Case Report: Omicron BA.2 Subvariant of SARS-CoV-2 Outcompetes BA.1 in Two Co-infection Cases.

Trends from around the world suggest that the omicron BA.2 subvariant is increasing in proportion to the original BA.1 subvariant. Here we report two cases of co-infection with omicron BA.1 and omicron BA.2 in co-exposed individuals. In both individuals, genome sequencing and/or S-gene specific PCR identified omicron BA.1 at early time-points, which was replaced by omicron BA.2 at later time-points of the infection. The timeline of our data supports the proposition that BA.2 outcompetes BA.1 in a real-life scenario, and in time becomes the dominant variant in the upper respiratory tract of the host.

Early Initiation of Extracorporeal Blood Purification Using the AN69ST (oXiris®) Hemofilter as a Treatment Modality for COVID-19 Patients: a Single-Centre Case Series

Abstract Introduction: Severe coronavirus disease 2019 (COVID-19) is characterised by hyperinflammatory state, systemic coagulopathies, and multiorgan involvement, especially acute respiratory distress syndrome (ARDS). We here describe our preliminary clinical experience with COVID-19 patients treated via an early initiation of extracorporeal blood purification combined with systemic heparinisation and respiratory support. Methods: Fifteen patients were included; several biomarkers associated with COVID-19 severity were monitored. Personalised treatment was tailored according to the levels of interleukin (IL)-6, IL-8, tumour necrosis factor alpha, C-reactive protein (CRP), neutrophil-to-lymphocyte ratio, thrombocyte counts, D-dimers, and fibrinogen. Treatment consisted of respiratory support, extracorporeal blood purification using the AN69ST (oXiris®) hemofilter, and 300 U/kg heparin to maintain activation clotting time ≥ 180 seconds. Results: Ten patients presented with severe to critical disease (dyspnoea, hypoxia, respiratory rate > 30/min, peripheral oxygen saturation < 90%, or > 50% lung involvement on X-ray imaging). The median intensive care unit length of stay was 9.3 days (interquartile range 5.3-10.1); two patients developed ARDS and died after 5 and 26 days. Clinical improvement was associated with normalisation (increase) of thrombocytes and white blood cells, stable levels of IL-6 (< 50 ng/mL), and a decrease of CRP and fibrinogen. Conclusion: Continuous monitoring of COVID-19 severity biomarkers and radiological imaging is crucial to assess disease progression, uncontrolled inflammation, and to avert irreversible multiorgan failure. The combination of systemic heparin anticoagulation regimens and extracorporeal blood purification using cytokine-adsorbing hemofilters may reduce hyperinflammation, prevent coagulopathy, and support clinical recovery.

Early Initiation of Extracorporeal Blood Purification Using the AN69ST (oXiris®) Hemofilter as a Treatment Modality for COVID-19 Patients: a Single-Centre Case Series.

INTRODUCTION: Severe coronavirus disease 2019 (COVID-19) is characterised by hyperinflammatory state, systemic coagulopathies, and multiorgan involvement, especially acute respiratory distress syndrome (ARDS). We here describe our preliminary clinical experience with COVID-19 patients treated via an early initiation of extracorporeal blood purification combined with systemic heparinisation and respiratory support. METHODS: Fifteen patients were included; several biomarkers associated with COVID-19 severity were monitored. Personalised treatment was tailored according to the levels of interleukin (IL)-6, IL-8, tumour necrosis factor alpha, C-reactive protein (CRP), neutrophil-to-lymphocyte ratio, thrombocyte counts, D-dimers, and fibrinogen. Treatment consisted of respiratory support, extracorporeal blood purification using the AN69ST (oXiris®) hemofilter, and 300 U/kg heparin to maintain activation clotting time ≥ 180 seconds. RESULTS: Ten patients presented with severe to critical disease (dyspnoea, hypoxia, respiratory rate > 30/min, peripheral oxygen saturation < 90%, or > 50% lung involvement on X-ray imaging). The median intensive care unit length of stay was 9.3 days (interquartile range 5.3-10.1); two patients developed ARDS and died after 5 and 26 days. Clinical improvement was associated with normalisation (increase) of thrombocytes and white blood cells, stable levels of IL-6 (< 50 ng/mL), and a decrease of CRP and fibrinogen. CONCLUSION: Continuous monitoring of COVID-19 severity biomarkers and radiological imaging is crucial to assess disease progression, uncontrolled inflammation, and to avert irreversible multiorgan failure. The combination of systemic heparin anticoagulation regimens and extracorporeal blood purification using cytokine-adsorbing hemofilters may reduce hyperinflammation, prevent coagulopathy, and support clinical recovery.

