Transcriptome from Paired Samples Improves the Power of Comprehensive COVID-19 Host-Viral Characterization.

Previous transcriptome profiling studies showed significantly upregulated genes and altered biological pathways in acute COVID-19. However, changes in the transcriptional signatures during a defined time frame are not yet examined and described. The aims of this study included viral metagenomics and evaluation of the total expression in time-matched and tissue-matched paired COVID-19 samples with the analysis of the host splicing profile to reveal potential therapeutic targets. Prospective analysis of paired nasopharyngeal swabs (NPS) and blood (BL) samples from 18 COVID-19 patients with acute and resolved infection performed using Kallisto, Suppa2, Centrifuge, EdgeR, PantherDB, and L1000CDS2 tools. In NPS, we discovered 6 genes with changed splicing and 40 differentially expressed genes (DEG) that yielded 88 altered pathways. Blood samples yielded 15 alternatively spliced genes. Although the unpaired DEG analysis failed, pairing identified 78 genes and 242 altered pathways with meaningful clinical interpretation and new candidate drug combinations with up to 65% overlap. Metagenomics analyses showed SARS-CoV-2 dominance during and after the acute infection, with a significant reduction in NPS (0.008 vs. 0.002, p = 0.019). Even though both NPS and BL give meaningful insights into expression changes, this is the first demonstration of how the power of blood analysis is vastly maximized by pairing. The obtained results essentially showed that pairing is a determinant between a failed and a comprehensive study. Finally, the bioinformatics results prove to be a comprehensive tool for full-action insights, drug development, and infectious disease research when designed properly.

Kinesin-14 family proteins and microtubule dynamics define S. pombe mitotic and meiotic spindle assembly, and elongation.

To segregate the chromosomes faithfully during cell division, cells assemble a spindle that captures the kinetochores and pulls them towards opposite poles. Proper spindle function requires correct interplay between microtubule motors and non-motor proteins. Defects in spindle assembly or changes in spindle dynamics are associated with diseases, such as cancer or developmental disorders. Here, we compared mitotic and meiotic spindles in fission yeast. We show that, even though mitotic and meiotic spindles underwent the typical three phases of spindle elongation, they have distinct features. We found that the relative concentration of the kinesin-14 family protein Pkl1 is decreased in meiosis I compared to mitosis, while the concentration of the kinesin-5 family protein Cut7 remains constant. We identified the second kinesin-14 family protein Klp2 and microtubule dynamics as factors necessary for proper meiotic spindle assembly. This work defines the differences between mitotic and meiotic spindles in fission yeast Schizosaccharomyces pombe, and provides prospect for future comparative studies.This article has an associated First Person interview with the first author of the paper.

Evolutionary dynamics of Usutu virus: Worldwide dispersal patterns and transmission dynamics in Europe.

Background: Usutu virus (USUV) is an emerging mosquito-borne Flavivirus, with birds as the main zoonotic reservoir. Humans are accidental hosts and mostly develop mild or even asymptomatic infections, although severe complications such as encephalitis can also arise. Detailed characterization of the pathogen's phylogenetics may offer valuable insights into the prediction and prevention of potential epidemics; however, lack of uniformity and the number of available USUV sequences worldwide hamper comprehensive investigation. Aim: The study aimed to investigate USUV spatio-temporal dispersal inter- and intracontinentally and to estimate the dynamics of viral spread within Europe. Methods: Phylogeographic and phylodynamic analyses were done using advanced phylogenetic methods implemented in Beast 1.10.4 and Beast 2.6.4 software packages. Results: Herein, we report on a new USUV isolate from Culex pipiens collected in 2019 from Serbia. The results of this research revealed two newly described intercontinental migration events of USUV from Africa to Germany in the 1970s and from Africa to the Middle East (Israel) in the late 90s. Finally, phylodynamic analysis substantiated the ongoing active expansion of USUV in Europe. Conclusion: The data would imply a high potential for further USUV expansion in Europe. Detailed phylogenetic characterization of the pathogen may offer valuable insights into prediction and prevention of potential epidemics; however, lack of uniformity and number of available USUV sequences worldwide hampers comprehensive investigation. This study draws attention to the need for upscaling USUV surveillance.

