Respiratory Syncytial Virus

Respiratory Syncytial Virus (RSV)-driven bronchiolitis is one of the most common causes of pediatric hospitalization. Every year, we face 33.1 million episodes of RSV-driven lower respiratory tract infection without any available vaccine or cost-effective therapeutics since the discovery of RSV eighty years before. RSV is an enveloped RNA virus belonging to the pneumoviridae family of viruses. This chapter aims to elucidate the structure and functions of the RSV genome and proteins and the mechanism of RSV infection in host cells from entry to budding, which will provide current insight into the RSV-host relationship. In addition, this book chapter summarizes the recent research outcomes regarding the structure of RSV and the functions of all viral proteins along with the RSV life cycle and cell-to-cell spread

AI is a viable alternative to high throughput screening: a 318-target study

: High throughput screening (HTS) is routinely used to identify bioactive small molecules. This requires physical compounds, which limits coverage of accessible chemical space. Computational approaches combined with vast on-demand chemical libraries can access far greater chemical space, provided that the predictive accuracy is sufficient to identify useful molecules. Through the largest and most diverse virtual HTS campaign reported to date, comprising 318 individual projects, we demonstrate that our AtomNet® convolutional neural network successfully finds novel hits across every major therapeutic area and protein class. We address historical limitations of computational screening by demonstrating success for target proteins without known binders, high-quality X-ray crystal structures, or manual cherry-picking of compounds. We show that the molecules selected by the AtomNet® model are novel drug-like scaffolds rather than minor modifications to known bioactive compounds. Our empirical results suggest that computational methods can substantially replace HTS as the first step of small-molecule drug discovery

<i><span style="font-size:20.5pt;mso-bidi-font-size:14.5pt;font-family:"Times New Roman","serif"">In vitro </span></i><span style="font-size:20.5pt;mso-bidi-font-size:14.5pt; font-family:"Times New Roman","serif"">propagation of emetic nut <i>Randia dumetorum </i>(Lamb.) </span>

1479-1481<span style="font-size: 14.5pt;mso-bidi-font-size:8.5pt;font-family:" times="" new="" roman","serif""="">An efficient protocol for in <span style="font-size:14.5pt;mso-bidi-font-size:8.5pt;font-family: " times="" new="" roman","serif""="">vitro <span style="font-size:14.5pt; mso-bidi-font-size:8.5pt;font-family:" times="" new="" roman","serif""="">shoot multiplication of Randia dumetorum (Emetic nut) has been developed. The seeds of R. dumetorum were germinated in vitro in MS medium in 5 weeks. Subsequent propagation using shoot tip as an <span style="font-size: 14.5pt;mso-bidi-font-size:8.5pt;font-family:" times="" new="" roman","serif""="">explant was carried out in MS medium along with different concentrations and combinations of BAP (0.5-2.0) and NAA (0.0-2.0). Maximum shoot multiplication was obtained (12.7 shoots per shoot tip) in MS medium containing  1mg/L BAP and 1mg/L NAA. Micropropagated shoots were rooted in <span style="font-size:15.5pt;mso-bidi-font-size: 9.5pt;font-family:" times="" new="" roman","serif";mso-bidi-font-style:italic"="">1/2MS medium supplemented with 1 mg/l IBA. This is the first <span style="font-size:14.5pt;mso-bidi-font-size:8.5pt;line-height:115%; font-family:" times="" new="" roman","serif";mso-fareast-font-family:"times="" roman";="" mso-ansi-language:en-us;mso-fareast-language:en-us;mso-bidi-language:ar-sa"="">report of in<span style="font-size: 14.0pt;mso-bidi-font-size:8.0pt;line-height:115%;font-family:" times="" new="" roman","serif";="" mso-fareast-font-family:"times="" roman";mso-ansi-language:en-us;mso-fareast-language:="" en-us;mso-bidi-language:ar-sa"=""> <span style="font-size:14.5pt; mso-bidi-font-size:8.5pt;line-height:115%;font-family:" times="" new="" roman","serif";="" mso-fareast-font-family:"times="" roman";mso-ansi-language:en-us;mso-fareast-language:="" en-us;mso-bidi-language:ar-sa"="">vitro <span style="font-size:14.5pt; mso-bidi-font-size:8.5pt;line-height:115%;font-family:" times="" new="" roman","serif";="" mso-fareast-font-family:"times="" roman";mso-ansi-language:en-us;mso-fareast-language:="" en-us;mso-bidi-language:ar-sa"="">plant propagation of R. dumetorum. In vitro grown plantlets showed a survival rate of 70% after 2 months of tranplantation to natural environment.</span

Multicolor Stimulated Emission Depletion (STED) Microscopy to Generate High-resolution Images of Respiratory Syncytial Virus Particles and Infected Cells

Human respiratory syncytial virus (RSV) infection in human lung epithelial A549 cells induces filopodia, cellular protrusions consisting of F-actin, that extend to neighboring uninfected cells (Mehedi et al., 2016). High-resolution imaging via stimulated emission depletion (STED) microscopy revealed filamentous RSV particles along these filopodia, suggesting that filopodia facilitate RSV cell-to-cell spread (Mehedi et al., 2016). In this protocol, we describe how to fix, permeabilize, immunostain, and mount RSV-infected A549 cells for STED imaging. We show that STED increases resolution compared to confocal microscopy, which can be further improved by image processing using deconvolution software

