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Aedes aegypti is a major vector for arboviruses such as dengue, chikungunya and Zika viruses. During acquisition of a viremic bloodmeal, an arbovirus infects mosquito midgut cells before disseminating to secondary tissues, including the salivary glands. Once virus is released into the salivary ducts it can be transmitted to another vertebrate host. The midgut is surrounded by a basal lamina (BL) in the extracellular matrix, consisting of a proteinaceous mesh composed of collagen IV and laminin. BL pore size exclusion limit prevents virions from passing through. Thus, the BL probably requires remodelling via enzymatic activity to enable efficient virus dissemination. Matrix metalloproteinases (MMPs) are extracellular endopeptidases that are involved in remodelling of the extracellular matrix. Here, we describe and characterize the nine Ae. aegypti encoded MMPs, AeMMPs 1-9, which share common features with other invertebrate and vertebrate MMPs. Expression profiling in Ae. aegypti revealed that Aemmp4 and Aemmp6 were upregulated during metamorphosis, whereas expression of Aemmp1 and Aemmp2 increased during bloodmeal digestion. Aemmp1 expression was also upregulated in the presence of a bloodmeal containing chikungunya virus. Using polyclonal antibodies, AeMMP1 and AeMMP2 were specifically detected in tissues associated with the mosquito midgut.

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Synapse formation in the developing brain depends on the coordinated activity of synaptogenic proteins, some of which have been implicated in a number of neurodevelopmental disorders. Here, we show that the sushi repeat-containing protein X-linked 2 (SRPX2) gene encodes a protein that promotes synaptogenesis in the cerebral cortex. In humans, SRPX2 is an epilepsy- and language-associated gene that is a target of the foxhead box protein P2 (FoxP2) transcription factor. We also show that FoxP2 modulates synapse formation through regulating SRPX2 levels and that SRPX2 reduction impairs development of ultrasonic vocalization in mice. Our results suggest FoxP2 modulates the development of neural circuits through regulating synaptogenesis and that SRPX2 is a synaptogenic factor that plays a role in the pathogenesis of language disorders.

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Abstract : Oral administration of Toxoplasma gondii oocysts to cats (i.e., monoxenous transmission) typically induces patent infections in fewer than half of test subjects. In the present study, oral administration of T. gondii oocysts to 5 kittens induced a patent infection in 2 of them, but only 1 kitten shed enough oocysts to enable further study. Those monoxenously-produced oocysts were administered to another kitten, which produced a second generation of monoxenous oocysts, and then those were used to induce a third generation of monoxenous oocysts. These results provide a rationale to develop a strain of T. gondii that has efficient direct transmission. The isolate of T. gondii that was able to be passaged in this manner has been designated the Dubey strain and cultured tachyzoites have been donated to a repository.

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Many viruses express proteins which prevent the host cell death that their infection would otherwise provoke. Some insect viruses suppress host apoptosis through the expression of caspase inhibitors belonging to the P35 superfamily. Although a number of P35 relatives have been identified, Autographa californica (Ac) P35 and Spodoptera littoralis (Spli) P49 have been the most extensively characterized. AcP35 was found to inhibit caspases via a suicide substrate mechanism: the caspase cleaves AcP35 within its 'reactive site loop' then becomes trapped, irreversibly bound to the cleaved inhibitor. The Maruca vitrata multiple nucleopolyhedrovirus encodes a P35 family member (MaviP35) that exhibits 81% identity to AcP35. We found that this relative shared with AcP35 the ability to inhibit mammalian and insect cell death. Caspase-mediated cleavage within the MaviP35 reactive site loop occurred at a sequence distinct from that in AcP35, and the inhibitory profiles of the two P35 relatives differed. MaviP35 potently inhibited human caspases 2 and 3, DCP-1, DRICE and CED-3 in vitro, but (in contrast to AcP35) only weakly suppressed the proteolytic activity of the initiator human caspases 8, 9 and 10. Although MaviP35 inhibited the AcP35-resistant caspase DRONC in yeast, and was sensitive to cleavage by DRONC in vitro, MaviP35 failed to inhibit the proteolytic activity of bacterially produced DRONC in vitro.