Detection of the Xanthi Chryso-like Virus in New Geographical Area and a Novel Arthropod Carrier.

Here, we report on a serendipitous finding of a chryso-like virus associated with Culex pipiens mosquitos in the course of study aimed to detect and characterize West Nile virus (WNV) circulating in mosquitos in Serbia, Southern Europe. Upon initial detection of unexpected product in a PCR protocol for partial WNV NS5 gene amplification, further confirmation and identification was obtained through additional PCR and Sanger sequencing experiments. Bioinformatic and phylogenetic analysis identified the obtained sequences as Xanthi chryso-like virus (XCLV). The finding is particular for the fact that it associates XCLV with a new potential vector species and documents a novel geographical area of its distribution.

The First Molecular Characterization of Serbian SARS-CoV-2 Isolates From a Unique Early Second Wave in Europe.

March 6, 2020 is considered as the official date of the beginning of the COVID-19 epidemic in Serbia. In late spring and early summer 2020, Europe recorded a decline in the rate of SARS-CoV-2 infection and subsiding of the first wave. This trend lasted until the fall, when the second wave of the epidemic began to appear. Unlike the rest of Europe, Serbia was hit by the second wave of the epidemic a few months earlier. Already in June 2020, newly confirmed cases had risen exponentially. As the COVID-19 pandemic is the first pandemic in which there has been instant sharing of genomic information on isolates around the world, the aim of this study was to analyze whole SARS-CoV-2 viral genomes from Serbia, to identify circulating variants/clade/lineages, and to explore site-specific mutational patterns in the unique early second wave of the European epidemic. This analysis of Serbian isolates represents the first publication from Balkan countries, which demonstrates the importance of specificities of local transmission especially when preventive measures differ among countries. One hundred forty-eight different genome variants among 41 Serbian isolates were detected in this study. One unique and seven extremely rare mutations were identified, with locally specific continuous dominance of the 20D clade. At the same time, amino acid substitutions of newly identified variants of concern were found in our isolates from October 2020. Future research should be focused on functional characterization of novel mutations in order to understand the exact role of these variations.

Comparison and Sensitivity Evaluation of Three Different Commercial Real-Time Quantitative PCR Kits for SARS-CoV-2 Detection.

Real-time reverse transcription polymerase chain reaction (RT-qPCR) is the most sensitive and specific assay and, therefore, is the "gold standard" diagnostic method for the diagnosis of SARS-CoV-2 infection. The aim of this study was to compare and analyze the detection performance of three different commercially available SARS-CoV-2 nucleic acid detection kits: Sansure Biotech, GeneFinderTM, and TaqPathTM on 354 randomly selected samples from hospitalized COVID-19 patients. All PCR reactions were performed using the same RNA isolates and one real-time PCR machine. The final result of the three evaluated kits was not statistically different (p = 0.107), and also had a strong positive association and high Cohen's κ coefficient. In contrast, the average Ct values that refer to the ORF1ab and N gene amplification were significantly different (p < 0.001 and p < 0.001, respectively), with the lowest obtained by the TaqPathTM for the ORF1ab and by the Sansure Biotech for the N gene. The results show a high similarity in the analytical sensitivities for SARS-CoV-2 detection, which indicates that the diagnostic accuracy of the three assays is comparable. However, the SanSure Biotech kit showed a bit better diagnostic performance. Our findings suggest that the imperative for improvement should address the determination of cut-off Ct values and rapid modification of the primer sets along with the appearance of new variants.

Non-polymerizing long-pitch actin dimers that interact with myosin.

The production of a soluble actomyosin complex would be a significant step toward elucidating molecular interactions responsible for biological movement. We took a systematic approach to producing soluble long-pitch actin dimers that are polymerization-deficient yet retain biological protein-protein interactions, including myosin binding. Actin mutant proteins and chemical crosslinking were combined with different polymerization inhibition strategies, including ADP-ribosylation, or the use of a polymerization-deficient actin mutant protein. While all of the long-pitch actin dimers retained interactions reflective of F-actin activity, each displayed different interactions with myosin. Myosin did not interact productively with long-pitch actin dimers capped with DNase-I, and led to filament formation of unmodified long-pitch actin dimers or dimers possessing a polymerization-deficient actin subunit. However, ADP-ribosylated long-pitch actin dimers interacted with myosin, giving this dimer great potential for producing a soluble actomyosin complex, which could greatly improve our understanding of the molecular basis of movement in cells, tissues, and organisms.

