Split aminoacyl-tRNA synthetases for proximity-induced stop codon suppression.

Synthetic biology tools for regulating gene expression have many useful biotechnology and therapeutic applications. Most tools developed for this purpose control gene expression at the level of transcription, and relatively few methods are available for regulating gene expression at the translational level. Here, we design and engineer split orthogonal aminoacyl-tRNA synthetases (o-aaRS) as unique tools to control gene translation in bacteria and mammalian cells. Using chemically induced dimerization domains, we developed split o-aaRSs that mediate gene expression by conditionally suppressing stop codons in the presence of the small molecules rapamycin and abscisic acid. By activating o-aaRSs, these molecular switches induce stop codon suppression, and in their absence stop codon suppression is turned off. We demonstrate, in Escherichia coli and in human cells, that split o-aaRSs function as genetically encoded AND gates where stop codon suppression is controlled by two distinct molecular inputs. In addition, we show that split o-aaRSs can be used as versatile biosensors to detect therapeutically relevant protein-protein interactions, including those involved in cancer, and those that mediate severe acute respiratory syndrome-coronavirus-2 infection.

Two-step fitness selection for intra-host variations in SARS-CoV-2.

Spontaneous mutations introduce uncertainty into coronavirus disease 2019 (COVID-19) control procedures and vaccine development. Here, we perform a spatiotemporal analysis on intra-host single-nucleotide variants (iSNVs) in 402 clinical samples from 170 affected individuals, which reveals an increase in genetic diversity over time after symptom onset in individuals. Nonsynonymous mutations are overrepresented in the pool of iSNVs but underrepresented at the single-nucleotide polymorphism (SNP) level, suggesting a two-step fitness selection process: a large number of nonsynonymous substitutions are generated in the host (positive selection), and these substitutions tend to be unfixed as SNPs in the population (negative selection). Dynamic iSNV changes in subpopulations with different gender, age, illness severity, and viral shedding time displayed a varied fitness selection process among populations. Our study highlights that iSNVs provide a mutational pool shaping the rapid global evolution of the virus.

Persistent High Percentage of HLA-DR+CD38high CD8+ T Cells Associated With Immune Disorder and Disease Severity of COVID-19.

Background: The global outbreak of coronavirus disease 2019 (COVID-19) has turned into a worldwide public health crisis and caused more than 100,000,000 severe cases. Progressive lymphopenia, especially in T cells, was a prominent clinical feature of severe COVID-19. Activated HLA-DR+CD38+ CD8+ T cells were enriched over a prolonged period from the lymphopenia patients who died from Ebola and influenza infection and in severe patients infected with SARS-CoV-2. However, the CD38+HLA-DR+ CD8+ T population was reported to play contradictory roles in SARS-CoV-2 infection. Methods: A total of 42 COVID-19 patients, including 32 mild or moderate and 10 severe or critical cases, who received care at Beijing Ditan Hospital were recruited into this retrospective study. Blood samples were first collected within 3 days of the hospital admission and once every 3-7 days during hospitalization. The longitudinal flow cytometric data were examined during hospitalization. Moreover, we evaluated serum levels of 45 cytokines/chemokines/growth factors and 14 soluble checkpoints using Luminex multiplex assay longitudinally. Results: We revealed that the HLA-DR+CD38+ CD8+ T population was heterogeneous, and could be divided into two subsets with distinct characteristics: HLA-DR+CD38dim and HLA-DR+CD38hi. We observed a persistent accumulation of HLA-DR+CD38hi CD8+ T cells in severe COVID-19 patients. These HLA-DR+CD38hi CD8+ T cells were in a state of overactivation and consequent dysregulation manifested by expression of multiple inhibitory and stimulatory checkpoints, higher apoptotic sensitivity, impaired killing potential, and more exhausted transcriptional regulation compared to HLA-DR+CD38dim CD8+ T cells. Moreover, the clinical and laboratory data supported that only HLA-DR+CD38hi CD8+ T cells were associated with systemic inflammation, tissue injury, and immune disorders of severe COVID-19 patients. Conclusions: Our findings indicated that HLA-DR+CD38hi CD8+ T cells were correlated with disease severity of COVID-19 rather than HLA-DR+CD38dim population.

COVID-19 reinfection in the presence of neutralizing antibodies.

After a short recovery period, COVID-19 reinfections could occur in convalescent patients, even those with measurable levels of neutralizing antibodies. Effective vaccinations and protective public health measures are recommended for the convalescent COVID-19 patients.

Atrial Arrhythmias in Patients with Severe COVID-19.

