SARS Coronavirus 3b Accessory Protein Modulates Transcriptional Activity of RUNX1b

BACKGROUND: The causative agent of severe acute respiratory syndrome, SARS coronavirus (SARS-CoV) genome encodes several unique group specific accessory proteins with unknown functions. Among them, accessory protein 3b (also known as ORF4) was lately identified as one of the viral interferon antagonist. Recently our lab uncovered a new role for 3b in upregulation of AP-1 transcriptional activity and its downstream genes. Thus, we believe that 3b might play an important role in SARS-CoV pathogenesis and therefore is of considerable interest. The current study aims at identifying novel host cellular interactors of the 3b protein. METHODOLOGY/PRINCIPAL FINDINGS: In this study, using yeast two-hybrid and co-immunoprecipitation techniques, we have identified a host transcription factor RUNX1b (Runt related transcription factor, isoform b) as a novel interacting partner for SARS-CoV 3b protein. Chromatin immunoprecipitaion (ChIP) and reporter gene assays in 3b expressing jurkat cells showed recruitment of 3b on the RUNX1 binding element that led to an increase in RUNX1b transactivation potential on the IL2 promoter. Kinase assay and pharmacological inhibitor treatment implied that 3b also affect RUNX1b transcriptional activity by regulating its ERK dependent phosphorylation levels. Additionally, mRNA levels of MIP-1α, a RUNX1b target gene upregulated in SARS-CoV infected monocyte-derived dendritic cells, were found to be elevated in 3b expressing U937 monocyte cells. CONCLUSIONS/SIGNIFICANCE: These results unveil a novel interaction of SARS-CoV 3b with the host factor, RUNX1b, and speculate its physiological relevance in upregulating cytokines and chemokine levels in state of SARS virus infection

Village health, sanitation, and nutrition committee: Do the village level functionaries aware of their roles?

Background: National Health Mission intended to achieve decentralization and community participation through creating and supporting Village Health Sanitation and Nutrition Committees (VHSNCs). The services offered through VHSNC include maternal and child health, family planning, sanitation, communicable diseases and health promotion, and nutrition. The study was carried out to assess awareness on the implementation of the functioning of VHSNC implementation among village-level functionaries in the Dehradun district. Methods: A community-based cross-sectional study was conducted in the doiwala and Raipur blocks of Dehradun district from June 2019 to July 2019. Members of VHSNC with at least 3 months of membership were included in the study. A pretested semi-structured questionnaire was used to collect sociodemographic variables and questions about their awareness and responsibilities in implementing VHSNC. Data were collected by personal interviews using Epi-Collect and analyzed by SPSS 23. Result: Out of 69 members, 64 (92.4%) had formal education until high school. Only around 50% of members knew about the essential documents related to the VHSNC. Sixty-five members (98.48%) believed that the committee had the primary role in health-related services in the village, 54 (81.82%) members also marked providing safe drinking water as a function, 48 (72.73%) were in favor of access to clean public toilet and sanitation facilities while some others added role in Public Distribution System. Conclusion: Around half of the members were partially or completely aware of the functioning of VHSNCs. Providing them with further awareness is required

Inhibition of N-terminal lysines acetylation and transcription factor assembly by epirubicin induced deranged cell homeostasis.

Epirubicin (EPI), an anthracycline antitumour antibiotic, is a known intercalating and DNA damaging agent. Here, we study the molecular interaction of EPI with histones and other cellular targets. EPI binding with histone core protein was predicted with spectroscopic and computational techniques. The molecular distance r, between donor (histone H3) and acceptor (EPI) was estimated using Förster's theory of non-radiation energy transfer and the detailed binding phenomenon is expounded. Interestingly, the concentration dependent reduction in the acetylated states of histone H3 K9/K14 was observed suggesting more repressed chromatin state on EPI treatment. Its binding site near N-terminal lysines is further characterized by thermodynamic determinants and molecular docking studies. Specific DNA binding and inhibition of transcription factor (Tf)-DNA complex formation implicates EPI induced transcriptional inhibition. EPI also showed significant cell cycle arrest in drug treated cells. Chromatin fragmentation and loss of membrane integrity in EPI treated cells is suggestive of their commitment to cell death. This study provides an analysis of nucleosome dynamics during EPI treatment and provides a novel insight into its action

Aggressive Fibromatosis in Neck.

Aggressive fibromatosis (AF) is a locally aggressive infiltrative low-grade benign tumor that accounts for approximately less than 3% of all soft tissue tumors. In the head and neck region this tumor tends to be more aggressive and associated with significant morbidity. Aggressive surgery is a viable management option and may be successfully used as a single modality treatment, or in combination with radiotherapy. We report a rare case of AF in a 38 year old female, who presented with a painless mass over the left supraclavicular fossa, extending inferiorly into the thoracic inlet, which was excised successfully in toto with the help of cardiothoracic vascular surgeon (CTVS)

3b expression increases phosphorylated RUNX1b levels through ERK activation.

<p>A. HEK293 cells were transfected with vector, 3b and RUNX1b expression plasmids. ERK immunoprecipitated from vector and 3b lysates were subjected to kinase assay with RUNX1b beads. Phosphorylated RUNX1b was visualized by autoradiography. Input levels of immunoprecipitated ERK and phospho ERK levels in lysates were probed by western blotting. Graph depicts fold increase in the levels of phosphorylated RUNX1b procured after three independent experiments. Bar represents mean±SD of values obtained by densitometry. #, <i>p</i><0.05. B. Jurkat cells were transfected with WT IL2-Luc in the presence or absence of Myc-3b and treated with DMSO or U0126. Relative luciferase activity was measured and is shown as the mean±SD of three independent experiments performed in triplicates. *, <i>p</i><0.005. Phospho ERK levels in lysates were probed by western blotting.</p

3b increases RUNX1b transactivation potential on the mouse IL2 promoter.

<p>A. HEK293 cells were transfected with WT IL2-Luc plasmid alone or with Flag-RUNX1b, Flag-CBFβ and indicated amounts of Myc-3b. Relative luciferase activities were calculated 48 h post transfection. B. Jurkat cells were transfected with WT IL2-Luc and indicated amounts of Myc-3b. Relative luciferase activities were calculated 48 h post-transfection. 3b expression was probed using α-myc antibody C. Jurkat cells were transfected with WT IL2-Luc or mutant IL2-Luc plasmid in the presence or absence of Myc-3b. Results in each panel are represented as mean±S.D. of triplicate cultures. Bar values represent fold increase in luciferase activity. *, <i>p</i><0.005.</p