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Genetic studies in Drosophila melanogaster have revealed that inhibitor of apoptosis (IAP) proteins and IAP antagonists such as reaper play a pivotal role in controlling cell death in insects. Interestingly, although the sequences and structures of IAPs are highly conserved, the sequence of IAP antagonists diverged very rapidly during evolution, making their identification difficult. Using a customized bioinformatics approach, we identified an IAP antagonist, IAP-binding motif 1 (Ibm1), from the genome of the silkworm Bombyx mori. This is the first reaper/grim orthologue identified in a nondipteran insect. Previous analysis indicated that both Reaper and Grim induce cell death through their N-terminal IBM as well as the Grim_helix3 (GH3) domain. Functional studies indicated that Ibm1 binds to an IAP protein from B. mori, BmIAP1, and induces apoptosis in insect cells via the IAP-binding motif, a seven amino acid sequence that is highly conserved in all IAP antagonists. Interestingly, Ibm1 also contains a region that is a statistically significant match to the GH3 domain. Mutational analysis indicated that the GH3-like motif in Ibm1 has an important supportive role in IAP-antagonist function and can trigger cell death under certain conditions.

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Controlled trials with a common protocol were conducted in Idaho, Illinois and Tennessee to evaluate anthelmintic effectiveness of Quest Gel (QG; 2% moxidectin) against lumenal parasites in horses. Candidate horses were required to have naturally acquired nematode infections, as confirmed by presence of strongylid eggs in feces. At each site, 24 equids were blocked on the basis of pretreatment strongyle fecal egg counts (EPG) and randomly assigned to treatments within blocks. Within each block of two animals, one received QG on Day 0 at a dosage of 0.4 mg moxidectin/kg b.w. and one was an untreated control. Body weights measured the day before treatment served as the basis for calculating treatment doses. Horses assigned to treatment with QG received the prescribed dose administered orally with the commercially packaged Sure Dial syringe. Horses were necropsied 12-14 days after treatment, and lumenal parasites and digesta were harvested separately from each of five organs, including the stomach, small intestine, cecum, ventral colon and dorsal colon. Parasites from stomachs and small intestines were identified to genus, species and stage. Micro- (i.e., < 1.5 cm) and macroparasites (i.e., > 1.5 cm) in aliquots from the cecum, ventral colon and dorsal colon were examined in aliquots of approximately 200 parasites until at least 600 parasites had been identified to genus, species and stage or until all parasites in the 5% aliquot were examined, whichever occurred first. Data were combined across sites and analyzed by mixed model analysis of variance to assess the fixed effect of treatment and random effects of site and block within site. Because QG does not contain a cestocide, efficacy of QG against tapeworms was not significant (P > 0.05). Based on geometric means, however, efficacy of QG was greater than 90% (P < 0.05) against 38 species and developmental stages of cyathostomes, strongyles, bots, larval pinworms and ascarids encountered in at least 6 of 36 control horses in the combined data set. None of the horses treated with moxidectin exhibited evidence of adverse effects. Study results demonstrate QG, administered to horses with naturally acquired endoparasite infections at a dosage of 0.4 mg moxidectin/kg b.w., was highly effective against a broad range of equine parasitic infections.

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Although mitochondrial proteins play well-defined roles in caspase activation in mammalian cells, the role of mitochondrial factors in caspase activation in Drosophila is unclear. Using cell-free extracts, we demonstrate that mitochondrial factors play no apparent role in Drosophila caspase activation. Cytosolic extract from apoptotic S2 cells, in which caspases were inhibited, induced caspase activation in cytosolic extract from normal S2 cells. Mitochondrial extract did not activate caspases, nor did it influence caspase activation by cytosolic extract. Silencing of Hid, Reaper, or Grim reduced caspase activation by apoptotic cell extract. Furthermore, a peptide representing the amino terminus of Hid was sufficient to activate caspases in cytosolic extract, and this activity was not enhanced by addition of mitochondria or mitochondrial lysate. The Hid peptide also induced apoptosis when introduced into S2 cells. These results suggest that caspase activation in Drosophila is regulated solely by cytoplasmic factors and does not involve any mitochondrial factors.

