Genome-Wide Characterization of SARS-CoV-2 Cytopathogenic Proteins in the Search of Antiviral Targets

Therapeutic inhibition of critical viral functions is important for curtailing coronavirus disease 2019 (COVID-19). We sought to identify antiviral targets through the genome-wide characterization of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) proteins that are crucial for viral pathogenesis and that cause harmful cytopathogenic effects.
All 29 viral proteins were tested in a fission yeast cell-based system using inducible gene expression. Twelve proteins, including eight nonstructural proteins (NSP1, NSP3, NSP4, NSP5, NSP6, NSP13, NSP14, and NSP15) and four accessory proteins (ORF3a, ORF6, ORF7a, and ORF7b), were identified that altered cellular proliferation and integrity and induced cell death. Cell death correlated with the activation of cellular oxidative stress. Of the 12 proteins, ORF3a was chosen for further study in mammalian cells because it plays an important role in viral pathogenesis and its activities are linked to lung tissue damage and a cytokine storm.
In human pulmonary and kidney epithelial cells, ORF3a induced cellular oxidative stress associated with apoptosis and necrosis and caused activation of proinflammatory response with production of the cytokines tumor necrosis factor alpha (TNF-α), interleukin-6 (IL-6), and IFN-β1, possibly through the activation of nuclear factor kappa B (NF-κB).
To further characterize the mechanism, we tested a natural ORF3a Beta variant, Q57H, and a mutant with deletion of the highly conserved residue, ΔG188. Compared with wild-type ORF3a, the ΔG188 variant yielded more robust activation of cellular oxidative stress, cell death, and innate immune response. Since cellular oxidative stress and inflammation contribute to cell death and tissue damage linked to the severity of COVID-19, our findings suggest that ORF3a is a promising, novel therapeutic target against COVID-19.
 IMPORTANCE The ongoing COVID-19 pandemic caused by SARS-CoV-2 has claimed over 5.5 million lives with more than 300 million people infected worldwide. While vaccines are effective, the emergence of new viral variants could jeopardize vaccine protection. Treatment of COVID-19 by antiviral drugs provides an alternative to battle against the disease. The goal of this study was to identify viral therapeutic targets that can be used in antiviral drug discovery. Utilizing a genome-wide functional analysis in a fission yeast cell-based system, we identified 12 viral candidates, including ORF3a, which cause cellular oxidative stress, inflammation, apoptosis, and necrosis that contribute to cytopathogenicity and COVID-19. Our findings indicate that antiviral agents targeting ORF3a could have a great impact on COVID-19.

Copolymers Containing 1-Methyl-2-phenyl-imidazole Moieties as Permanent Dipole Generating Units: Synthesis, Spectroscopic, Electrochemical, and Photovoltaic Properties

New donor-acceptor conjugated alternating or random copolymers containing 1-methyl-2-phenylbenzimidazole and benzothiadiazole (P1), diketopyrrolopyrrole (P4), or both acceptors (P2) are reported. The specific feature of these copolymers is the presence of a permanent dipole-bearing moiety (1-methyl-2-phenyl imidazole (MPI)) fused with the 1,4-phenylene ring of the polymer main chain.
For comparative reasons, polymers of the same main chain but deprived of the MPI group were prepared, namely, P5 with diketopyrrolopyrrole and P3 with both acceptors. The presence of the permanent dipole results in an increase of the optical band gap from 1.51 eV in P3 to 1.57 eV in P2 and from 1.49 eV in P5 to 1.55 eV in P4. It also has a measurable effect on the ionization potential (IP) and electrochemical band gap (EgCV), leading to their decrease from 5.00 and 1.83 eV in P3 to 4.92 and 1.79 eV in P2 as well as from 5.09 and 1.87 eV in P5 to 4.94 and 1.81 eV in P4.
 Moreover, the presence of permanent dipole lowers the exciton binding energy (Eb) from 0.32 eV in P3 to 0.22 eV in P2 and from 0.38 eV in P5 to 0.26 eV in P4. These dipole-induced changes in the polymer properties should be beneficial for photovoltaic applications. Bulk heterojunction solar cells fabricated from these polymers (with PC71BM acceptor) show low series resistance (rs), indicating good electrical transport properties. The measured power conversion efficiency (PCE) of 0.54% is limited by the unfavorable morphology of the active layer.

