The Dengue Virus Nonstructural Protein 1 (NS1) Interacts with the Putative Epigenetic Regulator DIDO1 to Promote Flavivirus Replication in Mosquito Cells.

Dengue virus (DENV) NS1 is a multifunctional protein essential for viral replication. To gain insights into NS1 functions in mosquito cells, the protein interactome of DENV NS1 in C6/36 cells was investigated using a proximity biotinylation system and mass spectrometry. A total of 817 mosquito targets were identified as protein-protein interacting with DENV NS1. Approximately 14% of them coincide with interactomes previously obtained in vertebrate cells, including the oligosaccharide transferase complex, the chaperonin containing TCP-1, vesicle localization, and ribosomal proteins. Notably, other protein pathways not previously reported in vertebrate cells, such as epigenetic regulation and RNA silencing, were also found in the NS1 interactome in mosquito cells. Due to the novel and strong interactions observed for NS1 and the epigenetic regulator DIDO1 (Death-Inducer Obliterator 1), the role of DIDO1 in viral replication was further explored. Interactions between NS1 and DIDO1 were corroborated in infected mosquito cells, by colocalization and proximity ligation assays. Silencing DIDO1 expression results in a significant reduction in DENV and ZIKV replication and progeny production. Comparison of transcription analysis of mock or DENV infected cells silenced for DIDO1 revealed variations in multiple gene expression pathways, including pathways associated with DENV infection such as RNA surveillance, IMD, and Toll. These results suggest that DIDO1 is a host factor involved in the negative modulation of the antiviral response necessary for flavivirus replication in mosquito cells. Our findings uncover novel mechanisms of NS1 to promote DENV and ZIKV replication, and add to the understanding of NS1 as a multifunctional protein. IMPORTANCE Dengue is the most important mosquito-borne viral disease to humans. Dengue virus NS1 is a multifunctional protein essential for replication and modulation of innate immunity. To gain insights into NS1 functions, the protein interactome of dengue virus NS1 in Aedes albopictus cells was investigated using a proximity biotinylation system and mass spectrometry. Several protein pathways, not previously observed in vertebrate cells, such as transcription and epigenetic regulation, were found as part of the NS1 interactome in mosquito cells. Among those, DIDO1 was found to be a necessary host factor for dengue and Zika virus replication in mosquito cells. Transcription analysis of infected mosquito cells silenced for DIDO1 revealed alterations of the IMD and Toll pathways, part of the antiviral response in mosquitoes. The results suggest that DIDO1 is a host factor involved in modulation of the antiviral response and necessary for flavivirus replication.

Flavivirus infections induce a Golgi stress response in vertebrate and mosquito cells.

The stress of the Golgi apparatus is an autoregulatory mechanism that is induced to compensate for greater demand in the Golgi functions. No examples of Golgi stress responses due to physiological stimuli are known. Furthermore, the impact on this organelle of viral infections that occupy the vesicular transport during replication is unknown. In this work, we evaluated if a Golgi stress response is triggered during dengue and Zika viruses replication, two flaviviruses whose replicative cycle is heavily involved with the Golgi complex, in vertebrate and mosquito cells. Using GM-130 as a Golgi marker, and treatment with monensin as a positive control for the induction of the Golgi stress response, a significant expansion of the Golgi cisternae was observed in BHK-21, Vero E6 and mosquito cells infected with either virus. Activation of the TFE3 pathway was observed in the infected cells as indicated by the translocation from the cytoplasm to the nucleus of TFE3 and increased expression of pathway targeted genes. Of note, no sign of activation of the stress response was observed in CRFK cells infected with Feline Calicivirus (FCV), a virus released by cell lysis, not requiring vesicular transport. Finally, dilatation of the Golgi complex and translocation of TFE3 was observed in vertebrate cells expressing dengue and Zika viruses NS1, but not NS3. These results indicated that infections by dengue and Zika viruses induce a Golgi stress response in vertebrate and mosquito cells due to the increased demand on the Golgi complex imposed by virion and NS1 processing and secretion.

Identifying potential synergies and trade-offs for meeting food security and climate change objectives in sub-Saharan Africa.

Potential interactions between food production and climate mitigation are explored for two situations in sub-Saharan Africa, where deforestation and land degradation overlap with hunger and poverty. Three agriculture intensification scenarios for supplying nitrogen to increase crop production (mineral fertilizer, herbaceous legume cover crops--green manures--and agroforestry--legume improved tree fallows) are compared to baseline food production, land requirements to meet basic caloric requirements, and greenhouse gas emissions. At low population densities and high land availability, food security and climate mitigation goals are met with all intensification scenarios, resulting in surplus crop area for reforestation. In contrast, for high population density and small farm sizes, attaining food security and reducing greenhouse gas emissions require mineral fertilizers to make land available for reforestation; green manure or improved tree fallows do not provide sufficient increases in yields to permit reforestation. Tree fallows sequester significant carbon on cropland, but green manures result in net carbon dioxide equivalent emissions because of nitrogen additions. Although these results are encouraging, agricultural intensification in sub-Saharan Africa with mineral fertilizers, green manures, or improved tree fallows will remain low without policies that address access, costs, and lack of incentives. Carbon financing for small-holder agriculture could increase the likelihood of success of Reducing Emissions from Deforestation and Forest Degradation in Developing Countries programs and climate change mitigation but also promote food security in the region.