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The baculoviruses make up a large, diverse family of DNA viruses that have evolved a number of fascinating mechanisms to manipulate their insect hosts. One of these is the ability to regulate apoptosis during infection by expressing proteins that can inhibit caspase activation and/or activity, including the caspase inhibitor P35 and its relatives, and the inhibitor of apoptosis (IAP) proteins. Experimental manipulations of the expression of these antiapoptotic genes, either by genetic deletions or by RNAi, have shed light on the effectiveness of apoptosis in combating baculovirus infection. The results of these experiments indicate that apoptosis can be an extremely powerful response to baculovirus infection, reducing viral replication, infectivity, and the ability of the virus to spread within the insect host even if a successful infection is established. Apoptosis is especially effective when it is combined with other innate antiviral defenses, which are largely unexplored in insects to date.

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BACKGROUND: More than 30 million needle syringes are distributed per year in Australia as a component of harm-reduction strategies for injecting drug users (IDU). Discarded needle syringes create considerable anxiety within the community, but the extent of needlestick injuries and level of blood-borne virus transmission risk is unclear. We have undertaken a review of studies of blood-borne virus survival as the basis for advice and management of community needlestick injuries. METHODS: A Medline review of published articles on blood-borne virus survival and outcome from community injuries. RESULTS: Hepatitis B virus (HBV), hepatitis C virus (HCV) and human immunodeficiency virus (HIV) can all survive outside the human body for several weeks, with virus survival influenced by virus titer, volume of blood, ambient temperature, exposure to sunlight and humidity. HBV has the highest virus titers in untreated individuals and is viable for the most prolonged periods in needle syringes stored at room temperature. However, prevalence of HBV and HIV are only 1-2% within the Australian IDU population. In contrast, prevalence of HCV is 50-60% among Australian IDUs and virus survival in needle syringes has been documented for prolonged periods. There have been no published cases of blood-borne virus transmission following community needlestick injury in Australia. CONCLUSION: The risk of blood-borne virus transmission from syringes discarded in community settings appears to be very low. Despite this, procedures to systematically follow up individuals following significant needlestick exposures sustained in the community setting should be developed.

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The PstI-I region of the Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) genome was previously shown to be a frequent target of spontaneous deletions during serial virus passage in TN-368 cells (Kumar and Miller, Virus Res. 7 (1987) 335). Analysis of two of these serial passage mutants showed that a portion of the Ac-iap1 gene was deleted. To directly test the effect of loss of Ac-iap1, three different deletions in Ac-iap1 were introduced into recombinant viruses and the ability of these viruses to replicate was examined in two cell lines, TN-368 and SF-21, as well as in two species of insect larvae, Trichoplusia ni and Spodoptera frugiperda. The mutant viruses were indistinguishable from wild type or control revertant virus in their ability to infect larvae of either species. Moreover, no effect was seen on the rate of replication or the overall amounts of budded or occluded virus produced in cultured cells. However, in co-infection experiments using TN-368 cells, it was consistently observed that mutants lacking a functional Ac-iap1 gene out-competed control viruses carrying Ac-iap1. Interestingly, this replication advantage was only evident in the TN-368 cell line, the cell line used for the original serial passage experiments, and not in SF-21 cells.

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Since 1991, when a baculovirus was first shown to inhibit apoptosis of its host insect cells, considerable contributions to our knowledge of apoptosis have arisen from the study of these viruses and the anti-apoptotic genes they encode. Baculovirus anti-apoptotic genes include p35, which encodes the most broadly acting caspase inhibitor protein known, and iap (inhibitor of apoptosis) genes, which were the first members of an evolutionarily conserved gene family involved in regulation of apoptosis and cytokinesis in organisms ranging from yeast to humans. Baculoviruses also provide an ideal system to study the effects of an apoptotic response on viral pathogenesis in an animal host. In this review, I discuss a number of interesting recent developments in the areas of apoptotic regulation by baculoviruses and the effects of apoptosis on baculovirus replication and pathogenesis.

