Corrigendum: Case Series: Maraviroc and pravastatin as a therapeutic option to treat long COVID/Post-acute sequalae of COVID (PASC).

[This corrects the article DOI: 10.3389/fmed.2023.1122529.].

Gene dosage compensation: Origins, criteria to identify compensated genes, and mechanisms including sensor loops as an emerging systems-level property in cancer.

The gene dosage compensation hypothesis presents a mechanism through which the expression of certain genes is modulated to compensate for differences in the dose of genes when additional chromosomes are present. It is one of the means through which cancer cells actively cope with the potential damaging effects of aneuploidy, a hallmark of most cancers. Dosage compensation arises through several processes, including downregulation or overexpression of specific genes and the relocation of dosage-sensitive genes. In cancer, a majority of compensated genes are generally thought to be regulated at the translational or post-translational level, and include the basic components of a compensation loop, including sensors of gene dosage and modulators of gene expression. Post-translational regulation is mostly undertaken by a general degradation or aggregation of remaining protein subunits of macromolecular complexes. An increasingly important role has also been observed for transcriptional level regulation. This article reviews the process of targeted gene dosage compensation in cancer and other biological conditions, along with the mechanisms by which cells regulate specific genes to restore cellular homeostasis. These mechanisms represent potential targets for the inhibition of dosage compensation of specific genes in aneuploid cancers. This article critically examines the process of targeted gene dosage compensation in cancer and other biological contexts, alongside the criteria for identifying genes subject to dosage compensation and the intricate mechanisms by which cells orchestrate the regulation of specific genes to reinstate cellular homeostasis. Ultimately, our aim is to gain a comprehensive understanding of the intricate nature of a systems-level property. This property hinges upon the kinetic parameters of regulatory motifs, which we have termed "gene dosage sensor loops." These loops have the potential to operate at both the transcriptional and translational levels, thus emerging as promising candidates for the inhibition of dosage compensation in specific genes. Additionally, they represent novel and highly specific therapeutic targets in the context of aneuploid cancer.

Case series: Maraviroc and pravastatin as a therapeutic option to treat long COVID/Post-acute sequelae of COVID (PASC).

Post-acute sequelae of COVID (PASC), or long COVID, is a multisystem complication of SARS-CoV-2 infection that continues to debilitate millions worldwide thus highlighting the public health importance of identifying effective therapeutics to alleviate this illness. One explanation behind PASC may be attributed to the recent discovery of persistent S1 protein subunit of SARS-CoV-2 in CD16+ monocytes up to 15 months after infection. CD16+ monocytes, which express both CCR5 and fractalkine receptors (CX3CR1), play a role in vascular homeostasis and endothelial immune surveillance. We propose targeting these receptors using the CCR5 antagonist, maraviroc, along with pravastatin, a fractalkine inhibitor, could disrupt the monocytic-endothelial-platelet axis that may be central to the etiology of PASC. Using five validated clinical scales (NYHA, MRC Dyspnea, COMPASS-31, modified Rankin, and Fatigue Severity Score) to measure 18 participants' response to treatment, we observed significant clinical improvement in 6 to 12 weeks on a combination of maraviroc 300 mg per oral twice a day and pravastatin 10 mg per oral daily. Subjective neurological, autonomic, respiratory, cardiac and fatigue symptoms scores all decreased which correlated with statistically significant decreases in vascular markers sCD40L and VEGF. These findings suggest that by interrupting the monocytic-endothelial-platelet axis, maraviroc and pravastatin may restore the immune dysregulation observed in PASC and could be potential therapeutic options. This sets the framework for a future double-blinded, placebo-controlled randomized trial to further investigate the drug efficacy of maraviroc and pravastatin in treating PASC.

Multistability in Macrophage Activation Pathways and Metabolic Implications.

Macrophages are innate immune cells with a dynamic range of reversible activation states including the classical pro-inflammatory (M1) and alternative anti-inflammatory (M2) states. Deciphering how macrophages regulate their transition from one state to the other is key for a deeper understanding of inflammatory diseases and relevant therapies. Common regulatory motifs reported for macrophage transitions, such as positive or double-negative feedback loops, exhibit a switchlike behavior, suggesting the bistability of the system. In this review, we explore the evidence for multistability (including bistability) in macrophage activation pathways at four molecular levels. First, a decision-making module in signal transduction includes mutual inhibitory interactions between M1 (STAT1, NF-KB/p50-p65) and M2 (STAT3, NF-KB/p50-p50) signaling pathways. Second, a switchlike behavior at the gene expression level includes complex network motifs of transcription factors and miRNAs. Third, these changes impact metabolic gene expression, leading to switches in energy production, NADPH and ROS production, TCA cycle functionality, biosynthesis, and nitrogen metabolism. Fourth, metabolic changes are monitored by metabolic sensors coupled to AMPK and mTOR activity to provide stability by maintaining signals promoting M1 or M2 activation. In conclusion, we identify bistability hubs as promising therapeutic targets for reverting or blocking macrophage transitions through modulation of the metabolic environment.

Immune-Based Prediction of COVID-19 Severity and Chronicity Decoded Using Machine Learning.

