Replication blocking lesions present a unique substrate for homologous recombination

Homologous recombination (HR) plays a critical role in the restart of blocked replication forks, but how this is achieved remains poorly understood. We show that mutants in the single Rad51 paralog in Caenorhabditis elegans, rfs-1, permit discrimination between HR substrates generated at DNA double-strand breaks (DSBs), or following replication fork collapse from HR substrates assembled at replication fork barriers (RFBs). Unexpectedly, RFS-1 is dispensable for RAD-51 recruitment to meiotic and ionizing radiation (IR)-induced DSBs and following replication fork collapse, yet, is essential for RAD-51 recruitment to RFBs formed by DNA crosslinking agents and other replication blocking lesions. Deletion of rfs-1 also suppresses the accumulation of toxic HR intermediates in him-6; top-3 mutants and accelerates deletion formation at presumed endogenous RFBs formed by poly G/C tracts in the absence of DOG-1. These data suggest that RFS-1 is not a general mediator of HR-dependent DSB repair, but acts specifically to promote HR at RFBs. HR substrates generated at conventional DSBs or following replication fork collapse are therefore intrinsically different from those produced during normal repair of blocked replication forks.     

Sex allocation theory reveals a hidden cost of neonicotinoid exposure in a parasitoid wasp

Sex allocation theory has proved to be one the most successful theories in evolutionary ecology. However, its role in more applied aspects of ecology has been limited. Here we show how sex allocation theory helps uncover an otherwise hidden cost of neonicotinoid exposure in the parasitoid wasp Nasonia vitripennis. Female N. vitripennis allocate the sex of their offspring in line with Local Mate Competition (LMC) theory. Neonicotinoids are an economically important class of insecticides, but their deployment remains controversial, with evidence linking them to the decline of beneficial species. We demonstrate for the first time to our knowledge, that neonicotinoids disrupt the crucial reproductive behaviour of facultative sex allocation at sub-lethal, field-relevant doses in N. vitripennis. The quantitative predictions we can make from LMC theory show that females exposed to neonicotinoids are less able to allocate sex optimally and that this failure imposes a significant fitness cost. Our work highlights that understanding the ecological consequences of neonicotinoid deployment requires not just measures of mortality or even fecundity reduction among non-target species, but also measures that capture broader fitness costs, in this case offspring sex allocation. Our work also highlights new avenues for exploring how females obtain information when allocating sex under LMC.     

The recovery of coral genetic diversity in the Sunda Strait following the 1883 eruption of Krakatau

Surveys of microsatellite variation show that genetic diversity has largely recovered in two reef-building corals, Pocillopora damicornis and Seriatopora hystrix (Scleractinia: Pocilloporidae), on reefs which were decimated by the eruption of the volcano Krakatau in 1883. Assignment methods and gene flow estimates indicate that the recolonization of Krakatau occurred mainly from the closest upstream reef system, Pulau Seribu, but that larval input from other regions has also occurred. This pattern is clearer in S. hystrix, which is traditionally the more dispersal-limited species. Despite these observed patterns of larval dispersal, self-recruitment appears to now be the most important factor in supplying larvae to coral populations in Krakatau. This suggests that the colonization of devastated reefs can occur quickly through larval dispersal; however, their survival requires local sources of larvae for self-recruitment. This research supports the observation that the recovery of genetic diversity in coral reef animals can occur on the order of decades and centuries rather than millennia. Conservation measures aimed at sustaining coral reef populations in Krakatau and elsewhere should include both the protection of upstream source populations for larval replenishment should disaster occur as well as the protection of large adult colonies to serve as local larval sources.     

Attenuation of Vesicular Stomatitis Virus Encephalitis through MicroRNA Targeting

