Discovery of a Novel Human Pegivirus in Blood Associated with Hepatitis C Virus Co-Infection

Hepatitis C virus (HCV) and human pegivirus (HPgV), formerly GBV-C, are the only known human viruses in the Hepacivirus and Pegivirus genera, respectively, of the family Flaviviridae. We present the discovery of a second pegivirus, provisionally designated human pegivirus 2 (HPgV-2), by next-generation sequencing of plasma from an HCV-infected patient with multiple bloodborne exposures who died from sepsis of unknown etiology. HPgV-2 is highly divergent, situated on a deep phylogenetic branch in a clade that includes rodent and bat pegiviruses, with which it shares <32% amino acid identity. Molecular and serological tools were developed and validated for high-throughput screening of plasma samples, and a panel of 3 independent serological markers strongly correlated antibody responses with viral RNA positivity (99.9% negative predictive value). Discovery of 11 additional RNA-positive samples from a total of 2440 screened (0.45%) revealed 93–94% nucleotide identity between HPgV-2 strains. All 12 HPgV-2 RNA-positive cases were identified in individuals also testing positive for HCV RNA (12 of 983; 1.22%), including 2 samples co-infected with HIV, but HPgV-2 RNA was not detected in non-HCV-infected individuals (p<0.0001), including those singly infected by HIV (p = 0.0075) or HBV (p = 0.0077), nor in volunteer blood donors (p = 0.0082). Nine of the 12 (75%) HPgV-2 RNA positive samples were reactive for antibodies to viral serologic markers, whereas only 28 of 2,429 (1.15%) HPgV-2 RNA negative samples were seropositive. Longitudinal sampling in two individuals revealed that active HPgV-2 infection can persist in blood for at least 7 weeks, despite the presence of virus-specific antibodies. One individual harboring both HPgV-2 and HCV RNA was found to be seronegative for both viruses, suggesting a high likelihood of simultaneous acquisition of HCV and HPgV-2 infection from an acute co-transmission event. Taken together, our results indicate that HPgV-2 is a novel bloodborne infectious virus of humans and likely transmitted via the parenteral route.     

Drosophila Spaghetti and Doubletime Link the Circadian Clock and Light to Caspases,Apoptosis and Tauopathy

While circadian dysfunction and neurodegeneration are correlated, the mechanism for this is not understood. It is not known if age-dependent circadian dysfunction leads to neurodegeneration or vice-versa, and the proteins that mediate the effect remain unidentified. Here, we show that the knock-down of a regulator (spag) of the circadian kinase Dbt in circadian cells lowers Dbt levels abnormally, lengthens circadian rhythms and causes expression of activated initiator caspase (Dronc) in the optic lobes during the middle of the day or after light pulses at night. Likewise, reduced Dbt activity lengthens circadian period and causes expression of activated Dronc, and a loss-of-function mutation in Clk also leads to expression of activated Dronc in a light-dependent manner. Genetic epistasis experiments place Dbt downstream of Spag in the pathway, and Spag-dependent reductions of Dbt are shown to require the proteasome. Importantly, activated Dronc expression due to reduced Spag or Dbt activity occurs in cells that do not express the spag RNAi or dominant negative Dbt and requires PDF neuropeptide signaling from the same neurons that support behavioral rhythms. Furthermore, reduction of Dbt or Spag activity leads to Dronc-dependent Drosophila Tau cleavage and enhanced neurodegeneration produced by human Tau in a fly eye model for tauopathy. Aging flies with lowered Dbt or Spag function show markers of cell death as well as behavioral deficits and shortened lifespans, and even old wild type flies exhibit Dbt modification and activated caspase at particular times of day. These results suggest that Dbt suppresses expression of activated Dronc to prevent Tau cleavage, and that the circadian clock defects confer sensitivity to expression of activated Dronc in response to prolonged light. They establish a link between the circadian clock factors, light, cell death pathways and Tau toxicity, potentially via dysregulation of circadian neuronal remodeling in the optic lobes.     

FtsZ-ZapA-ZapB interactome of Escherichia coli

Bacterial cell division relies on the formation and contraction of the Z ring, coordinated and regulated by a dynamic protein complex called the divisome. The cell division factor ZapA interacts directly with FtsZ and thereby increases FtsZ protofilament association and Z-ring stability. Here, we investigated ZapB interaction with ZapA and its effect on Z-ring formation and FtsZ protofilament bundling. The combination of the ftsZ84 allele that encodes an FtsZ variant that polymerizes inefficiently with a zapB null mutant resulted in a synthetic defective phenotype. Overproduction of ZapA led to the formation of aberrant FtsZ helical structures and delocalization of ZapB. The N-terminal end of ZapB was essential for ZapB-ZapA interaction, and amino acid changes close to the N terminus of ZapB exhibited reduced interaction with ZapA. Sedimentation assays showed that ZapB interacts strongly with ZapA and reduces ZapA's interaction with FtsZ in vitro. The morphology of the structures formed by ZapA and ZapB together was similar to the cables formed by ZapB in the presence of CaCl(2), a known ZapB bundling agent. The in vivo and in vitro data support a model in which ZapA interacts strongly with ZapB and the ZapA-ZapB interaction is favored over ZapA-FtsZ.     

