Unambiguous detection of SARS-CoV-2 subgenomic mRNAs with single cell RNA sequencing

Single cell RNA sequencing (scRNAseq) studies have provided critical insight into the pathogenesis of Severe Acute Respiratory Syndrome CoronaVirus 2 (SARS-CoV-2), the causative agent of COronaVIrus Disease 2019 (COVID-19). scRNAseq workflows are generally designed for the detection and quantification of eukaryotic host mRNAs and not viral RNAs. The performance of different scRNAseq methods to study SARS-CoV-2 RNAs has not been thoroughly evaluated. Here, we compare different scRNAseq methods for their ability to quantify and detect SARS-CoV-2 RNAs with a focus on subgenomic mRNAs (sgmRNAs), which are produced only during active viral replication and not present in viral particles. We present a data processing strategy, single cell CoronaVirus sequencing (scCoVseq), which quantifies reads unambiguously assigned to sgmRNAs or genomic RNA (gRNA). Compared to standard 10X Genomics Chromium Next GEM Single Cell 3′ (10X 3′) and Chromium Next GEM Single Cell V(D)J (10X 5′) sequencing, we find that 10X 5′ with an extended R1 sequencing strategy maximizes the unambiguous detection of sgmRNAs by increasing the number of reads spanning leader-sgmRNA junction sites. Differential gene expression testing and KEGG enrichment analysis of infected cells compared with bystander or mock cells showed an enrichment for COVID19-associated genes, supporting the ability of our method to accurately identify infected cells. Our method allows for quantification of coronavirus sgmRNA expression at single-cell resolution, and thereby supports high resolution studies of the dynamics of coronavirus RNA synthesis.

Bandgaps of Atomically Precise Graphene Nanoribbons and Occam’s Razor

Rationalization of energy gaps of atomically precise AGNRs, “bulk” (ΔΕac) or “zigzag-end” (ΔΕzz), could be challenging and controversial concerning their magnitude, origin, substrate influence (ΔΕsb), and spin-polarization, among others. Hereby, a simple self-consistent and “economical” interpretation is presented, based on “appropriate” DFT (and TDDFT) calculations, general symmetry principles, and plausibility arguments, which is fully consistent with current experimental measurements for 5-, 7-, and 9-AGNRs within less than 1%, although at variance with some prevailing views or interpretations for ΔΕac, ΔΕzz, and ΔΕsb. Thus, an excellent agreement between experiment and theory emerges, provided some established stereotypes are reconsidered and/or abandoned. The primary source of discrepancies is the finite length of AGNRs together with inversion-symmetry conflict and topological end/edge states, which invariably mix with other “bulk” states making their unambiguous detection/distinction difficult. This can be further tested by eliminating end-states (and ΔΕzz), by eliminating empty (non-aromatic) end-rings

Abstract P494: Truncated Titin Protein In Dilated Cardiomyopathy

Truncating variations in the gene coding for titin (TTNtv) have been known to cause dilated cardiomyopathy for nearly 20 years. Efforts to detect direct evidence of either haploinsufficiency or dominant negative mechanisms have thus far failed, leaving the mechanism open to controversy. By analyzing a collection of 184 post-transplant human hearts, 22 of which bear TTNtv’s, we show evidence supporting both haploinsufficient (lack of sufficient full length titin to maintain normal cardiomyocyte contractility) and dominant-negative (toxic gain of function due to truncated titin) mechanisms. Using allele specific proteomics as well as epitope specific agarose gel immunoblotting we show that TTNtv are present in human myocardium at the expected molecular weight and bear only the epitopes expected to be present in TTNtv protein. TTNtv associate with the sarcomere bearing insoluble fraction of human myocardium but are more weakly attached to the sarcomere than full length titin, consistent with their lack of thick filament and M-line attachment sites. We further show that DCM hearts bearing TTNtv have less full length titin than non-TTNtv bearing DCM hearts, by both total protein and in ratio to sarcomeric proteins, indicating TTN haploinsufficiency is also present in TTNtv hearts. This unambiguous detection of TTNtv protein in the myocardium of DCM combined with a reduction in full length titin supports a combined dominant negative and haploinsufficient mechanism of pathogenesis of TTNtv induced DCM.

