Genetic recombination during coinfection of two mutants of human respiratory syncytial virus

Recombination between coinfecting viruses had not been documented previously for a nonsegmented negative-strand RNA virus (mononegavirus). We investigated the potential of intermolecular recombination by respiratory syncytial virus (RSV) by coinfecting HEp-2 cells with two recombinant RSV (rRSV) mutants lacking either the G gene (DeltaG/HEK) or the NS1 and NS2 genes (DeltaNS1/2). These viruses replicate inefficiently and form pinpoint plaques in HEp-2 cells. Therefore, potential recombined viruses with a growth and/or plaque formation advantage should easily be identified and differentiated from the two parental viruses. Further identification of potential recombinants was aided by the inclusion of point mutation markers in the F and L genes of DeltaG/HEK and the design of reverse transcription-PCR (RT-PCR) primers capable of detecting these markers. Independent coinfections and control single infections by these two rRSV mutants were performed. In one of six coinfections, an RSV variant was identified that produced plaques slightly larger than those of wild-type RSV in HEp-2 cells. RT-PCR and sequencing provided evidence that this variant was a recombined RSV (rec-RSV). The rec-RSV appeared to have been generated by a polymerase jump from the DeltaG/HEK genome to that of DeltaNS1/2 and back again in the vicinity of the SH-G-F genes. This apparently involved nonhomologous and homologous recombination events, respectively. The recombined genome was identical to that of the DeltaG/HEK mutant except that all but the first 12 nucleotides of the SH gene were deleted and replaced by an insert consisting of the last 91 nucleotides of the G gene and its downstream intergenic region. This insert could have come only from the coinfecting DeltaNS1/2 virus. This resulted in the formation of a short chimeric SH:G gene. Northern and Western blot analysis confirmed that the rec-RSV did not express the normal SH and G mRNAs and proteins but did express the aberrant SH:G mRNA. This provides an experimental demonstration of intermolecular recombination yielding a viable, helper-independent mononegavirus. However, the isolation of only a single rec-RSV under these optimized conditions supports the idea that RSV recombination is rare indeed

Searching for the Imprints of AGN Feedback on the Lyman Alpha Forest Around Luminous Red Galaxies

We explore the potential of using the low-redshift Lyman-$\alpha$ (Ly$\alpha$) forest surrounding luminous red galaxies (LRGs) as a tool to constrain active galactic nuclei (AGN) feedback models. Our analysis is based on snapshots from the Illustris and IllustrisTNG simulations at a redshift of $z=0.1$. These simulations offer an ideal platform for studying the influence of AGN feedback on the gas surrounding galaxies, as they share the same initial conditions and underlying code but incorporate different feedback prescriptions. Both simulations show significant impacts of feedback on the temperature and density of the gas around massive halos. Following our previous work, we adjusted the UV background in both simulations to align with the observed number density of Ly$\alpha$ lines ($\rm dN/dz$) in the intergalactic medium and study the Ly$\alpha$ forest around massive halos hosting LRGs, at impact parameters ($r_{\perp}$) ranging from 0.1 to 100 pMpc. Our findings reveal that $\rm dN/dz$, as a function of $r_{\perp}$, is approximately 1.5 to 2 times higher in IllustrisTNG compared to Illustris up to $r_{\perp}$ of $\sim 10$ pMpc. To further assess whether existing data can effectively discern these differences, we search for archival data containing spectra of background quasars probing foreground LRGs. Through a feasibility analysis based on this data, we demonstrate that ${\rm dN/dz} (r_{\perp})$ measurements can distinguish between feedback models of IllustrisTNG and Illustris with a precision exceeding 12$\sigma$. This underscores the potential of ${\rm dN/dz} (r_{\perp})$ measurements around LRGs as a valuable benchmark observation for discriminating between different feedback models.Comment: 21 pages (including 4 page appendix), Submitted to MNRA

Respiratory Syncytial Virus Matrix Protein-Chromatin Association Is Key to Transcriptional Inhibition in Infected Cells