Spiders are special: fear and disgust evoked by pictures of arthropods

Because all spiders are predators and most subdue their prey with poison, it has been suggested that fear of spiders is an evolutionary adaptation. However, it has not been sufficiently examined whether other arthropods similarly elicit fear or disgust. Our aim was to examine if all arthropods are rated similarly, if only potentially dangerous arthropods (spiders, bees/wasps) elicit comparable responses, or if spiders are rated in a unique way. We presented pictures of arthropods (15 spiders, 15 beetles, 15 bees/wasps, and 15 butterflies/moths) to 76 students who rated each picture for fear, disgust, and how dangerous they thought the animal is. They also categorized each animal into one of the four animal groups. In addition, we assessed the participants' fear of spiders and estimates for trait anxiety. The ratings showed that spiders elicit significantly greater fear and disgust than any other arthropod group, and spiders were rated as more dangerous. Fear and disgust ratings of spider pictures significantly predicted the questionnaire scores for fear of spiders, whereas dangerousness ratings of spiders and ratings of other arthropods do not provide any predictive power. Thus, spider fear is in fact spider specific. Our results demonstrate that potential harmfulness alone cannot explain why spiders are feared so frequently.     

Roles of Myosin Va and Rab3D in Membrane Remodeling of Immature Secretory Granules

Neuroendocrine secretory granules (SGs) are formed at the trans-Golgi network (TGN) as immature intermediates. In PC12 cells, these immature SGs (ISGs) are transported within seconds to the cell cortex, where they move along actin filaments and complete maturation. This maturation process comprises acidification-dependent processing of cargo proteins, condensation of the SG matrix, and removal of membrane and proteins not destined to mature SGs (MSGs) into ISG-derived vesicles (IDVs). We investigated the roles of myosin Va and Rab3 isoforms in the maturation of ISGs in neuroendocrine PC12 cells. The expression of dominant-negative mutants of myosin Va or Rab3D blocked the removal of the endoprotease furin from ISGs. Furthermore, expression of mutant Rab3D, but not of mutant myosin Va, impaired cargo processing of SGs. In conclusion, our data suggest an implication of myosin Va and Rab3D in the maturation of SGs where they participate in overlapping but not identical tasks.     

A mechanism for ParB-dependent waves of ParA,a protein related to DNA segregation during cell division in prokaryotes

Prokaryotic plasmids encode partitioning (par) loci involved in segregation of DNA to daughter cells at cell division. A functional fusion protein consisting of Walker-type ParA ATPase and green fluorescent protein (Gfp) oscillates back and forth within nucleoid regions with a wave period of about 20 minutes. A model is discussed which is based on cooperative non-specific binding of ParA to the nucleoid, and local ParB initiated generation of ParA oligomer degradation products, which act autocatalytically on the degradation reaction. The model yields self-initiated spontaneous pattern formation, based on Turing's mechanism, and these patterns are destroyed by the degradation products, only to initiate a new pattern at the opposite nucleoid region. A recurrent wave thus emerges. This may be a particular example of a more general class of pattern forming mechanisms, based on protein oligomerization upon a template (membranes, DNA a.o.) with resulting enhanced NTPase function in the oligomer state, which may bring the oligomer into an unstable internal state. An effector initializes destabilization of the oligomer to yield degradation products, which act as seeds for further degradation in an autocatalytic process. We discuss this mechanism in relation to recent models for MinDE oscillations in E.coli and to microtubule degradation in mitosis. The study points to an ancestral role for the presented pattern types in generating bipolarity in prokaryotes and eukaryotes.     

Biphenyls as potent vitronectin receptor antagonists. Part 2: biphenylalanine ureas

Vitronectin receptor (alpha(V)beta(3)) antagonism has been implicated in a variety of disease states, like restenosis, osteoporosis and cancer. In this work, we present the development of a novel class of biphenyl vitronectin receptor antagonists. Identified from a focused combinatorial library based on para-bromo phenylalanine, these compounds show nanomolar affinity to the vitronectin receptor and display unprecedented SAR. Their binding mode can be rationalized by computational docking studies using the X-ray structure of alpha(V)beta(3).