Vesicular stomatitis virus (VSV) has long been regarded as a promising recombinant vaccine platform and oncolytic agent but has not yet been tested in humans because it causes encephalomyelitis in rodents and primates. Recent studies have shown that specific tropisms of several viruses could be eliminated by engineering microRNA target sequences into their genomes, thereby inhibiting spread in tissues expressing cognate microRNAs. We therefore sought to determine whether microRNA targets could be engineered into VSV to ameliorate its neuropathogenicity. Using a panel of recombinant VSVs incorporating microRNA target sequences corresponding to neuron-specific or control microRNAs (in forward and reverse orientations), we tested viral replication kinetics in cell lines treated with microRNA mimics, neurotoxicity after direct intracerebral inoculation in mice, and antitumor efficacy. Compared to picornaviruses and adenoviruses, the engineered VSVs were relatively resistant to microRNA-mediated inhibition, but neurotoxicity could nevertheless be ameliorated significantly using this approach, without compromise to antitumor efficacy. Neurotoxicity was most profoundly reduced in a virus carrying four tandem copies of a neuronal mir125 target sequence inserted in the 3′-untranslated region of the viral polymerase (L) gene.Vesicular stomatitis virus (VSV) is a nonsegmented, negative-strand rhabdovirus widely used as a vaccine platform as well as an anticancer therapeutic. While VSV is predominantly a pathogen of livestock (34), it has a very broad species tropism. The cellular tropism of VSV is determined predominantly at postentry steps, since the G glycoprotein of the virus mediates entry into most tissues in nearly all animal species (10).Though viral entry can take place in nearly all cell types, in vivo models of VSV infection have revealed that the virus is highly sensitive to the innate immune response, limiting its pathogenesis (4). VSV is intensively responsive to type I interferon (IFN), as the double-stranded RNA (dsRNA)-dependent PKR (2), the downstream effector of pattern recognition receptors MyD88 (32), and other molecules mediate shutdown of viral translation and allow the adaptive immune response to clear the virus. The vulnerability of the virus to the type I IFN response, typically defective in many cancers, has been exploited to generate tumor-selective replication (49), such that the virus is now poised to enter phase I trials. However, the virus remains potently neurotoxic, causing lethal encephalitis not only in rodent models (7, 22, 53) but also in nonhuman primates (25).VSV very often infiltrates the central nervous system (CNS) through infection of the olfactory nerves (41). When administered intranasally, the virus replicates rapidly in the nasal epithelium and is transmitted to olfactory neurons, from which it then moves retrograde axonally to the brain and replicates robustly, causing neuropathogenesis. While intranasal inoculation does cause neuropathy in mice, neurotoxicity following viral administration also occurs when the virus is delivered intravascularly (47), intraperitoneally (42), and (not surprisingly) intracranially (13). Previously, other groups have modified the VSV genome to be more sensitive to cellular IFNs (49) and have actually encoded IFN in the virus (36). However, the former can result in attenuation of the virus, such that it has reduced anticancer potential, while the latter still results in lethal encephalitis (unpublished results). In order to mitigate the effects of VSV infection on the brain without perturbing the potent oncolytic activity of the virus, we utilized a microRNA (miRNA) targeting paradigm, whereby viral replication is restricted in the brain without altering the tropism of the virus for other tissues.To redirect the tissue tropism of anticancer therapeutics, we (26) and others (11, 14, 55) have previously exploited the tissue-specific expression of cellular miRNAs. miRNAs are ∼22-nucleotide (nt) regulatory RNAs that regulate a diverse and expansive array of cellular activities. Through recognition of sequence-complementary target elements, miRNAs can either translationally suppress or catalytically degrade both cellular (6) and viral (50) RNAs. We have determined that cellular miRNAs can potentially regulate numerous steps of a virus life cycle and that this regulation of the virus by endogenous miRNAs can then abrogate toxicities of replication-competent viruses (27; E. J. Kelly et al., unpublished data).miRNAs are known to be highly upregulated in many different tissues, including (but not limited to) muscle (40), lung (44), liver (15, 44), spleen (44, 46), and kidney (51). In addition, the brain has a number of upregulated miRNAs, with each different subtype of cell having a unique miRNA profile. miR-125 is highly upregulated in all cells in the brain (neurons, astrocytes, and glia cells), while miR-124 is found predominantly in neuronal cells (48). Glial cells and glioblastomas are thought to have decreased expression of miR-128 compared to neurons (17), while miR-134 is particularly abundant in dendrites of neurons in the hippocampus (43). In addition to these miRNAs, the tumor suppressor miRNA let-7 and miRs 9, 26, and 29 (51) are also found to be enriched in the brain, with expression varying not only between different cell types and regions of the brain but also temporally (48).MicroRNAs have previously been exploited to modulate the tissue tropism of nonreplicating lentiviral vectors (8, 9), as well as curbing known toxicities of replication-competent picornaviruses (5, 26), adenoviruses (11), herpes simplex virus 1 (33), and influenza A virus (39). In addition, a recombinant VSV encoding a tumor suppressor target was found to be responsive to sequence-complementary miRNAs in vitro, possibly by affecting expression of the matrix (M) protein (14), and evidence from Dicer-deficient mice suggests that endogenously expressed microRNA targets within the P and L genes of VSV could restrict enhanced pathogenicity of the virus (37). However, in vivo protection from neuropathogenesis by this means has not been demonstrated for VSV.Here we evaluate the efficiencies of different brain-specific miRNAs for shutting down gene expression and extensively characterize the ability of miRNA targeting to attenuate the neurotoxicity of vesicular stomatitis virus in vivo. We constructed and evaluated recombinant VSVs with miRNA target (miRT) insertions at different regions of the viral genome, with special focus upon those affecting viral L expression. In addition, we looked at the regulatory efficiency of different brain-specific miRNAs and the impact of miRT orientation on VSV replication and determined the impact of the virus on oncolytic activity in vivo.     