Preparation of Naringenin Nanosuspension and Its Antitussive and Expectorant Effects

Naringenin (NRG) is a natural flavonoid compound abundantly present in citrus fruits and has the potential to treat respiratory disorders. However, the clinical therapeutic effect of NRG is limited by its low bioavailability due to poor solubility.
To enhance the solubility, naringenin nanosuspensions (NRG-NSps) were prepared by applying tocopherol polyethylene glycol succinate (TPGS) as the nanocarrier via the media-milling method. The particle size, morphology, and drug-loading content of NRG-NSps were examined, and the stability was evaluated by detecting particle size changes in different physiological media. NRG-NSps exhibited a flaky appearance with a mean diameter of 216.9 nm, and the drug-loading content was 66.7%. NRG-NSps exhibited good storage stability and media stability.
NRG-NSps presented a sustainable release profile, and the cumulative drug-release rate approached approximately 95% within 7 d. NRG-NSps improved the antitussive effect significantly compared with the original NRG, the cough frequency was decreased from 22 to 15 times, and the cough incubation period was prolonged from 85.3 to 121.6 s. Besides, NRG-NSps also enhanced expectorant effects significantly, and phenol red secretion was increased from 1.02 to 1.45 μg/mL. These results indicate that NRG-NSps could enhance the bioavailability of NRG significantly and possess a potential clinical application.
Keywords: antitussive effect; bioavailability; expectorant effect; media-milling method; naringenin nanosuspension.

Distinct effects of volatile and intravenous anaesthetics on presynaptic calcium dynamics in mouse hippocampal GABAergic neurones

Background: General anaesthetics have marked effects on synaptic transmission, but their neuronal and circuit-level effects remain unclear. The volatile anaesthetic isoflurane differentially inhibits synaptic vesicle exocytosis in specific neuronal subtypes, but whether other common anaesthetics also have neurone-subtype-specific actions is unknown.
Methods: We used the genetically encoded fluorescent Ca2+ sensor GCaMP6f to compare the pharmacological effects of isoflurane, sevoflurane, propofol, and ketamine on presynaptic excitability in hippocampal glutamatergic neurones and in hippocampal parvalbumin-, somatostatin-, and vasoactive intestinal peptide-expressing (PV+, SST+, and VIP+, respectively) GABAergic interneurones.
Results: Isoflurane and sevoflurane depressed activity-driven presynaptic Ca2+ transients in a neurone-type-specific manner, with greater potency for inhibition of glutamate and SST+ compared with PV+ and VIP+ neurone presynaptic activation. In contrast, clinical concentrations of propofol (1 μM) or ketamine (15 μM) had no significant effects on presynaptic activation. Propofol potentiated evoked Ca2+ entry in PV+ interneurones but only at a supraclinical concentration (3 μM).

Formulated DMEM

from Addexbio
C0003-01 | RT 500 mL Bottle: 98.00 EUR

Optimized DMEM

from Addexbio
C0003-02 | RT 500 mL Bottle: 100.00 EUR

Specialized DMEM

from Addexbio
C0003-03 | RT 500 mL Bottle: 104.00 EUR

DMEM, High Glucose

from Abbexa
abx098878-500ml | 500 ml: 175.00 EUR

DMEM, High Glucose

from GenDepot
CM001-050 | 500ml: 72.00 EUR

DMEM, High Glucose

from GenDepot
CM001-300 | 6x500ml: 158.00 EUR

DMEM, High Glucose

from GenDepot
CM001-310 | 10x500ml: 201.00 EUR

DMEM, High Glucose

from GenDepot
CM001-320 | 20x500ml: 313.00 EUR

DMEM, High Glucose

from GenDepot
CM001-350 | 50x500ml: 571.00 EUR

DMEM, High Glucose

from GenDepot
CM002-050 | 500ml: 72.00 EUR

DMEM, High Glucose

from GenDepot
CM002-300 | 6x500ml: 158.00 EUR

DMEM, High Glucose

from GenDepot
CM002-310 | 10x500ml: 201.00 EUR

DMEM, High Glucose

from GenDepot
CM002-320 | 20x500ml: 313.00 EUR

DMEM, High Glucose

from GenDepot
CM002-350 | 50x500ml: 571.00 EUR

DMEM, High Glucose

from GenDepot
CM003-050 | 500ml: 74.00 EUR

DMEM, High Glucose

from GenDepot
CM003-300 | 6x500ml: 165.00 EUR

DMEM, High Glucose

from GenDepot
CM003-310 | 10x500ml: 205.00 EUR

DMEM, High Glucose

from GenDepot
CM003-320 | 20x500ml: 317.00 EUR
Conclusions: Anaesthetic-agent-selective effects on presynaptic Ca2+ entry have functional implications for hippocampal circuit function during i.v. or volatile anaesthetic-mediated anaesthesia. Hippocampal interneurones have distinct subtype-specific sensitivities to volatile anaesthetic actions on presynaptic Ca2+, which are similar between isoflurane and sevoflurane.
Keywords: calcium; interneurone; isoflurane; ketamine; mechanisms of anaesthesia; presynaptic; propofol; sevoflurane.

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