Femtosecond time-delay X-ray holography

Extremely intense and ultrafast X-ray pulses from free-electron lasers offer unique opportunities to study fundamental aspects of complex transient phenomena in materials. Ultrafast time-resolved methods usually require highly synchronized pulses to initiate a transition and then probe it after a precisely defined time delay. In the X-ray regime, these methods are challenging because they require complex optical systems and diagnostics. Here we propose and apply a simple holographic measurement scheme, inspired by Newton's 'dusty mirror' experiment, to monitor the X-ray-induced explosion of microscopic objects. The sample is placed near an X-ray mirror; after the pulse traverses the sample, triggering the reaction, it is reflected back onto the sample by the mirror to probe this reaction. The delay is encoded in the resulting diffraction pattern to an accuracy of one femtosecond, and the structural change is holographically recorded with high resolution. We apply the technique to monitor the dynamics of polystyrene spheres in intense free-electron-laser pulses, and observe an explosion occurring well after the initial pulse. Our results support the notion that X-ray flash imaging can be used to achieve high resolution, beyond radiation damage limits for biological samples. With upcoming ultrafast X-ray sources we will be able to explore the three-dimensional dynamics of materials at the timescale of atomic motion.     

Postcranial evidence from early Homo from Dmanisi, Georgia

The Plio-Pleistocene site of Dmanisi, Georgia, has yielded a rich fossil and archaeological record documenting an early presence of the genus Homo outside Africa. Although the craniomandibular morphology of early Homo is well known as a result of finds from Dmanisi and African localities, data about its postcranial morphology are still relatively scarce. Here we describe newly excavated postcranial material from Dmanisi comprising a partial skeleton of an adolescent individual, associated with skull D2700/D2735, and the remains from three adult individuals. This material shows that the postcranial anatomy of the Dmanisi hominins has a surprising mosaic of primitive and derived features. The primitive features include a small body size, a low encephalization quotient and absence of humeral torsion; the derived features include modern-human-like body proportions and lower limb morphology indicative of the capability for long-distance travel. Thus, the earliest known hominins to have lived outside of Africa in the temperate zones of Eurasia did not yet display the full set of derived skeletal features.     

Intracellular bacterial growth is controlled by a kinase network around PKB/AKT1

With the emergence of multidrug resistant (MDR) bacteria, it is imperative to develop new intervention strategies. Current antibiotics typically target pathogen rather than host-specific biochemical pathways. Here we have developed kinase inhibitors that prevent intracellular growth of unrelated pathogens such as Salmonella typhimurium and Mycobacterium tuberculosis. An RNA interference screen of the human kinome using automated microscopy revealed several host kinases capable of inhibiting intracellular growth of S. typhimurium. The kinases identified clustered in one network around AKT1 (also known as PKB). Inhibitors of AKT1 prevent intracellular growth of various bacteria including MDR-M. tuberculosis. AKT1 is activated by the S. typhimurium effector SopB, which promotes intracellular survival by controlling actin dynamics through PAK4, and phagosome-lysosome fusion through the AS160 (also known as TBC1D4)-RAB14 pathway. AKT1 inhibitors counteract the bacterial manipulation of host signalling processes, thus controlling intracellular growth of bacteria. By using a reciprocal chemical genetics approach, we identified kinase inhibitors with antibiotic properties and their host targets, and we determined host signalling networks that are activated by intracellular bacteria for survival.     

The structural basis of Holliday junction resolution by T7 endonuclease I

The four-way (Holliday) DNA junction is the central intermediate in homologous recombination, a ubiquitous process that is important in DNA repair and generation of genetic diversity. The penultimate stage of recombination requires resolution of the DNA junction into nicked-duplex species by the action of a junction-resolving enzyme, examples of which have been identified in a wide variety of organisms. These enzymes are nucleases that are highly selective for the structure of branched DNA. The mechanism of this selectivity has, however, been unclear in the absence of structural data. Here we present the crystal structure of the junction-resolving enzyme phage T7 endonuclease I in complex with a synthetic four-way DNA junction. Although the enzyme is structure-selective, significant induced fit occurs in the interaction, with changes in the structure of both the protein and the junction. The dimeric enzyme presents two binding channels that contact the backbones of the junction's helical arms over seven nucleotides. These interactions effectively measure the relative orientations and positions of the arms of the junction, thereby ensuring that binding is selective for branched DNA that can achieve this geometry.     

Rag mutations reveal robust alternative end joining

Mammalian cells repair DNA double-strand breaks (DSBs) through either homologous recombination or non-homologous end joining (NHEJ). V(D)J recombination, a cut-and-paste mechanism for generating diversity in antigen receptors, relies on NHEJ for repairing DSBs introduced by the Rag1-Rag2 protein complex. Animals lacking any of the seven known NHEJ factors are therefore immunodeficient. Nevertheless, DSB repair is not eliminated entirely in these animals: evidence of a third mechanism, 'alternative NHEJ', appears in the form of extremely rare V(D)J junctions and a higher rate of chromosomal translocations. The paucity of these V(D)J events has suggested that alternative NHEJ contributes little to a cell's overall repair capacity, being operative only (and inefficiently) when classical NHEJ fails. Here we find that removing certain portions of murine Rag proteins reveals robust alternative NHEJ activity in NHEJ-deficient cells and some alternative joining activity even in wild-type cells. We propose a two-tier model in which the Rag proteins collaborate with NHEJ factors to preserve genomic integrity during V(D)J recombination.