The morbidity and mortality caused by the globally prevalent human respiratory pathogen respiratory syncytial virus (RSV) approaches that world-wide of influenza. We previously demonstrated that the RSV matrix (M) protein shuttles, in signal-dependent fashion, between host cell nucleus and cytoplasm, and that this trafficking is central to RSV replication and assembly. Here we analyze in detail the nuclear role of M for the first time using a range of novel approaches, including quantitative analysis of de novo cell transcription in situ in the presence or absence of RSV infection or M ectopic expression, as well as in situ DNA binding. We show that M, dependent on amino acids 110–183, inhibits host cell transcription in RSV-infected cells as well as cells transfected to express M, with a clear correlation between nuclear levels of M and the degree of transcriptional inhibition. Analysis of bacterially expressed M protein and derivatives thereof mutated in key residues within M’s RNA binding domain indicates that M can bind to DNA as well as RNA in a cell-free system. Parallel results for point-mutated M derivatives implicate arginine 170 and lysine 172, in contrast to other basic residues such as lysine 121 and 130, as critically important residues for inhibition of transcription and DNA binding both in situ and in vitro. Importantly, recombinant RSV carrying arginine 170/lysine 172 mutations shows attenuated infectivity in cultured cells and in an animal model, concomitant with altered inflammatory responses. These findings define an RSV M-chromatin interface critical for host transcriptional inhibition in infection, with important implications for anti-RSV therapeutic development

Broadband Observations of the Compton-thick Nucleus of NGC 3393

We present new NuSTAR and Chandra observations of NGC 3393, a galaxy reported to host the smallest separation dual AGN resolved in the X-rays. While past results suggested a 150 pc separation dual AGN, three times deeper Chandra imaging, combined with adaptive optics and radio imaging suggest a single, heavily obscured, radio-bright AGN. Using VLA and VLBA data, we find an AGN with a two-sided jet rather than a dual AGN and that the hard X-ray, UV, optical, NIR, and radio emission are all from a single point source with a radius <0.2". We find that the previously reported dual AGN is most likely a spurious detection resulting from the low number of X-ray counts (<160) at 6-7 keV and Gaussian smoothing of the data on scales much smaller than the PSF (0.25" vs. 0.80" FWHM). We show that statistical noise in a single Chandra PSF generates spurious dual peaks of the same separation (0.55$\pm$0.07" vs. 0.6") and flux ratio (39$\pm$9% vs. 32% of counts) as the purported dual AGN. With NuSTAR, we measure a Compton-thick source (NH=$2.2\pm0.4\times10^{24}$ cm$^{-2}$) with a large torus half-opening angle, {\theta}=79 which we postulate results from feedback from strong radio jets. This AGN shows a 2-10 keV intrinsic to observed flux ratio of 150. Using simulations, we find that even the deepest Chandra observations would severely underestimate the intrinsic luminosity of NGC 3393 above z>0.2, but would detect an unobscured AGN of this luminosity out to high redshift (z=5).Comment: Accepted for publication in ApJ. 15 Figures and 4 table

High-speed intravascular photoacoustic imaging of lipid-laden atherosclerotic plaque enabled by a 2-kHz barium nitrite raman laser

Lipid deposition inside the arterial wall is a key indicator of plaque vulnerability. An intravascular photoacoustic (IVPA) catheter is considered a promising device for quantifying the amount of lipid inside the arterial wall. Thus far, IVPA systems suffered from slow imaging speed (~50 s per frame) due to the lack of a suitable laser source for high-speed excitation of molecular overtone vibrations. Here, we report an improvement in IVPA imaging speed by two orders of magnitude, to 1.0 s per frame, enabled by a custom-built, 2-kHz master oscillator power amplifier (MOPA)-pumped, barium nitrite [Ba(NO3)2] Raman laser. This advancement narrows the gap in translating the IVPA technology to the clinical setting

A respiratory syncytial virus (RSV) vaccine based on parainfluenza virus 5 (PIV5)

AbstractHuman respiratory syncytial virus (RSV) is a leading cause of severe respiratory disease and hospitalizations in infants and young children. It also causes significant morbidity and mortality in elderly and immune compromised individuals. No licensed vaccine currently exists. Parainfluenza virus 5 (PIV5) is a paramyxovirus that causes no known human illness and has been used as a platform for vector-based vaccine development. To evaluate the efficacy of PIV5 as a RSV vaccine vector, we generated two recombinant PIV5 viruses – one expressing the fusion (F) protein and the other expressing the attachment glycoprotein (G) of RSV strain A2 (RSV A2). The vaccine strains were used separately for single-dose vaccinations in BALB/c mice. The results showed that both vaccines induced RSV antigen-specific antibody responses, with IgG2a/IgG1 ratios similar to those seen in wild-type RSV A2 infection. After challenging the vaccinated mice with RSV A2, histopathology of lung sections showed that the vaccines did not exacerbate lung lesions relative to RSV A2-immunized mice. Importantly, both F and G vaccines induced protective immunity. Therefore, PIV5 presents an attractive platform for vector-based vaccines against RSV infection

Analysis of the evolution of COVID-19 disease understanding through temporal knowledge graphs

