivity. Because both unmodified and modified proteins are produced by a general process that is the same for any protein of interest and does not require any chromatography, the time, effort, and cost associated with protein purification and modification is greatly reduced.

We also developed an innovative and unique method that attaches a tunable number of drug molecules to any recombinant protein of interest in a site-specific manner. Although the ability of sortase A to carry out native peptide ligation is widely used, we demonstrated that Sortase A is also capable of attaching small molecules to proteins through an isopeptide bond at lysine side chains within a unique amino acid sequence. This reaction —isopeptide ligation— is a new site-specific conjugation method that is orthogonal to all available protein-small conjugation technologies and is the first site-specific conjugation method that attaches the payload to lysine residues. We show that isopeptide ligation can be applied broadly to peptides, proteins, and antibodies using a variety of small molecule cargoes to efficiently generate stable conjugates. We thoroughly assessed the site-selectivity of this reaction using a variety of analytical methods and showed that in many cases the reaction is site-specific for lysines in flexible, disordered regions of the substrate proteins. Finally, we showed that isopeptide ligation can be used to create clinically-relevant antibody-drug conjugates that have potent cytotoxicity towards cancerous cells

Plato's Cretan Colony: Theology and Religion in the Political Philosophy of the Laws

The Laws is generally regarded as Plato’s attempt to engage with the practical realities of political life, as opposed to the more idealistic, or utopian, vision of the Republic. Yet modern scholars have often felt disquieted at the central role of religion in the Laws’ second-best city and regime. There are essentially the two dominant interpretations on offer today: either religion supports a repressive theocracy, which controls every aspect of the citizens’ lives to such an extent that even philosophy itself is discouraged, or religion is an example of the kind of noble lie, which the philosopher must deceive the citizens into believing—viz., that a god, not a man, is the author of the regime’s laws. I argue that neither of these interpretations do justice to the dialogue’s intricately dramatic structure, and therefore to Plato’s treatment of civil religion. What I propose is a third position in which Plato both takes seriously the social and political utility of religion, and views theology as a legitimate, and even necessary, subject of philosophical inquiry without going so far as to advocate theocracy as the second best form of regime.

I conclude that a proper focus on the dialogue form, combined with a careful historical analysis of Plato’s use of social and political institutions, reveals an innovative yet traditional form of civil religion, purified of the harmful influence of the poets, based on the authority of the oracle at Delphi, and grounded on a philosophical conception of god as the eternal source of order, wisdom, and all that is good. Through a union of traditional Delphic theology and Platonic natural theology, Plato gives the city of the Laws a common cult acceptable to philosopher and non-philosopher alike, and thus, not only bridges the gap between religion and philosophy, but also creates a sense of community, political identity, and social harmony—the prerequisites for political order and stability. The political theology of the Laws, therefore, provides a rational defense of the rule of law (νόμος) re-conceived as the application of divine Reason (νοῦς) to human affairs.

Mammalian genes induce partially reprogrammed pluripotent stem cells in non-mammalian vertebrate and invertebrate species.

Cells are fundamental units of life, but little is known about evolution of cell states. Induced pluripotent stem cells (iPSCs) are once differentiated cells that have been re-programmed to an embryonic stem cell-like state, providing a powerful platform for biology and medicine. However, they have been limited to a few mammalian species. Here we found that a set of four mammalian transcription factor genes used to generate iPSCs in mouse and humans can induce a partially reprogrammed pluripotent stem cell (PRPSCs) state in vertebrate and invertebrate model organisms, in mammals, birds, fish, and fly, which span 550 million years from a common ancestor. These findings are one of the first to show cross-lineage stem cell-like induction, and to generate pluripotent-like cells for several of these species with in vivo chimeras. We suggest that the stem-cell state may be highly conserved across a wide phylogenetic range. DOI:http://dx.doi.org/10.7554/eLife.00036.001.

Prayer and Depression: Women in Rural Pakistan

Background: Mental health, specifically depression, is a burden of disease in Pakistan. Religion and depression have not been studied in Pakistan currently, specially within a subset of a rural population. Methods: A secondary-data analysis was conducted using logistic regression for a non-parametrically distributed data set. The setting was in rural Pakistan, near Rawalpindi, and the sample size data was collected from the SHARE (South Asian Hub for Advocacy, Research, and Education). The measures used were the phq9 scaled for depression, prayer number, mother’s education, mother’s age, and if the mothers work. Results: This study demonstrated that there was no association between prayer and depression in this cohort. The mean prayer number between depressed and non-depressed women was 1.22 and 1.42, respectively, and a Wilcoxan rank sum test indicated that this was not significant. Conclusions: The primary finding indicates that increased frequency of prayer is not associated with a decreased rate of depression. This may be due to prayer number not being a significant enough measure. The implications of these findings stress the need for more depression intervention in rural Pakistan.