Patient-Centered Discussion on End-of-Life Care for Patients with Advanced COPD

Exacerbations of chronic obstructive pulmonary disease (COPD) may lead to a rapid decline in health and subsequent death, an unfortunate tyranny of having COPD—an irreversible health condition of 16 million individuals in the USA totaling 60 million in the world. While COPD is the third largest leading cause of death, causing 3.23 million deaths worldwide in 2019 (according to the WHO), most patients with COPD do not receive adequate treatment at the end stages of life. Although death is inevitable, the trajectory towards end-of-life is less predictable in severe COPD. Thus, clinician-patient discussion for end-of-life and palliative care could bring a meaningful life-prospective to patients with advanced COPD. Here, we summarized the current understanding and treatment of COPD. This review also highlights the importance of patient-centered discussion and summarizes current status of managing patients with advanced COPD

Ebola Virus RNA Editing Depends on the Primary Editing Site Sequence and an Upstream Secondary Structure

<div><p>Ebolavirus (EBOV), the causative agent of a severe hemorrhagic fever and a biosafety level 4 pathogen, increases its genome coding capacity by producing multiple transcripts encoding for structural and nonstructural glycoproteins from a single gene. This is achieved through RNA editing, during which non-template adenosine residues are incorporated into the EBOV mRNAs at an editing site encoding for 7 adenosine residues. However, the mechanism of EBOV RNA editing is currently not understood. In this study, we report for the first time that minigenomes containing the glycoprotein gene editing site can undergo RNA editing, thereby eliminating the requirement for a biosafety level 4 laboratory to study EBOV RNA editing. Using a newly developed dual-reporter minigenome, we have characterized the mechanism of EBOV RNA editing, and have identified cis-acting sequences that are required for editing, located between 9 nt upstream and 9 nt downstream of the editing site. Moreover, we show that a secondary structure in the upstream cis-acting sequence plays an important role in RNA editing. EBOV RNA editing is glycoprotein gene-specific, as a stretch encoding for 7 adenosine residues located in the viral polymerase gene did not serve as an editing site, most likely due to an absence of the necessary cis-acting sequences. Finally, the EBOV protein VP30 was identified as a trans-acting factor for RNA editing, constituting a novel function for this protein. Overall, our results provide novel insights into the RNA editing mechanism of EBOV, further understanding of which might result in novel intervention strategies against this viral pathogen.</p></div

The hepta-uridine stretch in the editing site is necessary but not sufficient for RNA editing.

<p>Dual-reporter minigenome (45 nt-7A-58 nt) assays were performed in the presence (with L) or absence (without L; negative control) of the viral polymerase. The minigenomes contained either an unaltered 110 nt stretch from the GP translated region flanking the editing site, or variants with point mutations or deletions as shown in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003677#ppat-1003677-g002" target="_blank">Figure 2B</a>. Cells were analyzed for eGFP expression (from unedited and edited mRNA) and mCherry expression by fluorescence microscopy (from edited mRNA only).</p

Model for EBOV RNA editing.

<p>The EBOV RNA-dependent RNA polymerase (L) transcribes vRNA into mRNA. A stem-loop structure in the mRNA directly upstream of the primary editing site causes the polymerase to pause, and enables insertion of non-templated adenosine residues due to stuttering. VP30 then resolves the stem loop, and allows continued faithful transcription.</p

The secondary structure of the cis-acting sequence upstream of the editing site is important for RNA editing.

<p>(A) Predicted stem-loops in the 45 nt upstream of the editing site. The Mfold RNA secondary structure prediction webserver was used for secondary structure analysis of the region upstream of the editing site within the nascent mRNA. Bases that were mutated to destabilize secondary structures are marked with an asterisk. (B) The editing site proximal, but not the editing site distal stem-loop is required for editing. Mutations were introduced into the minigenome to destabilize the predicted first (45 nt-7A-58 nt (C3U, A18C and G24A)) or second (45 nt-7A-58 nt (G38A and G39A) or 45 nt-7A-58 nt (G39A and C44U)) stem-loop. Dual-reporter minigenome assays were performed using these minigenomes, and the mean fluorescent intensity (MFI) of eGFP (expressed from unedited and edited mRNA) and mCherry (expressed from edited mRNA only) in eGFP-positive cells was measured by flow cytometry. The intensity of each reporter in context of an unaltered minigenome was defined as 100%. Mean and standard deviation from three independent experiments are shown.</p

Mutations and deletions in the dual-reporter minigenome.

<p>(A) Dual reporter minigenome. A cartoon showing the structure of the minigenome, along with unedited and edited mRNA transcripts as well as the resulting reporter protein (eGFP and mCherry) expression. (B) Mutated minigenomes. Overview of the deletions and point mutations in the dual-reporter minigenome. Shown is the minigenome region corresponding to the GP translated region. Point mutations are indicated in red.</p