4-1BB signaling synergizes with programmed death ligand 1 blockade to augment CD8 T cell responses during chronic viral infection

Previous studies have identified the inhibitory role that the programmed death 1 (PD-1) pathway plays during chronic infection. Blockade of this pathway results in rescue of viral-specific CD8 T cells, as well as reduction of viral loads in mice chronically infected with lymphocytic choriomeningitis virus (LCMV). We tested the effect of combining PD ligand 1 (PD-L1) blockade with an agonistic regimen that induces 4-1BB costimulation during chronic LCMV infection. There is a boosting effect in the rescue of LCMV-specific CD8 T cell responses after dual treatment with PD-L1 blockade and 4-1BB agonistic Abs when the amount and timing of 4-1BB costimulation are carefully controlled. When PD-L1-blocking Abs are given together with a single low dose of anti-4-1BB agonistic Abs, there is an enhanced and stable expansion of viral-specific CD8 T cells. Conversely, when blocking Abs to PD-L1 are given with a repetitive high dose of anti-4-1BB, there is an initial synergistic expansion of viral-specific CD8 T cells by day 7, followed by dramatic apoptosis by day 14. Viral control paralleled CD8 T cell kinetics after dual treatment. By day 7 posttreatment, viral titers were lower in both of the combined regimens (compared with PD-L1 blockade alone). However, whereas the high dose of anti-4-1BB plus PD-L1 blockade resulted in rebound of viral titers to original levels, the low dose of anti-4-1BB plus PD-L1 blockade resulted in a stable reduction of viral loads. These findings demonstrate the importance of carefully manipulating the balance between activating and inhibitory signals to enhance T cell responses during chronic infection.     

Phylogenetic ecology of widespread uncultured clades of the Kingdom Euryarchaeota

Despite its widespread distribution and high levels of phylogenetic diversity, microbes are poorly understood creatures. We applied a phylogenetic ecology approach in the Kingdom Euryarchaeota (Archaea) to gain insight into the environmental distribution and evolutionary history of one of the most ubiquitous and largely unknown microbial groups. We compiled 16S rRNA gene sequences from our own sequence libraries and public genetic databases for two of the most widespread mesophilic Euryarchaeota clades, Lake Dagow Sediment (LDS) and Rice Cluster-V (RC-V). The inferred population history indicated that both groups have undergone specific nonrandom evolution within environments, with several noteworthy habitat transition events. Remarkably, the LDS and RC-V groups had enormous levels of genetic diversity when compared with other microbial groups, and proliferation of sequences within each single clade was accompanied by significant ecological differentiation. Additionally, the freshwater Euryarchaeota counterparts unexpectedly showed high phylogenetic diversity, possibly promoted by their environmental adaptability and the heterogeneous nature of freshwater ecosystems. The temporal phylogenetic diversification pattern of these freshwater Euryarchaeota was concentrated both in early times and recently, similarly to other much less diverse but deeply sampled archaeal groups, further stressing that their genetic diversity is a function of environment plasticity. For the vast majority of living beings on Earth (i.e. the uncultured microorganisms), how they differ in the genetic or physiological traits used to exploit the environmental resources is largely unknown. Inferring population history from 16S rRNA gene-based molecular phylogenies under an ecological perspective may shed light on the intriguing relationships between lineage, environment, evolution and diversity in the microbial world.     

Strategic egg allocation in the zebra fish, Danio rerio

Females across a range of taxa have been shown to differentiallyallocate their reproductive resources according to the attractivenessof their mate. Previous studies demonstrated a female preferencefor larger males in the zebra fish but have so far failed touncover a size-mediated difference in male mating success, possiblydue to the effects of male–male competition. By controllingfor male–male competition in the present study, we showthat females strategically allocate their reproductive resources(i.e., eggs) toward larger males. When females were mated sequentiallywith a large and small male, they released a greater numberof eggs to the second male when he was large than when he wassmall. Furthermore, there was also a trend for females to releasea greater proportion of their eggs to the first male when hewas large. Across females, the total number of eggs laid byeach female increased with the average standard length of themale pair, whereas the number of eggs laid to the second malealso increased with his standard length. This study representsone of the first attempts at identifying differential allocationin a resource-free egg scatterer and suggests that female preferencesmay play a greater role in the reproductive success of malesin this species than previously envisaged.     