Analysis of Organophosphorus-Based Nerve Agent Degradation Products by Gas Chromatography-Mass Spectrometry (GC-MS): Current Derivatization Reactions in the Analytical Chemist’s Toolbox

The field of gas chromatography-mass spectrometry (GC-MS) in the analysis of chemical warfare agents (CWAs), specifically those involving the organophosphorus-based nerve agents (OPNAs), is a continually evolving and dynamic area of research. The ever-present interest in this field within analytical chemistry is driven by the constant threat posed by these lethal CWAs, highlighted by their use during the Tokyo subway attack in 1995, their deliberate use on civilians in Syria in 2013, and their use in the poisoning of Sergei and Yulia Skripal in Great Britain in 2018 and Alexei Navalny in 2020. These events coupled with their potential for mass destruction only serve to stress the importance of developing methods for their rapid and unambiguous detection. Although the direct detection of OPNAs is possible by GC-MS, in most instances, the analytical chemist must rely on the detection of the products arising from their degradation. To this end, derivatization reactions mainly in the form of silylations and alkylations employing a vast array of reagents have played a pivotal role in the efficient detection of these products that can be used retrospectively to identify the original OPNA.

Detection of the Ether Using the Global Positioning System

In this paper the latest evidence for the existence of a cosmic ether obtained using modern technology is reviewed. The synchronized clocks of the GPS are applied in the search for ether drift by direct measurement of light travel times in the East-West direction. This method reveals that light travels faster West than East and therefore indicates the existence of an Earth-bound ether which we identify as the Earth-centered Inertial (ECI) frame for light transmission. The GPS clocks are then applied in the search for ether drift by direct measurement of light travel times in a modified Michelson-Morley experiment. The East-West light speed difference enables the unambiguous detection of ether drift and the direct confirmation of the existence of a preferred frame. The range equation of the GPS that operates in the Earth-centered inertial (ECI) frame is employed to demonstrate ether drift for rotational motion and Time Transfer technology involving a geo-stationary GPS satellite provides further confirmation of ether drift resulting from the rotating Earth. Finally, using a model applicable in the sun-centered inertial (SCI) frame with Coordinated Universal Time, light speed variation arising from the Earth's orbital motion for light reflected from planets and spacecraft and received at the surface of the Earth is demonstrated. The evidence then is that modern technology has detected ether drift for rotational and orbital motion from the frame of the moving Earth.

Analyzing Assay Redundancy in Metabolomics using Unique Ion Signatures

Targeted, untargeted and data-independent acquisition (DIA) metabolomics workflows are often hampered by ambiguous identification based on either MS1 information alone or relatively few MS2 fragment ions. While DIA methods have enjoyed popularity in proteomics, it is less clear whether they are suitable for metabolomics workflows due to their large precursor isolation windows and complex co-isolation patterns. Here, we quantitatively investigate the conditions necessary for unique metabolite identification in complex backgrounds using precursor and fragment ion mass-to-charge separation, comparing three benchmarked MS methods (MS1, MRM, DIA). We simulated MS1, MRM and DIA using the NIST LC-MS library as a complex background (8274 compounds at collision energy=35) and compared these methods with regards to unambiguous detection using unique ion signatures. Our simulations show that data generated with DIA with 25 Da mass windows outperformed MS1-only and MRM-based methods by a factor of 13.6-fold and 8.7-fold, respectively. Additionally, we use saturation analysis to show that for highly complex samples, a large portion of MS1-only detection (44.9%) is ambiguous while MRM (6.6%) and DIA (0.6%) present lower ambiguity. Our analysis demonstrates the power of using both high resolution precursor and high resolution fragment ion m/z for unambiguous compound detection. This work also establishes DIA as an emerging MS acquisition method with high selectivity for metabolomics, outperforming both DDA and MRM with regards to unique compound identification potential.

