A minimal view of single-particle imaging with X-ray lasers

The ability to serially interrogate single biomolecules with femtosecond X-ray pulses from free-electron lasers has ushered in the possibility of determining the three-dimensional structure of biomolecules without crystallization. However, the complexity of imaging a sample''s structure from very many of its noisy and incomplete diffraction data can be daunting. In this review, we introduce a simple analogue of this imaging workflow, use it to describe a structure reconstruction algorithm based on the expectation maximization principle, and consider the effects of extraneous noise. Such a minimal model can aid experiment and algorithm design in future studies.     

Towards a Case Definition for Devil Facial Tumour Disease: What Is It?

In the mid 1990s an emerging disease characterised by the development of proliferative lesions around the face of Tasmanian devils (Sarcophilus harrisii) was observed. A multi-disciplinary approach was adopted to define the condition. Histopathological and transmission electron microscopic examination combined with immunohistochemistry help define Devil Facial Tumour Disease (DFTD) as a neoplastic condition of cells of neuroendocrine origin. Cytogenetic analysis of neoplastic tissue revealed it to be markedly different from normal devil tissue and having a consistent karyotype across all tumours examined. Combined with evidence for Major histocompatability (MHC) gene analysis there is significant evidence to confirm the tumour is a transmissible neoplasm.     

Granzymes and caspase 3 play important roles in control of gammaherpesvirus latency

Gammaherpesviruses can establish lifelong latent infections in lymphoid cells of their hosts despite active antiviral immunity. Identification of the immune mechanisms which regulate gammaherpesvirus latent infection is therefore essential for understanding how gammaherpesviruses persist for the lifetime of their host. Recently, an individual with chronic active Epstein-Barr virus infection was found to have mutations in perforin, and studies using murine gammaherpesvirus 68 (gammaHV68) as a small-animal model for gammaherpesvirus infection have similarly revealed a critical role for perforin in regulating latent infection. These results suggest involvement of the perforin/granzyme granule exocytosis pathway in immune regulation of gammaherpesvirus latent infection. In this study, we examined gammaHV68 infection of knockout mice to identify specific molecules within the perforin/granzyme pathway which are essential for regulating gammaherpesvirus latent infection. We show that granzymes A and B and the granzyme B substrate, caspase 3, are important for regulating gammaHV68 latent infection. Interestingly, we show for the first time that orphan granzymes encoded in the granzyme B gene cluster are also critical for regulating viral infection. The requirement for specific granzymes differs for early versus late forms of latent infection. These data indicate that different granzymes play important and distinct roles in regulating latent gammaherpesvirus infection.     

Translation elongation factor eEF1A2 is essential for post-weaning survival in mice

Translation elongation factor eEF1A, formerly known as EF-1 alpha, exists as two variant forms; eEF1A1, which is almost ubiquitously expressed, and eEF1A2, whose expression is restricted to muscle and brain at the level of whole tissues. Expression analysis of these genes has been complicated by a general lack of availability of antibodies that specifically recognize each variant form. Wasted mice (wst/wst) have a 15.8-kilobase deletion that abolishes activity of eEF1A2, but before this study it was unknown whether the deletion also affected neighboring genes. We have generated a panel of anti-peptide antibodies and used them to show that eEF1A2 is expressed at high levels in specific cell types in tissues previously thought not to express this variant, such as pancreatic islet cells and enteroendocrine cells in colon crypts. Expression of eEF1A1 and eEF1A2 is shown to be generally mutually exclusive, and we relate the expression pattern of eEF1A2 to the phenotype seen in wasted mice. We then carried out a series of transgenic experiments to establish whether the expression of other genes is affected by the deletion in wasted mice. We show that aspects of the phenotype such as motor neuron degeneration relate precisely to the relative expression of eEF1A1 and eEF1A2, whereas the immune system abnormalities are likely to result from a stress response. We conclude that loss of eEF1A2 function is solely responsible for the abnormalities seen in these mice.     

Evidence for transposition of dispersed repetitive DNA families in yeast.