Composition of Transcription Machinery and Its Crosstalk with Nucleoid-Associated Proteins and Global Transcription Factors.

The coordination of bacterial genomic transcription involves an intricate network of interdependent genes encoding nucleoid-associated proteins (NAPs), DNA topoisomerases, RNA polymerase subunits and modulators of transcription machinery. The central element of this homeostatic regulatory system, integrating the information on cellular physiological state and producing a corresponding transcriptional response, is the multi-subunit RNA polymerase (RNAP) holoenzyme. In this review article, we argue that recent observations revealing DNA topoisomerases and metabolic enzymes associated with RNAP supramolecular complex support the notion of structural coupling between transcription machinery, DNA topology and cellular metabolism as a fundamental device coordinating the spatiotemporal genomic transcription. We analyse the impacts of various combinations of RNAP holoenzymes and global transcriptional regulators such as abundant NAPs, on genomic transcription from this viewpoint, monitoring the spatiotemporal patterns of couplons-overlapping subsets of the regulons of NAPs and RNAP sigma factors. We show that the temporal expression of regulons is by and large, correlated with that of cognate regulatory genes, whereas both the spatial organization and temporal expression of couplons is distinctly impacted by the regulons of NAPs and sigma factors. We propose that the coordination of the growth phase-dependent concentration gradients of global regulators with chromosome configurational dynamics determines the spatiotemporal patterns of genomic expression.

Workflow for the Implementation of Precision Genomics in Healthcare.

To enable the implementation of precise genomics in a local healthcare system, we devised a pipeline for filtering and reporting of relevant genetic information to healthy individuals based on exome or genome data. In our analytical pipeline, the first tier of filtering is variant-centric, and it is based on the selection of annotated pathogenic, protective, risk factor, and drug response variants, and their one-by-one detailed evaluation. This is followed by a second-tier gene-centric deconstruction and filtering of virtual gene lists associated with diseases, and VUS-centric filtering according to ACMG pathogenicity criteria and pre-defined deleteriousness criteria. By applying this filtering protocol, we were able to provide valuable insights regarding the carrier status, pharmacogenetic profile, actionable cardiovascular and cancer predispositions, and potentially pathogenic variants of unknown significance to our patients. Our experience demonstrates that genomic profiling can be implemented into routine healthcare and provide information of medical significance.

A Pediatric Case of Glioblastoma Multiforme Associated With a Novel Germline p.His112CysfsTer9 Mutation in the MLH1 Gene Accompanied by a p.Arg283Cys Mutation in the TP53 Gene: A Case Report.

Targeted gene panel testing has the power to interrogate hundreds of genes and evaluate the genetic risk for many types of hereditary cancers simultaneously. We screened a 13-year-old male patient diagnosed with glioblastoma multiforme with the aim to get further insights into the biology of his condition. Herein, we applied gene panel sequencing and identified a heterozygous frameshift mutation c.333_334delTC; p.His112CysfsTer9 in the MLH1 gene in blood and tumor tissue accompanied by a known heterozygous missense variant of unknown significance c.847C > T; p.Arg283Cys in the TP53 gene. Parental screening revealed the presence of the same TP53 variant in the father and the same MLH1 variant in the mother, who was in fact undergoing treatment for early-stage breast cancer at the time of her son's unfortunate diagnosis. This case reports for the first time the co-occurrence of a genetic mutation in the MLH1 gene of the mismatch repair pathway, commonly associated with the Lynch syndrome, accompanied by a rare variant in the TP53 gene. This report underlines the need for broad panel gene testing in lieu of single-gene or syndrome-focused gene screening and evaluation of the effects of multiple pathogenic or modifier variants on the phenotypic spectrum of the disease.