Searching for signatures of chaos in γ-ray light curves of selected Fermi-LAT blazars

ABSTRACT Blazar variability appears to be stochastic in nature. However, a possibility of low-dimensional chaos was considered in the past, but with no unambiguous detection so far. If present, it would constrain the emission mechanism by suggesting an underlying dynamical system. We rigorously searched for signatures of chaos in Fermi-Large Area Telescope light curves of 11 blazars. The data were comprehensively investigated using the methods of nonlinear time-series analysis: phase-space reconstruction, fractal dimension, and maximal Lyapunov exponent (mLE). We tested several possible parameters affecting the outcomes, in particular the mLE, in order to verify the spuriousness of the outcomes. We found no signs of chaos in any of the analysed blazars. Blazar variability is either truly stochastic in nature or governed by high-dimensional chaos that can often resemble randomness.

Homogeneity in the early chemical evolution of the Sextans dwarf spheroidal galaxy

We present the high-resolution spectroscopic analysis of two new extremely metal-poor star (EMPS) candidates in the dwarf spheroidal galaxy Sextans. These targets were preselected from medium-resolution spectra centered around the Ca II triplet in the near-infrared and were followed-up at higher resolution with VLT/UVES. We confirm their low metallicities with [Fe/H] = −2.95 and [Fe/H] = −3.01, which place them among the most metal-poor stars known in Sextans. The abundances of 18 elements, including C, Na, the α, Fe-peak, and neutron-capture elements, are determined. In particular, we present the first unambiguous detection of Zn in a classical dwarf at extremely low metallicity. Previous indications were made of a large scatter in the abundance ratios of the Sextans stellar population around [Fe/H] ∼ − 3 when compared to other galaxies, particularly with very low observed [α/Fe] ratios. We took the opportunity of reanalyzing the full sample of EMPS in Sextans and find a [α/Fe] Milky Way-like plateau and a ∼0.2 dex dispersion at fixed metallicity.

The Taxon Hypothesis Paradigm—On the Unambiguous Detection and Communication of Taxa

Here, we describe the taxon hypothesis (TH) paradigm, which covers the construction, identification, and communication of taxa as datasets. Defining taxa as datasets of individuals and their traits will make taxon identification and most importantly communication of taxa precise and reproducible. This will allow datasets with standardized and atomized traits to be used digitally in identification pipelines and communicated through persistent identifiers. Such datasets are particularly useful in the context of formally undescribed or even physically undiscovered species if data such as sequences from samples of environmental DNA (eDNA) are available. Implementing the TH paradigm will to some extent remove the impediment to hastily discover and formally describe all extant species in that the TH paradigm allows discovery and communication of new species and other taxa also in the absence of formal descriptions. The TH datasets can be connected to a taxonomic backbone providing access to the vast information associated with the tree of life. In parallel to the description of the TH paradigm, we demonstrate how it is implemented in the UNITE digital taxon communication system. UNITE TH datasets include rich data on individuals and their rDNA ITS sequences. These datasets are equipped with digital object identifiers (DOI) that serve to fix their identity in our communication. All datasets are also connected to a GBIF taxonomic backbone. Researchers processing their eDNA samples using UNITE datasets will, thus, be able to publish their findings as taxon occurrences in the GBIF data portal. UNITE species hypothesis (species level THs) datasets are increasingly utilized in taxon identification pipelines and even formally undescribed species can be identified and communicated by using UNITE. The TH paradigm seeks to achieve unambiguous, unique, and traceable communication of taxa and their properties at any level of the tree of life. It offers a rapid way to discover and communicate undescribed species in identification pipelines and data portals before they are lost to the sixth mass extinction.