Dispersed repetitive DNA sequences from yeast (Saccharomyces cerevisiae) nuclear DNA have been isolated as molecular hybrids in lambdagt. Related S. cerevisiae strains show marked alterations in the size of the restriction fragments containing these repetitive DNAs. "Ty1" is one such family of repeated sequences in yeast and consists of a 5.6 kilobase (kb) sequence including a noninverted 0.25 kb sequence of another repetitious family, "delta", on each end. There are about 35 copies of Ty1 and at least 100 copies of delta (not always associated with Ty1) in the haploid genome. A few Ty1 elements are tandem and/or circular, but most are disperse and show (along with delta) some sequence divergence between repeat units. Sequence alterations involving Ty1 elements have been found during the continual propagation of a single yeast clone over the course of a month. One region with a large number of delta sequences (SUP4) also shows a high frequency of sequence alterations when different strains are compared. One of the differences between two such strains involves the presence or absence of a Ty1 element. The novel joint is at one inverted pair of delta sequences.     

Drosophila ref(2)P is required for the parkin-mediated suppression of mitochondrial dysfunction in pink1 mutants

Autophagy is a critical regulator of organellar homeostasis, particularly of mitochondria. Upon the loss of membrane potential, dysfunctional mitochondria are selectively removed by autophagy through recruitment of the E3 ligase Parkin by the PTEN-induced kinase 1 (PINK1) and subsequent ubiquitination of mitochondrial membrane proteins. Mammalian sequestrome-1 (p62/SQSTM1) is an autophagy adaptor, which has been proposed to shuttle ubiquitinated cargo for autophagic degradation downstream of Parkin. Here, we show that loss of ref(2)P, the Drosophila orthologue of mammalian P62, results in abnormalities, including mitochondrial defects and an accumulation of mitochondrial DNA with heteroplasmic mutations, correlated with locomotor defects. Furthermore, we show that expression of Ref(2)P is able to ameliorate the defects caused by loss of Pink1 and that this depends on the presence of functional Parkin. Finally, we show that both the PB1 and UBA domains of Ref(2)P are crucial for mitochondrial clustering. We conclude that Ref(2)P is a crucial downstream effector of a pathway involving Pink1 and Parkin and is responsible for the maintenance of a viable pool of cellular mitochondria by promoting their aggregation and autophagic clearance.     

Mutability of DNA polymerase I: implications for the creation of mutant DNA polymerases

DNA polymerases of the Family A catalyze the addition of deoxynucleotides to a primer with high efficiency, processivity, and selectivity-properties that are critical to their function both in nature and in the laboratory. These polymerases tolerate many amino acid substitutions, even in regions that are evolutionarily conserved. This tolerance can be exploited to create DNA polymerases with novel properties and altered substrate specificities, using rational design and molecular evolution. These efforts have focused mainly on the Family A DNA polymerises -Taq, E. coli Pol I, and T7 - because they are widely utilized in biotechnology today. The redesign of polymerases often requires knowledge of the function of specific residues in the protein, including those located in six evolutionarily conserved regions. The most well characterized of these are motifs A and B, which regulate the fidelity of replication and the incorporation of nucleotide analogs such as dideoxynucleotides. Regions that remain to be more thoroughly characterized are motif C, which is critical for catalysis, and motifs 1, 2 and 6, all of which bind to DNA primer or template. Several recently identified mutants with abilities to incorporate nucleotides with bulky adducts have mutations that are not located within conserved regions and warrant further study. Analysis of these mutants will help advance our understanding of how DNA polymerases select bases with high fidelity.     

Neurite Outgrowth is Dependent on the Association of c-Src and Lipid Rafts

Regulation of neurite outgrowth is an important aspect not only for proper development of the nervous system but also for tissue regeneration after nerve injury and the treatment of neuropathological conditions. Here, we report that neurite outgrowth in cortical neuron and neuro 2A (N2A) cell was dependent on intact lipid rafts, as well as the enhanced localization of c-Src in the lipid rafts. Src inhibition or lipid rafts disruption could specifically block c-Src phosphorylation profile, pY416 Src increase and pY529 Src decrease, they also resulted in pY529 Src and c-terminal Src kinase (Csk) partition out of lipid rafts. Thus, we concluded that c-Src signal cascades within the lipid rafts is crucial for efficient neurite outgrowth.