EEG discriminant analyses of mild head trauma.

Measures of EEG power spectral analyses were obtained from a total of 608 mild head trauma patients and 108 age-matched normal subjects. A training-set discriminant function was first developed from 264 mild head-injured patients and 83 age-matched controls yielding an overall discriminant classification accuracy of 94.8%. The first independent cross-validation of the discriminant function using 130 mild head trauma patients and 21 age-matched normals yielded a discriminant classification accuracy of 96.2% for the trauma patients and 90.5% for the normals. A second independent cross-validation of the discriminant function using 51 patients and measures of test-retest reliability from 93 patients yielded classification accuracies ranging between 77.8% and 92.3%. A third independent cross-validation of 70 mild head-injured patients tested at a different location with a different EEG computer system yielded a discriminant accuracy of 92.8%. The discriminating EEG power spectral analyses indicated 3 classes of neurophysiological variables which are attributable to mechanical head injury: (1) increased coherence and decreased phase in frontal and frontal-temporal regions; (2) decreased power differences between anterior and posterior cortical regions; and (3) reduced alpha power in posterior cortical regions.

Myelin basic protein, oligoclonal bands, and IgG in cerebrospinal fluid as indicators of multiple sclerosis.

There currently are three clinical laboratory procedures for use with cerebrospinal fluid that assist in the diagnosis of multiple sclerosis: measurement of myelin basic protein and IgG, and demonstration of an oligoclonal band. We compared characteristics of these procedures, using CSF samples from 166 patients identified as having (54 patients) or not having (112 patients) multiple sclerosis. We find that oligoclonal band demonstration is the most useful single test in helping to establish the presence of multiple sclerosis; IgG quantitation is the least helpful. Myelin basic protein should be quantitated for following the activity of multiple sclerosis; it may be applied only selectively in the context of screening. The incidence of false-positive results reinforces the view that the diagnosis of multiple sclerosis must be made in clinical context. These laboratory procedures are not suitable for use as screening tests.

Two agarose electrophoretic systems for demonstration of oligoclonal bands in cerebrospinal fluid compared.

Demonstration of oligoclonal bands by electrophoresis of cerebrospinal fluid is an important aid in establishing the diagnosis of multiple sclerosis. Electrophoretic systems vary in their effectiveness in doing so. We compared two systems in this respect. For a thin-layer agarose system, sensitivity was less (47%) than for a high-resolution agarose system (87%). Each system had good specificity (92 and 85%, respectively). Interpretation of electrophoretic patterns for cerebrospinal fluid should be available in clinical laboratories. Further, the best available system should be used for demonstration of oligoclonal bands.

The use of the electroencephalogram in patients admitted for alcohol abuse with seizures.

This study of 174 seizure presenting alcohol abusers revealed that the EEG is overwhelmingly normal by visual inspection. When the EEG is abnormal, however, usefulness is significant. The probable EEG findings in this group are presented and discussed here both clinically and experimentally. The literature is reviewed and while much divergent opinion is revealed, it basically is in agreement with the conclusion as set forth under E, significant findings.

Ultrastructural differentiation during Drosophila neurogenesis in vitro.

Drosophila melanogaster field neuroblasts differentiate in vitro, and each gives rise to a cluster of about 18 daughter neurons. Electron microscopic observations of single clusters show that axons from daughter neurons form a neuropile within the cluster of cell bodies. The neuropile increases in size and complexity for several hours, during which time chemical, and probably electrotonic, synapses form between neurites. Clear vesicles with diameters of about 35 nm and dense core vesicles with diameters of about 60 and 160 nm were detected. The development of the neuropile indicates that the prerequisite cell recognition phenomena were manifested during differentiation in vitro, and the complexity of the neuropile suggests it may have attained the capacity to process information.

Ultrastructural differentiation during embryonic Drosophila myogenesis in vitro.

Cultures of embryonic Drosophila melanogaster cells were examined by electron microscopy and events in myogenesis were recorded. Thick and thin myofilaments, T-tubules and sarcoplasmic reticulum all appeared at about the same time, 10.5 hr. This was about 5 hr after the final division of myoblasts and about the time that muscle cells were elongating, aligning and fusing. Sarcoplasm typical of insect muscle was detected by 18.5 hr, as were myotendonal and tendocuticular junctions. Two populations of myocytes were detected, the cytoplasm of one more electron-dense than the other. The only previous report of myofibrilogenesis in invertebrate embryos had described novel mechanisms. In Drosophila embryonic material, however, the sequence of myofibrilogenesis resembled that in postembryonic insect or vertebrate material.

Differentiation of neuromuscular junctions in cultures of embryonic Drosophila cells.

Cultures were prepared of embryonic cells from Drosophila melanogaster. Neurons and myocytes differentiated in vitro from their respective stem cells. Electron microscopy showed that neuromuscular junctions formed where axons contacted myocytes. Electrical stimuli were applied to axons and these caused contractions of innervated myocytes. This is the first report of insect or other invertebrate neuromuscular junctions differentiating in vitro. In addition, this is the first system reported in which the neurons, myocytes, and junctions are completely differentiated in vitro from neuroblasts and myoblasts.