Actin polymerization is controlled by residue size at position 204.

Previous work has shown that purified double mutant A204C/C374A yeast actin is polymerization-deficient in vitro under physiological concentrations. To understand the importance of the 204 residue in subdomain 4, a series of actin proteins with a single mutation at this position were created with Cys-374 retained. Only yeast expressing A204G-, A204S-, or A204C-actin were viable. The A204G and A204S strains were sensitive to cold temperature and hyperosmolarity, whereas the A204C strain showed more profound effects on growth under these conditions. Cells expressing A204C-actin exhibited anomalies previously observed for A204C/C374A actin, including abnormal actin structures. A204G- and A204S-actin proteins had 12- and 13-fold increased critical concentrations, respectively, relative to wild-type. Only at very high concentrations could A204C actin polymerize when ATP was bound; when hydrolyzed, the ADP-containing A204C filaments depolymerized, demonstrating a profound difference in critical concentration between ATP and ADP states with A204C actin. A correlation between size of the residue substituted at position 204 and energy minimization of actin filament models was observed. We propose that the region surrounding residue 204 is involved in interactions that change depending on the phosphorylation state of the bound nucleotide that might reflect different conformations of F-actin subunits.

Cysteine engineering of actin self-assembly interfaces.

The Holmes model of filamentous actin (F-actin) and recent structural studies suggest specific atomic interactions between F-actin subunits. We tested these interactions through a cysteine-engineering approach with the goal of inhibiting filament formation by introducing chemical groups at sites important for polymerization. We substituted surface amino acids on the actin molecule with cysteine residues and tested the effect of producing these actin mutant proteins in a yeast expression system. The intrinsic folding and polymerization characteristics of the cysteine-engineered actin proteins were measured. The effect of chemical modification of the introduced cysteine residues on the polymerization of the actin mutant proteins was also examined. Modification of cysteine residues with large hydrophobic reagents resulted in polymerization inhibition. We examined the finding that the D288C actin protein does not polymerize under oxidizing conditions and forms protein aggregates when magnesium and EGTA are present. Chemical crosslinking experiments revealed the presence of a lower dimer when only D288C actin was present. When both D288C and A204C actin were present, crosslinking experiments support the proximity of Asp288 on the barbed end of one subunit to Ala204 on the pointed end of a neighboring subunit in the Holmes model of F-actin.

Stimulation of actin polymerization by vacuoles via Cdc42p-dependent signaling.

We have previously shown that actin ligands inhibit the fusion of yeast vacuoles in vitro, which suggests that actin remodeling is a subreaction of membrane fusion. Here, we demonstrate the presence of vacuole-associated actin polymerization activity, and its dependence on Cdc42p and Vrp1p. Using a sensitive in vitro pyrene-actin polymerization assay, we found that vacuole membranes stimulated polymerization, and this activity increased when vacuoles were preincubated under conditions that support membrane fusion. Vacuoles purified from a VRP1-gene deletion strain showed reduced polymerization activity, which could be recovered when reconstituted with excess Vrp1p. Cdc42p regulates this activity because overexpression of dominant-negative Cdc42p significantly reduced vacuole-associated polymerization activity, while dominant-active Cdc42p increased activity. We also used size-exclusion chromatography to directly examine changes in yeast actin induced by vacuole fusion. This assay confirmed that actin undergoes polymerization in a process requiring ATP. To further confirm the need for actin polymerization during vacuole fusion, an actin polymerization-deficient mutant strain was examined. This strain showed in vivo defects in vacuole fusion, and actin purified from this strain inhibited in vitro vacuole fusion. Affinity isolation of vacuole-associated actin and in vitro binding assays revealed a polymerization-dependent interaction between actin and the SNARE Ykt6p. Our results suggest that actin polymerization is a subreaction of vacuole membrane fusion governed by Cdc42p signal transduction.