Expression of CCR5 and its cognate ligands have been implicated in COVID-19 pathogenesis, consequently therapeutics directed against CCR5 are being investigated. Here, we explored the role of CCR5 and its ligands across the immunologic spectrum of COVID-19. We used a bioinformatics approach to predict and model the immunologic phases of COVID so that effective treatment strategies can be devised and monitored. We investigated 224 individuals including healthy controls and patients spanning the COVID-19 disease continuum. We assessed the plasma and isolated peripheral blood mononuclear cells (PBMCs) from 29 healthy controls, 26 Mild-Moderate COVID-19 individuals, 48 Severe COVID-19 individuals, and 121 individuals with post-acute sequelae of COVID-19 (PASC) symptoms. Immune subset profiling and a 14-plex cytokine panel were run on all patients from each group. B-cells were significantly elevated compared to healthy control individuals (P<0.001) as was the CD14+, CD16+, CCR5+ monocytic subset (P<0.001). CD4 and CD8 positive T-cells expressing PD-1 as well as T-regulatory cells were significantly lower than healthy controls (P<0.001 and P=0.01 respectively). CCL5/RANTES, IL-2, IL-4, CCL3, IL-6, IL-10, IFN-γ, and VEGF were all significantly elevated compared to healthy controls (all P<0.001). Conversely GM-CSF and CCL4 were in significantly lower levels than healthy controls (P=0.01). Data were further analyzed and the classes were balanced using SMOTE. With a balanced working dataset, we constructed 3 random forest classifiers: a multi-class predictor, a Severe disease group binary classifier and a PASC binary classifier. Models were also analyzed for feature importance to identify relevant cytokines to generate a disease score. Multi-class models generated a score specific for the PASC patients and defined as S1 = (IFN-γ + IL-2)/CCL4-MIP-1ß. Second, a score for the Severe COVID-19 patients was defined as S2 = (IL-6+sCD40L/1000 + VEGF/10 + 10*IL-10)/(IL-2 + IL-8). Severe COVID-19 patients are characterized by excessive inflammation and dysregulated T cell activation, recruitment, and counteracting activities. While PASC patients are characterized by a profile able to induce the activation of effector T cells with pro-inflammatory properties and the capacity of generating an effective immune response to eliminate the virus but without the proper recruitment signals to attract activated T cells.

A Fluorescent Real-Time Plaque Assay Enables Single-Cell Analysis of Virus-Induced Cytopathic Effect by Live-Cell Imaging.

Conventional plaque assays rely on the use of overlays to restrict viral infection allowing the formation of distinct foci that grow in time as the replication cycle continues leading to countable plaques that are visualized with standard techniques such as crystal violet, neutral red, or immunolabeling. This classical approach takes several days until large enough plaques can be visualized and counted with some variation due to subjectivity in plaque recognition. Since plaques are clonal lesions produced by virus-induced cytopathic effect, we applied DNA fluorescent dyes with differential cell permeability to visualize them by live-cell imaging. We could observe different stages of that cytopathic effect corresponding to an early wave of cells with chromatin-condensation followed by a wave of dead cells with membrane permeabilization within plaques generated by different animal viruses. This approach enables an automated plaque identification using image analysis to increase single plaque resolution compared to crystal violet counterstaining and allows its application to plaque tracking and plaque reduction assays to test compounds for both antiviral and cytotoxic activities. This fluorescent real-time plaque assay sums to those next-generation technologies by combining this robust classical method with modern fluorescence microscopy and image analysis approaches for future applications in virology.

Persistence of SARS CoV-2 S1 Protein in CD16+ Monocytes in Post-Acute Sequelae of COVID-19 (PASC) up to 15 Months Post-Infection.

The recent COVID-19 pandemic is a treatment challenge in the acute infection stage but the recognition of chronic COVID-19 symptoms termed post-acute sequelae SARS-CoV-2 infection (PASC) may affect up to 30% of all infected individuals. The underlying mechanism and source of this distinct immunologic condition three months or more after initial infection remains elusive. Here, we investigated the presence of SARS-CoV-2 S1 protein in 46 individuals. We analyzed T-cell, B-cell, and monocytic subsets in both severe COVID-19 patients and in patients with post-acute sequelae of COVID-19 (PASC). The levels of both intermediate (CD14+, CD16+) and non-classical monocyte (CD14Lo, CD16+) were significantly elevated in PASC patients up to 15 months post-acute infection compared to healthy controls (P=0.002 and P=0.01, respectively). A statistically significant number of non-classical monocytes contained SARS-CoV-2 S1 protein in both severe (P=0.004) and PASC patients (P=0.02) out to 15 months post-infection. Non-classical monocytes were sorted from PASC patients using flow cytometric sorting and the SARS-CoV-2 S1 protein was confirmed by mass spectrometry. Cells from 4 out of 11 severe COVID-19 patients and 1 out of 26 PASC patients contained ddPCR+ peripheral blood mononuclear cells, however, only fragmented SARS-CoV-2 RNA was found in PASC patients. No full length sequences were identified, and no sequences that could account for the observed S1 protein were identified in any patient. That non-classical monocytes may be a source of inflammation in PASC warrants further study.