The 2microm-plasmid-encoded Rep1 and Rep2 proteins interact with each other and colocalize to the Saccharomyces cerevisiae nucleus.

The efficient partitioning of the 2microm plasmid of Saccharomyces cerevisiae at cell division requires two plasmid-encoded proteins (Rep1p and Rep2p) and a cis-acting locus, REP3 (STB). By using protein hybrids containing fusions of the Rep proteins to green fluorescent protein (GFP), we show here that fluorescence from GFP-Rep1p or GFP-Rep2p is almost exclusively localized in the nucleus in a cir+ strain. Nuclear localization of GFP-Rep1p and GFP-Rep2p, though discernible, is less efficient in a cir(0) host. GFP-Rep2p or GFP-Rep1p is able to promote the stability of a 2microm circle-derived plasmid harboring REP1 or REP2, respectively, in a cir(0) background. Under these conditions, fluorescence from GFP-Rep2p or GFP-Rep1p is concentrated within the nucleus, as is the case in cir+ cells. This characteristic nuclear accumulation is not dependent on the expression of the FLP or RAF1 gene of the 2microm circle. Nuclear colocalization of Rep1p and Rep2p is consistent with the hypothesis that the two proteins directly or indirectly interact to form a functional bipartite or high-order protein complex. Immunoprecipitation experiments as well as baiting assays using GST-Rep hybrid proteins suggest a direct interaction between Rep1p and Rep2p which, in principle, may be modulated by other yeast proteins. Furthermore, these assays provide evidence for Rep1p-Rep1p and Rep2p-Rep2p associations as well. The sum of these interactions may be important in controlling the effective cellular concentration of the Rep1p-Rep2p complex.     

Characterization of Intracellular Ribonucleic Acid Specific for the Murine Sarcoma-Leukemia Virus Complex

Virus particles were continuously produced by a cell line (78A1) of rat embryo fibroblasts that had been transformed by the murine sarcoma-leukemia virus complex. Since most of the mature virions were found in the extracellular fluid and were not cell-associated, a measurable quantity of viral ribonucleic acid (RNA) could not be extracted from these cells. Cycloheximide, a protein inhibitor, was successfully used to accumulate viral RNA within the cells. This ribonuclease-sensitive RNA, with a sedimentation coefficient of 71S, had the same base composition as the high molecular weight RNA (S(20,w) = 71) isolated from purified virions released by the transformed cells.     

Chronic cigarette smoke causes oxidative damage and apoptosis to retinal pigmented epithelial cells in mice

The purpose of this study was to determine whether mice exposed to chronic cigarette smoke develop features of early age-related macular degeneration (AMD). Two month old C57Bl6 mice were exposed to either filtered air or cigarette smoke in a smoking chamber for 5 h/day, 5 days/week for 6 months. Eyes were fixed in 2.5% glutaraldehyde/2% paraformaldehyde and examined for ultrastructural changes by transmission electron microscopy. The contralateral eye was fixed in 2% paraformaldehyde and examined for oxidative injury to the retinal pigmented epithelium (RPE) by 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-OHdG) immunolabeling and apoptosis by TUNEL labeling. Mice exposed to cigarette smoke had immunolabeling for 8-OHdG in 85+/-3.7% of RPE cells counted compared to 9.5+/-3.9% in controls (p<0.00001). Bruch membrane was thicker in mice exposed to smoke (1086+/-332 nm) than those raised in air (543+/-132 nm; p = 0.0069). The two most pronounced ultrastructural changes (severity grading scale from 0-3) seen were a loss of basal infoldings (mean difference in grade = 1.98; p<0.0001), and an increase in intracellular vacuoles (mean difference in grade = 1.7; p<0.0001). Ultrastructural changes to Bruch membrane in cigarette-smoke exposed mice were smaller in magnitude but consistently demonstrated significantly higher grade injury in cigarette-exposed mice, including basal laminar deposits (mean difference in grade = 0.54; p<0.0001), increased outer collagenous layer deposits (mean difference in grade = 0.59; p = 0.002), and increased basal laminar deposit continuity (mean difference in grade = 0.4; p<0.0001). TUNEL assay showed a higher percentage of apoptotic RPE from mice exposed to cigarette smoke (average 8.0+/-1.1%) than room air (average 0+/-0%; p = 0.043). Mice exposed to chronic cigarette smoke develop evidence of oxidative damage with ultrastructural degeneration to the RPE and Bruch membrane, and RPE cell apoptosis. This model could be useful for studying the mechanism of smoke induced changes during early AMD.     

Nitrogen-deficiency-induced loss in photosynthesis and modulation of β-galactosidase activity during senescence of <Emphasis Type="Italic">Arabidopsis</Emphasis> leaves

Senescence-induced loss in the content of chlorophyll and the rate of oxygen evolution is remarkably enhanced when the leaves of Arabidopsis thaliana experience nitrogen-deficiency stress. On the other hand, the decline in the level of total soluble sugar during senescence is very slow and nitrogen deficiency does not exhibit any further change. The relative stability in the level of the sugar in the background of severe decline of photosynthesis may suggest the contribution of sugars from other sources to sustain its homeostasis to execute and complete energy-dependent senescence process and stress response. The possible participation of cell wall polysaccharides contributing to sugar homeostasis is predicted. Senescence-induced increase in the activity of β-galactosidase (EC 3.2.1.23) and its further enhancement in senescing leaves experiencing nitrogen stress support the proposition of participation of the enzyme for breakdown of the wall polysaccharides to sugars. The loss of photosynthesis as a possible signal for enhancement in the activity of β-galactosidase has been further examined in the excised leaves incubated in Okada and Shimura (OS) nutrient medium with and without nitrogen. Nitrogen limitation experienced by excised leaves causes rapid loss in photosynthesis with concomitant increase in the activity of the enzyme extracted both from soluble and cell wall fractions. The differential activity of the enzyme from soluble and cell wall fractions during development-dependent leaf senescence and premature senescence in excised leaves induced by nitrogen deficiency appears to be complex and needs to be resolved in the future.     