The number of confirmed COVID-19 cases has increased drastically; however, information regarding the impact of this disease on the occurrence of arrhythmias is scarce. The aim of this study was to determine the impact of COVID-19 on arrhythmia occurrence. This prospective study included patients with COVID-19 treated at the Leishenshan Temporary Hospital of Wuhan City, China, from February 24 to April 5, 2020. Demographic, comorbidity, and arrhythmias data were collected from patients with COVID-19 (n = 84) and compared with control data from patients with bacterial pneumonia (n = 84) infection. Furthermore, comparisons were made between patients with severe and nonsevere COVID-19 and between older and younger patients. Compared with patients with bacterial pneumonia, those with COVID-19 had higher total, mean, and minimum heart rates (all P < 0.01). Patients with severe COVID-19 (severe and critical type diseases) developed more atrial arrhythmias compared with those with nonsevere symptoms. Plasma creatine kinase isoenzyme (CKMB) levels (P=0.01) were higher in the severe group than in the nonsevere group, and there were more deaths in the severe group than in the nonsevere group (6 (15%) vs. 3 (2.30%); P=0.05). Premature atrial contractions (PAC) and nonsustained atrial tachycardia (NSAT) were significantly positively correlated with plasma CKMB levels but not with high-sensitive cardiac troponin I or myoglobin levels. Our data demonstrate that COVID-19 patients have higher total, mean, and minimum heart rates compared with those with bacterial pneumonia. Patients with severe or critical disease had more frequent atrial arrhythmias (including PAC and AF) and higher CKMB levels and mortality than those with nonsevere symptoms.

Carboxypeptidase B blocks ex vivo activation of the anaphylatoxin-neutrophil extracellular trap axis in neutrophils from COVID-19 patients.

BACKGROUND: Thrombosis and coagulopathy are highly prevalent in critically ill patients with COVID-19 and increase the risk of death. Immunothrombosis has recently been demonstrated to contribute to the thrombotic events in COVID-19 patients with coagulopathy. As the primary components of immunothrombosis, neutrophil extracellular traps (NETs) could be induced by complement cascade components and other proinflammatory mediators. We aimed to explore the clinical roles of NETs and the regulation of complement on the NET formation in COVID-19. METHODS: We recruited 135 COVID-19 patients and measured plasma levels of C5, C3, cell-free DNA and myeloperoxidase (MPO)-DNA. Besides, the formation of NETs was detected by immunofluorescent staining and the cytotoxicity to vascular endothelial HUVEC cells was evaluated by CCK-8 assay. RESULTS: We found that the plasma levels of complements C3 and MPO-DNA were positively related to coagulation indicator fibrin(-ogen) degradation products (C3: r = 0.300, p = 0.005; MPO-DNA: r = 0.316, p = 0.002) in COVID-19 patients. Besides, C3 was positively related to direct bilirubin (r = 0.303, p = 0.004) and total bilirubin (r = 0.304, p = 0.005), MPO-DNA was positively related to lactate dehydrogenase (r = 0.306, p = 0.003) and creatine kinase (r = 0.308, p = 0.004). By using anti-C3a and anti-C5a antibodies, we revealed that the complement component anaphylatoxins in the plasma of COVID-19 patients strongly induced NET formation. The pathological effect of the anaphylatoxin-NET axis on the damage of vascular endothelial cells could be relieved by recombinant carboxypeptidase B (CPB), a stable homolog of enzyme CPB2 which can degrade anaphylatoxins to inactive products. CONCLUSIONS: Over-activation in anaphylatoxin-NET axis plays a pathological role in COVID-19. Early intervention in anaphylatoxins might help prevent thrombosis and disease progression in COVID-19 patients.

Genomic surveillance of COVID-19 cases in Beijing.

The spread of SARS-CoV-2 in Beijing before May, 2020 resulted from transmission following both domestic and global importation of cases. Here we present genomic surveillance data on 102 imported cases, which account for 17.2% of the total cases in Beijing. Our data suggest that all of the cases in Beijing can be broadly classified into one of three groups: Wuhan exposure, local transmission and overseas imports. We classify all sequenced genomes into seven clusters based on representative high-frequency single nucleotide polymorphisms (SNPs). Genomic comparisons reveal higher genomic diversity in the imported group compared to both the Wuhan exposure and local transmission groups, indicating continuous genomic evolution during global transmission. The imported group show region-specific SNPs, while the intra-host single nucleotide variations present as random features, and show no significant differences among groups. Epidemiological data suggest that detection of cases at immigration with mandatory quarantine may be an effective way to prevent recurring outbreaks triggered by imported cases. Notably, we also identify a set of novel indels. Our data imply that SARS-CoV-2 genomes may have high mutational tolerance.