Trio Clinical Exome Sequencing in a Patient With Multicentric Carpotarsal Osteolysis Syndrome: First Case Report in the Balkans.

Exome sequencing can interrogate thousands of genes simultaneously and it is becoming a first line diagnostic tool in genomic medicine. Herein, we applied trio clinical exome sequencing (CES) in a patient presenting with undiagnosed skeletal disorder, minor facial abnormalities, and kidney hypoplasia; her parents were asymptomatic. Testing the proband and her parents led to the identification of a de novo mutation c.188C>T (p.Pro63Leu) in the MAFB gene, which is known to cause multicentric carpotarsal osteolysis syndrome (MCTO). The c.188C>T mutation lies in a hotspot amino acid stretch within the transactivation domain of MAFB, which is a negative regulator of RANKL-induced osteoclastogenesis. MCTO is an extremely rare autosomal dominant (AD) disorder that typically arises spontaneously and causes carpotarsal osteolysis, often followed by nephropathy. To the best of our knowledge, this is the first study reporting genetically diagnosed MCTO in the Balkans.

Isolation and Analysis of RNA Polymerase Supramolecular Complex with Associated Proteins.

Transcription machinery plays a central role in both the gene expression and nucleoid compaction. In this chapter we elaborate on the optimization of RNA polymerase purification protocol using a mild procedure with the purpose of preserving its native composition. This protocol combines protein extraction under non-denaturing conditions, heparin based affinity purification, and consequent BN-PAGE-SDS-PAGE separation. The outcome is an experimental procedure for screening RNA polymerase composition with associated proteins, in various bacterial strains or mutant backgrounds. With modifications in the column purification step, this procedure can be applied for isolation and identification of the components of other multi-protein complexes.

Structural coupling between RNA polymerase composition and DNA supercoiling in coordinating transcription: a global role for the omega subunit?

In growing bacterial cells, the global reorganization of transcription is associated with alterations of RNA polymerase composition and the superhelical density of the DNA. However, the existence of any regulatory device coordinating these changes remains elusive. Here we show that in an exponentially growing Escherichia coli rpoZ mutant lacking the polymerase ω subunit, the impact of the Eσ(38) holoenzyme on transcription is enhanced in parallel with overall DNA relaxation. Conversely, overproduction of σ(70) in an rpoZ mutant increases both overall DNA supercoiling and the transcription of genes utilizing high negative superhelicity. We further show that transcription driven by the Eσ(38) and Eσ(70) holoenzymes from cognate promoters induces distinct superhelical densities of plasmid DNA in vivo. We thus demonstrate a tight coupling between polymerase holoenzyme composition and the supercoiling regimen of genomic transcription. Accordingly, we identify functional clusters of genes with distinct σ factor and supercoiling preferences arranging alternative transcription programs sustaining bacterial exponential growth. We propose that structural coupling between DNA topology and holoenzyme composition provides a basic regulatory device for coordinating genome-wide transcription during bacterial growth and adaptation. IMPORTANCE Understanding the mechanisms of coordinated gene expression is pivotal for developing knowledge-based approaches to manipulating bacterial physiology, which is a problem of central importance for applications of biotechnology and medicine. This study explores the relationships between variations in the composition of the transcription machinery and chromosomal DNA topology and suggests a tight interdependence of these two variables as the major coordinating principle of gene regulation. The proposed structural coupling between the transcription machinery and DNA topology has evolutionary implications and suggests a new methodology for studying concerted alterations of gene expression during normal and pathogenic growth both in bacteria and in higher organisms.