ARTICLE | doi:10.20944/preprints202008.0633.v1
Subject: Biology, Other Keywords: giant phage; phiKZ; Pseudomonas aeruginosa; nucleoid; pseudo-nucleus; analytical electron microscopy; electron tomography; fluorescent in situ hybridization; stress response
Online: 28 August 2020 (10:29:39 CEST)
Bacteria develop various defense mechanisms against stresses, including the bacteriophage infection. The giant phiKZ phage infection induced the appearance of a pseudo-nucleus inside the bacterial cytoplasm. Here, we used FISH, electron tomography and analytical electron microscopy to study the morphology of this unique nucleus-like shell and to demonstrate the distribution of phiKZ and bacterial DNA in infected P. aeruginosa cells. The maturation of the pseudo-nucleus was traced in short intervals for 40 min after infection. This study was accompanied by the identification of phiKZ and bacterial DNA by real-time RCR. We demonstrated that phage DNA that isolated from the cytoplasm during all infection stages were compacted within the pseudo-nucleus in a specific structure. Bacterial DNA was diminished in the course of infection, but did not completely degrade until at least 40 min after phage application. The content of the total phage DNA, on the other hand, increased. EDX analysis confirmed these results and revealed that, during the infection, Sulfur content in the bacterial cytoplasm increased, which suggests the increase of DNA-binding Met-reach proteins synthesis, which could protect bacterial DNA from stress.
REVIEW | doi:10.20944/preprints202206.0022.v1
Subject: Life Sciences, Virology Keywords: SARS-CoV-2; COVID-19; Omicron; bioinformatics; immune escape; RBD mutations; vaccine development
Online: 1 June 2022 (14:12:36 CEST)
The evolution and the emergence of new mutations of viruses affect their transmissibility and/or pathogenicity features, depending on different evolutionary scenarios of virus adaptation to the host. A typical trade-off scenario of SARS-CoV-2 evolution has been proposed, which leads to the appearance of an Omicron strain with lowered lethality, yet enhanced transmissibility. This direction of evolution might be partly explained by virus adaptation to therapeutic agents and enhanced escape from vaccine-induced and natural immunity formed by other SARS-CoV-2 strains. Omicron’s high mutation rate in the Spike protein, as well as its previously described high genome mutation rate (Kandeel et al., 2021), revealed a gap between it and other SARS-CoV-2 strains, indicating the absence of a transitional evolutionary form to the Omicron strain. Therefore, Omicron has emerged as a new serotype and divergent from the evolutionary lineage of other SARS-CoV-2 strains. Omicron is a rapidly evolving variant of high concern, whose new subvariants continue to manifest. Its further understanding and the further monitoring of key mutations that provide virus immune escape and/or high affinity towards the receptor could be useful for vaccine and therapeutic development in order to control the evolutionary direction of the COVID-19 pandemic.
ARTICLE | doi:10.20944/preprints202206.0199.v1
Subject: Life Sciences, Biophysics Keywords: KCNQ1; Kv7.1; IKs; patch-clamp; inherited channelopathy; LQTS
Online: 14 June 2022 (08:42:22 CEST)
We identified a single nucleotide variation (SNV) (c.1264A>G) in the KCNQ1 gene in a 5-year-old boy who presented with a prolonged QT interval. His elder brother and mother, but not sister and father, also had this mutation. This missense mutation leads to a p.Lys422Glu (K422E) substitution in the Kv7.1 protein, never mentioned before. We inserted this substitution in an expression plasmid containing Kv7.1 cDNA and studied the electrophysiological characteristics of the mutated channel expressed in CHO-K1 using the whole-cell configuration of the patch-clamp technique. Expression of the mutant Kv7.1 channel in both homo- and heterozygous conditions, in the presence of auxiliary subunit KCNE1, results in a significant decrease in tail current densities compared to the expression of wild-type (WT) Kv7.1 and KCNE1. This study also indicates that K422E point mutation causes a dominant negative effect. The mutation was not associated with a trafficking defect, the mutant channel protein was confirmed to localize at the cell membrane. This mutation disrupts the poly-Lys strip in the proximal part of the highly conserved cytoplasmic A-B linker of Kv7.1, which was not shown before to be crucial for channel functioning.
ARTICLE | doi:10.20944/preprints202209.0024.v1
Subject: Life Sciences, Virology Keywords: flavivirus; tick-borne encephalitis; electron microscopy; EELS; nanoparticle tracking analysis; cryo-EM
Online: 1 September 2022 (10:54:41 CEST)
Tick-borne encephalitis virus (TBEV) is a RNA-containing enveloped virus, a member of the Flaviviridae family. Here we describe a detailed analysis of the size and structure of inactivated TBEV (the Sofyin-Chumakov TBEV strain, used in vaccines). Four analytical methods were used to analyze individual TBEV particles—negative staining TEM, cryo-EM, AFM, and NTA. All methods confirmed that the particles were monodisperse, and their mean size was ~50 nm. Cryo-EM data were used to obtain a 3D electron density model of the virus with clearly distinguishable E-proteins. STEM-EELS analysis detected phosphorous in the particles, which was interpreted as the RNA signal. Altogether, the described analytical procedures can be valuable for the further analysis of vaccine inactivated virus samples.