Characterization of the NiRAN domain from RNA-dependent RNA polymerase provides insights into a potential therapeutic target against SARS-CoV-2

Apart from the canonical fingers, palm and thumb domains, the RNA dependent RNA polymerases (RdRp) from the viral order Nidovirales possess two additional domains. Of these, the function of the Nidovirus RdRp associated nucleotidyl transferase domain (NiRAN) remains unanswered. The elucidation of the 3D structure of RdRp from the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), provided the first ever insights into the domain organisation and possible functional characteristics of the NiRAN domain. Using in silico tools, we predict that the NiRAN domain assumes a kinase or phosphotransferase like fold and binds nucleoside triphosphates at its proposed active site. Additionally, using molecular docking we have predicted the binding of three widely used kinase inhibitors and five well characterized anti-microbial compounds at the NiRAN domain active site along with their drug-likeliness. For the first time ever, using basic biochemical tools, this study shows the presence of a kinase like activity exhibited by the SARS-CoV-2 RdRp. Interestingly, a well-known kinase inhibitor- Sorafenib showed a significant inhibition and dampened viral load in SARS-CoV-2 infected cells. In line with the current global COVID-19 pandemic urgency and the emergence of newer strains with significantly higher infectivity, this study provides a new anti-SARS-CoV-2 drug target and potential lead compounds for drug repurposing against SARS-CoV-2.     

Modulatory Interactions of Resting-State Brain Functional Connectivity

The functional brain connectivity studies are generally based on the synchronization of the resting-state functional magnetic resonance imaging (fMRI) signals. Functional connectivity measures usually assume a stable relationship over time; however, accumulating studies have reported time-varying properties of strength and spatial distribution of functional connectivity. The present study explored the modulation of functional connectivity between two regions by a third region using the physiophysiological interaction (PPI) technique. We first identified eight brain networks and two regions of interest (ROIs) representing each of the networks using a spatial independent component analysis. A voxel-wise analysis was conducted to identify regions that showed modulatory interactions (PPI) with the two ROIs of each network. Mostly, positive modulatory interactions were observed within regions involved in the same system. For example, the two regions of the dorsal attention network revealed modulatory interactions with the regions related to attention, while the two regions of the extrastriate network revealed modulatory interactions with the regions in the visual cortex. In contrast, the two regions of the default mode network (DMN) revealed negative modulatory interactions with the regions in the executive network, and vice versa, suggesting that the activities of one network may be associated with smaller within network connectivity of the competing network. These results validate the use of PPI analysis to study modulation of resting-state functional connectivity by a third region. The modulatory effects may provide a better understanding of complex brain functions.     

Two conserved oligomer interfaces of NSP7 and NSP8 underpin the dynamic assembly of SARS-CoV-2 RdRP

Replication of the ∼30 kb-long coronavirus genome is mediated by a complex of non-structural proteins (NSP), in which NSP7 and NSP8 play a critical role in regulating the RNA-dependent RNA polymerase (RdRP) activity of NSP12. The assembly of NSP7, NSP8 and NSP12 proteins is highly dynamic in solution, yet the underlying mechanism remains elusive. We report the crystal structure of the complex between NSP7 and NSP8 of SARS-CoV-2, revealing a 2:2 heterotetrameric form. Formation of the NSP7-NSP8 complex is mediated by two distinct oligomer interfaces, with interface I responsible for heterodimeric NSP7-NSP8 assembly, and interface II mediating the heterotetrameric interaction between the two NSP7-NSP8 dimers. Structure-guided mutagenesis, combined with biochemical and enzymatic assays, further reveals a structural coupling between the two oligomer interfaces, as well as the importance of these interfaces for the RdRP activity of the NSP7-NSP8-NSP12 complex. Finally, we identify an NSP7 mutation that differentially affects the stability of the NSP7-NSP8 and NSP7-NSP8-NSP12 complexes leading to a selective impairment of the RdRP activity. Together, this study provides deep insights into the structure and mechanism for the dynamic assembly of NSP7 and NSP8 in regulating the replication of the SARS-CoV-2 genome, with important implications for antiviral drug development.     

Resting Network Plasticity Following Brain Injury

The purpose of this study was to examine neural network properties at separate time-points during recovery from traumatic brain injury (TBI) using graph theory. Whole-brain analyses of the topological properties of the fMRI signal were conducted in 6 participants at 3 months and 6 months following severe TBI. Results revealed alterations of network properties including a change in the degree distribution, reduced overall strength in connectivity, and increased “small-worldness” from 3 months to 6 months post injury. The findings here indicate that, during recovery from injury, the strength but not the number of network connections diminishes, so that over the course of recovery, the network begins to approximate what is observed in healthy adults. These are the first data examining functional connectivity in a disrupted neural system during recovery.