Synonymous mutations in the core gene are linked to unusual serological profile in hepatitis C virus infection

The biological role of the protein encoded by the alternative open reading frame (core+1/ARF) of the Hepatitis C virus (HCV) genome remains elusive, as does the significance of the production of corresponding antibodies in HCV infection. We investigated the prevalence of anti-core and anti-core+1/ARFP antibodies in HCV-positive blood donors from Cambodia, using peptide and recombinant protein-based ELISAs. We detected unusual serological profiles in 3 out of 58 HCV positive plasma of genotype 1a. These patients were negative for anti-core antibodies by commercial and peptide-based assays using C-terminal fragments of core but reacted by Western Blot with full-length core protein. All three patients had high levels of anti-core+1/ARFP antibodies. Cloning of the cDNA that corresponds to the core-coding region from these sera resulted in the expression of both core and core+1/ARFP in mammalian cells. The core protein exhibited high amino-acid homology with a consensus HCV1a sequence. However, 10 identical synonymous mutations were found, and 7 were located in the aa(99-124) region of core. All mutations concerned the third base of a codon, and 5/10 represented a T>C mutation. Prediction analyses of the RNA secondary structure revealed conformational changes within the stem-loop region that contains the core+1/ARFP internal AUG initiator at position 85/87. Using the luciferase tagging approach, we showed that core+1/ARFP expression is more efficient from such a sequence than from the prototype HCV1a RNA. We provide additional evidence of the existence of core+1/ARFP in vivo and new data concerning expression of HCV core protein. We show that HCV patients who do not produce normal anti-core antibodies have unusually high levels of anti-core+1/ARFP and harbour several identical synonymous mutations in the core and core+1/ARFP coding region that result in major changes in predicted RNA structure. Such HCV variants may favour core+1/ARFP production during HCV infection.     

The impact of insecticide resistance in the currant-lettuce aphid, Nasonovia ribisnigri, on pest management in lettuce

Abstract 1 This paper reports on experiments to determine how two different insecticide resistance phenotypes in the aphid Nasonovia ribisnigri (Mosley), which is a major pest of lettuce, change its susceptibility to pyrethroid insecticides and the carbamate pirimicarb. 2 A novel statistical approach determined how the effectiveness of different insecticides was changed by the two resistance phenotypes. This compared the between‐plant distribution of aphid numbers, as opposed to the mean number of aphids per plant. 3 Results from field cage experiments showed that the effect of the resistances differed. Pyrethroid resistance resulted in lower mortality immediately after application of pyrethroids, whereas resistance to pirimicarb shortened the time over which the chemical was effective. 4 The results of laboratory bioassays suggested that these two resistances were not found together in N. ribisnigri. However, the results reported here contradict this assertion. 5 Experiments with insecticide residues showed that reproduction of resistant N. ribisnigri was greater than that of susceptible N. ribisnigri on plants with ageing insecticide residues, even in circumstances where mortality of resistant and susceptible clones of N. ribisnigri were similar. 6 If more than a few aphids are found on a plant then a whole consignment can be rejected for processing. The results reported here suggest that the effect of both insecticide resistances in N. ribsinigri will be to increase the proportion of lettuce heads with an unacceptable number of aphids on them, leading to increased rejection of plants for processing.     

Heterogeneous gap junction remodeling stabilizes reentrant circuits in the epicardial border zone of the healing canine infarct: a computational study

The ventricular tachycardias (VTs) that originate in the 5-day epicardial border zone (EBZ) of the healing canine infarcted heart are due to reentrant excitation. In cells surviving in the EBZ, both sarcolemmal ionic channels and gap junction conductance and distribution are remodeled. We previously showed that the heterogeneities in sodium current (I(Na)) and L-type calcium channel current (I(CaL)) of the center and outer pathway cells result in a homogenization of the refractory period that in turn stabilizes reentrant VTs for approximately 10 beats. To understand how heterogeneities in transverse gap junctional conductance remodeling reported experimentally contribute to the stability of these tachycardias, we studied the dynamics of reentering waves in two-dimensional computer models of the EBZ. First we used a computer model with homogeneous ionic channel properties [infarcted border zone cell model (IZ)]. These simulations show that, in the absence of heterogeneities in ionic channel properties, reentrant waves tend to drift to localized regions of uncoupling and stabilize there. Second, we used a computer model with a more realistic representation of the heterogeneous EBZ, including cellular models for both the center (IZ(c)) and outer (IZ(o)) pathway cells. These simulations show that neither a region of uniform uncoupling nor a step transition between two regions with different side-to-side (transverse) cell coupling stabilizes reentry in this substrate. However, an area of localized uncoupling did stabilize reentry in such a model. We propose that in addition to the heterogeneities in I(Na) and I(CaL) properties, heterogeneities in gap junctional conductance in the EBZ causing regions of localized uncoupling stabilize VT in the EBZ. Previous experimental in situ activation maps of the 5-day EBZ show that the lines of block form in regions of slow transverse propagation. This is consistent with our findings that areas of localized uncoupling stabilize reentry.