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Although human c-IAP1 and c-IAP2 have been reported to possess antiapoptotic activity against a variety of stimuli in several mammalian cell types, we observed that full-length c-IAP1 and c-IAP2 failed to protect cells from apoptosis induced by Bax overexpression, tumor necrosis factor alpha treatment or Sindbis virus infection. However, deletion of the C-terminal RING domains of c-IAP1 and c-IAP2 restored antiapoptotic activity, indicating that this region negatively regulates the antiapoptotic function of the N-terminal BIR domain. This finding is consistent with the observation by others that the spacer region and RING domain of c-IAP1 functions as an E3 ligase, promoting autoubiquitination and degradation of c-IAP1. In addition, we found that c-IAP1 is cleaved during apoptosis to 52- and 35-kDa fragments. Both fragments contain the C-terminal end of c-IAP1 including the RING finger. In vitro cleavage of c-IAP1 with apoptotic cell extracts or with purified recombinant caspase-3 produced similar fragments. Furthermore, transfection of cells with the spacer-RING domain alone suppressed the antiapoptotic function of the N-terminal BIR domain of c-IAP1 and induced apoptosis. Optimal death-inducing activity of the spacer-RING required both the spacer region and the zinc-binding RING domain of c-IAP1 but did not require the caspase recruitment domain located within the spacer region. To the contrary, deletion of the caspase recruitment domain increased proapoptotic activity, apparently by stabilizing the C-terminal fragment.

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Sindbis virus infection of cultured cells and of neurons in mouse brains leads to programmed cell death exhibiting the classical characteristics of apoptosis. Although the mechanism by which Sindbis virus activates the cell suicide program is not known, we demonstrate here that Sindbis virus activates caspases, a family of death-inducing proteases, resulting in cleavage of several cellular substrates. To study the role of caspases in virus-induced apoptosis, we determined the effects of specific caspase inhibitors on Sindbis virus-induced cell death. CrmA (a serpin from cowpox virus) and zVAD-FMK (N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone) inhibited Sindbis virus-induced cell death, suggesting that cellular caspases facilitate apoptosis induced by Sindbis virus. Furthermore, CrmA significantly increased the rate of survival of infected mice. These inhibitors appear to protect cells by inhibiting the cellular death pathway rather than impairing virus replication or by inhibiting the nsP2 and capsid viral proteases. The specificity of CrmA indicates that the Sindbis virus-induced death pathway is similar to that induced by Fas or tumor necrosis factor alpha rather than being like the death pathway induced by DNA damage. Taken together, these data suggest a central role for caspases in Sindbis virus-induced apoptosis.

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The caspases are cysteine proteases that have been implicated in the execution of programmed cell death in organisms ranging from nematodes to humans. Many members of the Bcl-2 family, including Bcl-XL, are potent inhibitors of programmed cell death and inhibit activation of caspases in cells. Here, we report a direct interaction between caspases and Bcl-XL. The loop domain of Bcl-XL is cleaved by caspases in vitro and in cells induced to undergo apoptotic death after Sindbis virus infection or interleukin 3 withdrawal. Mutation of the caspase cleavage site in Bcl-XL in conjunction with a mutation in the BH1 homology domain impairs the death-inhibitory activity of Bcl-XL, suggesting that interaction of Bcl-XL with caspases may be an important mechanism of inhibiting cell death. However, once Bcl-XL is cleaved, the C-terminal fragment of Bcl-XL potently induces apoptosis. Taken together, these findings indicate that the recognition/cleavage site of Bcl-XL may facilitate protection against cell death by acting at the level of caspase activation and that cleavage of Bcl-XL during the execution phase of cell death converts Bcl-XL from a protective to a lethal protein.