The COVID-19 pandemic highlighted two critical barriers hindering rapid response to novel pathogens. These include inefficient use of existing biological knowledge about treatments, compounds, gene interactions, proteins, etc. to fight new diseases, and the lack of assimilation and analysis of the fast-growing knowledge about new diseases to quickly develop new treatments, vaccines, and compounds. Overcoming these critical challenges has the potential to revolutionize global preparedness for future pandemics. Accordingly, this article introduces a novel knowledge graph application that functions as both a repository of life science knowledge and an analytics platform capable of extracting time-sensitive insights to uncover evolving disease dynamics and, importantly, researchers' evolving understanding. Specifically, we demonstrate how to extract time-bounded key concepts, also leveraging existing ontologies, from evolving scholarly articles to create a single temporal connected source of truth specifically related to COVID-19. By doing so, current knowledge can be promptly accessed by both humans and machines, from which further understanding of disease outbreaks can be derived. We present key findings from the temporal analysis, applied to a subset of the resulting knowledge graph known as the temporal keywords knowledge graph, and delve into the detailed capabilities provided by this innovative approach

Quantitative localized proton-promoted dissolution kinetics of calcite using scanning electrochemical microscopy (SECM)

Scanning electrochemical microscopy (SECM) has been used to determine quantitatively the kinetics of proton-promoted dissolution of the calcite (101̅4) cleavage surface (from natural “Iceland Spar”) at the microscopic scale. By working under conditions where the probe size is much less than the characteristic dislocation spacing (as revealed from etching), it has been possible to measure kinetics mainly in regions of the surface which are free from dislocations, for the first time. To clearly reveal the locations of measurements, studies focused on cleaved “mirror” surfaces, where one of the two faces produced by cleavage was etched freely to reveal defects intersecting the surface, while the other (mirror) face was etched locally (and quantitatively) using SECM to generate high proton fluxes with a 25 μm diameter Pt disk ultramicroelectrode (UME) positioned at a defined (known) distance from a crystal surface. The etch pits formed at various etch times were measured using white light interferometry to ascertain pit dimensions. To determine quantitative dissolution kinetics, a moving boundary finite element model was formulated in which experimental time-dependent pit expansion data formed the input for simulations, from which solution and interfacial concentrations of key chemical species, and interfacial fluxes, could then be determined and visualized. This novel analysis allowed the rate constant for proton attack on calcite, and the order of the reaction with respect to the interfacial proton concentration, to be determined unambiguously. The process was found to be first order in terms of interfacial proton concentration with a rate constant k = 6.3 (± 1.3) × 10–4 m s–1. Significantly, this value is similar to previous macroscopic rate measurements of calcite dissolution which averaged over large areas and many dislocation sites, and where such sites provided a continuous source of steps for dissolution. Since the local measurements reported herein are mainly made in regions without dislocations, this study demonstrates that dislocations and steps that arise from such sites are not needed for fast proton-promoted calcite dissolution. Other sites, such as point defects, which are naturally abundant in calcite, are likely to be key reaction sites

Low levels of ATM in breast cancer patients with clinical radiosensitivity

BACKGROUND AND PURPOSE Adjuvant radiotherapy for cancer can result in severe adverse side effects for normal tissues. In this respect, individuals with anomalies of the ATM (ataxia telangiectasia) protein/gene are of particular interest as they may be at risk of both breast cancer and clinical radiosensitivity. The association of specific ATM gene mutations with these pathologies has been well documented, however, there is uncertainty regarding pathological thresholds for the ATM protein. RESULTS Semi-quantitative immuno-blotting provided a reliable and reproducible method to compare levels of the ATM protein for a rare cohort of 20 cancer patients selected on the basis of their severe adverse normal tissue reactions to radiotherapy. We found that 4/12 (33%) of the breast cancer patients with severe adverse normal tissue reactions following radiotherapy had ATM protein levels < 55% compared to the mean for non-reactor controls. CONCLUSIONS ATM mutations are generally considered low risk alleles for breast cancer and clinical radiosensitivity. From results reported here we propose a tentative ATM protein threshold of ~55% for high-risk of clinical radiosensitivity for breast cancer patients.The authors acknowledge grant support from the Royal Australian and New Zealand College of Radiologists

Evolution of collective and noncollective structures in Xe-123

An experiment involving a heavy-ion-induced fusion-evaporation reaction was carried out where high-spin states of 123Xe were populated in the 80Se (48Ca,5n) 123Xe reaction at 207 MeV beam energy. Gamma-ray coincidence events were recorded with the Gammasphere Ge detector array. The previously known level scheme was confirmed and enhanced with the addition of five new band structures and several interband transitions. Cranked Nilsson-Strutinsky (CNS) calculations were performed and compared with the experimental results in order to assign configurations to the bands.Additional co-authors: T Lauritsen, S Zhu, A Korichi, P Fallon, B M Nyakó, and J Timá