Optimization of scarless human stem cell genome editing

Efficient strategies for precise genome editing in human-induced pluripotent cells (hiPSCs) will enable sophisticated genome engineering for research and clinical purposes. The development of programmable sequence-specific nucleases such as Transcription Activator-Like Effectors Nucleases (TALENs) and Cas9-gRNA allows genetic modifications to be made more efficiently at targeted sites of interest. However, many opportunities remain to optimize these tools and to enlarge their spheres of application. We present several improvements: First, we developed functional re-coded TALEs (reTALEs), which not only enable simple one-pot TALE synthesis but also allow TALE-based applications to be performed using lentiviral vectors. We then compared genome-editing efficiencies in hiPSCs mediated by 15 pairs of reTALENs and Cas9-gRNA targeting CCR5 and optimized ssODN design in conjunction with both methods for introducing specific mutations. We found Cas9-gRNA achieved 7–8× higher non-homologous end joining efficiencies (3%) than reTALENs (0.4%) and moderately superior homology-directed repair efficiencies (1.0 versus 0.6%) when combined with ssODN donors in hiPSCs. Using the optimal design, we demonstrated a streamlined process to generated seamlessly genome corrected hiPSCs within 3 weeks

Repulsively bound atom pairs in an optical lattice

Throughout physics, stable composite objects are usually formed via attractive forces, which allow the constituents to lower their energy by binding together. Repulsive forces separate particles in free space. However, in a structured environment such as a periodic potential and in the absence of dissipation, stable composite objects can exist even for repulsive interactions. Here we report on the first observation of such an exotic bound state, comprised of a pair of ultracold atoms in an optical lattice. Consistent with our theoretical analysis, these repulsively bound pairs exhibit long lifetimes, even under collisions with one another. Signatures of the pairs are also recognised in the characteristic momentum distribution and through spectroscopic measurements. There is no analogue in traditional condensed matter systems of such repulsively bound pairs, due to the presence of strong decay channels. These results exemplify on a new level the strong correspondence between the optical lattice physics of ultracold bosonic atoms and the Bose-Hubbard model, a correspondence which is vital for future applications of these systems to the study of strongly correlated condensed matter systems and to quantum information.Comment: 5 pages, 4 figure

Inflammation Triggers Emergency Granulopoiesis through a Density-Dependent Feedback Mechanism

Normally, neutrophil pools are maintained by homeostatic mechanisms that require the transcription factor C/EBPα. Inflammation, however, induces neutrophilia through a distinct pathway of “emergency” granulopoiesis that is dependent on C/EBPβ. Here, we show in mice that alum triggers emergency granulopoiesis through the IL-1RI-dependent induction of G-CSF. G-CSF/G-CSF-R neutralization impairs proliferative responses of hematopoietic stem and progenitor cells (HSPC) to alum, but also abrogates the acute mobilization of BM neutrophils, raising the possibility that HSPC responses to inflammation are an indirect result of the exhaustion of BM neutrophil stores. The induction of neutropenia, via depletion with Gr-1 mAb or myeloid-specific ablation of Mcl-1, elicits G-CSF via an IL-1RI-independent pathway, stimulating granulopoietic responses indistinguishable from those induced by adjuvant. Notably, C/EBPβ, thought to be necessary for enhanced generative capacity of BM, is dispensable for increased proliferation of HSPC to alum or neutropenia, but plays a role in terminal neutrophil differentiation during granulopoietic recovery. We conclude that alum elicits a transient increase in G-CSF production via IL-1RI for the mobilization of BM neutrophils, but density-dependent feedback sustains G-CSF for accelerated granulopoiesis

Defiant scholarship: Dismantling coloniality in contemporary African geographies

Colonial epistemes persist in studies of African geographies. We argue that colonial continuities are revealed in (a) the status of human geography within African higher education; (b) the marginalization of Africa (particularly beyond Southern Africa) within the discipline of human geography; and (c) erasures of the functions of racialization in African societies. These are compounded by the relative marginalization of African knowledge within decolonial thought, including decolonial geographies and the disunities between the subfields of black geographies and African geographies. To challenge some of these dynamics, we introduce the concept of defiant scholarship in Africa, a form of scholarship that seeks to work against and outside of dominant grammars and prevailing registers and which draws from a powerful and extensive intellectual tradition across the African continent. Working from Walter Rodney's ‘guerrilla intellectuals’ and drawing on Walter Mignolo's ‘epistemic disobedience’, defiant scholarship cultivates those ways of thinking and those practices that are external to, in opposition to, and/or unconventional to the coloniality of knowledge. We ask what it means for our scholarship to be disobedient to colonial and capitalist epistemes, and, in so doing, we sketch the contours of an African geographies subdiscipline that is anti-racist, decolonial, and in active conversation with black geographies. The result of our engagement is a call for a reinvigoration of African geographies as we currently know and practice them