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Here we demonstrate that open reading frame 16 (ORF16) of the oncogenic herpesvirus saimiri protects cells from heterologous virus-induced apoptosis. The BH1 and BH2 homology domains are highly conserved in ORF16, and ORF16 heterodimerizes with Bcl-2 family members Bax and Bak. However, ORF16 lacks the core sequence of the conserved BH3 homology domain, suggesting that this region is not essential for anti-apoptotic activity. Conservation of a functional bcl-2 homolog among gammaherpesviruses suggests that inhibition of programmed cell death is important in the biology of these viruses.

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Caspases are a family of cysteine proteases implicated in the biochemical and morphological changes that occur during apoptosis (programmed cell death). The loop domain of Bcl-2 is cleaved at Asp34 by caspase-3 (CPP32) in vitro, in cells overexpressing caspase-3, and after induction of apoptosis by Fas ligation and interleukin-3 withdrawal. The carboxyl-terminal Bcl-2 cleavage product triggered cell death and accelerated Sindbis virus-induced apoptosis, which was dependent on the BH3 homology and transmembrane domains of Bcl-2. Inhibitor studies indicated that cleavage of Bcl-2 may further activate downstream caspases and contribute to amplification of the caspase cascade. Cleavage-resistant mutants of Bcl-2 had increased protection from interleukin-3 withdrawal and Sindbis virus-induced apoptosis. Thus, cleavage of Bcl-2 by caspases may ensure the inevitability of cell death.

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The iap family of anti-apoptotic genes, originally discovered in viruses, has grown considerably in the past two years with the addition of a number of evolutionary conserved cellular homologues. Although the mechanism(s) by which these novel proteins block cell death is still unknown, intriguing clues to their function have been revealed by the discovery of interactions between some of the IAP homologues and cellular proteins involved in carrying out apoptotic signalling. Here, Rollie Clem and Colin Duckett discuss how the various IAP proteins may function in regulating apoptosis.

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The baculovirus inhibitor of apoptosis gene, iap, can impede cell death in insect cells. Here we show that iap can also prevent cell death in mammalian cells. The ability of iap to regulate programmed cell death in widely divergent species raised the possibility that cellular homologs of iap might exist. Consistent with this hypothesis, we have isolated Drosophila and human genes which encode IAP-like proteins (dILP and hILP). Like IAP, both dILP and hILP contain amino-terminal baculovirus IAP repeats (BIRs) and carboxy-terminal RING finger domains. Human ilp encodes a widely expressed cytoplasmic protein that can suppress apoptosis in transfected cells. An analysis of the expressed sequence tag database suggests that hilp is one of several human genes related to iap. Together these data suggest that iap and related cellular genes play an evolutionarily conserved role in the regulation of apoptosis.

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Baculoviruses possess two different classes of genes with anti-apoptptic activity: p35 and iap. The p35 gene product (P35) is able to block apoptosis induced by a variety of stimuli in phylogenetically diverse organisms. P35 has recently been shown to be capable of inhibiting the ICE/ced-3 family of cysteine proteases, a family of enzymes which are implicated in cell death and which exhibit specificity for cleavage at aspartate residues. The products of the iap genes are a distinct class of proteins containing a carboxyl ring finger and tandem duplications of a unique motif known as the BIR motif. Homologues of the baculovirus iap genes have been identified in the human genome. Both classes of baculovirus anti-apoptotic genes will continue to be important tools in defining the pathways involved in apoptosis. Since our demonstration in 1991 that a baculovirus prevents host cells from undergoing apoptosis by expressing a gene known as p35(Clem et al., 1991), the study of baculovirus-induced apoptosis and the anti-apoptotic genes they possess has led to discoveries with far-reaching implications for viral pathogenesis, human disease, and the study of cell death. It is now known that a variety of eukaryotic viruses encode genes which allow them to control cellular apoptosis. Understanding the mechanism(s) by which these viral gene products act provides fundamental insights into the pathways regulating apoptosis. In this review, we discuss the inhibition of apoptosis by baculoviruses, concentrating mainly on the nature and mechanism of action of the two classes of baculovirus genes, p35 and iap, which are able to control apoptosis in a diversity ofeukaryotes.