Development of AAV-delivered broadly neutralizing anti-human ACE2 antibodies against SARS-CoV-2 variants.

The ongoing evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), resulting in the emergence of new variants that are resistant to existing vaccines and therapeutic antibodies, has raised the need for novel strategies to combat the persistent global COVID-19 epidemic. In this study, a monoclonal anti-human angiotensin-converting enzyme 2 (hACE2) antibody, ch2H2, was isolated and humanized to block the viral receptor-binding domain (RBD) binding to hACE2, the major entry receptor of SARS-CoV-2. This antibody targets the RBD-binding site on the N terminus of hACE2 and has a high binding affinity to outcompete the RBD. In vitro, ch2H2 antibody showed potent inhibitory activity against multiple SARS-CoV-2 variants, including the most antigenically drifted and immune-evading variant Omicron. In vivo, adeno-associated virus (AAV)-mediated delivery enabled a sustained expression of monoclonal antibody (mAb) ch2H2, generating a high concentration of antibodies in mice. A single administration of AAV-delivered mAb ch2H2 significantly reduced viral RNA load and infectious virions and mitigated pulmonary pathological changes in mice challenged with SARS-CoV-2 Omicron BA.5 subvariant. Collectively, the results suggest that AAV-delivered hACE2-blocking antibody provides a promising approach for developing broad-spectrum antivirals against SARS-CoV-2 and potentially other hACE2-dependent pathogens that may emerge in the future.

Impacts of Cancer-associated Mutations on the Structure-Activity Relationship of BAP1.

BRAC1 associated protein-1 (BAP1) is a major tumor suppressor involved in many cancers. The deubiquitinase (DUB) activity of BAP1 is essential for its nuclear localization, histone remodeling and proteostasis associated with mitochondrial calcium flux. Loss of the DUB activity due to catalytic mutations within the ubiquitin C-terminal hydrolase (UCH) domain of BAP1 (BAP1-UCH) directly contributes to oncogenesis. Nevertheless, it is non-trivial to rationalize how the other high-frequency but non-catalytic mutations within the BAP1-UCH lead to malignancies. Here we used multiplex spectroscopic, thermodynamic and biophysical analyses to investigate the impacts of eleven high-occurrence mutations within BAP1-UCH on the structure, folding and function. Several mutations significantly destabilize BAP1-UCH and increase its aggregation propensity. Hydrogen-deuterium exchange mass spectrometry data revealed allosteric destabilizations caused by mutations distant from the catalytic site. Our findings gave a comprehensive and multiscale account of the molecular basis of how these non-catalytic mutations within BAP1-UCH may be implicated in oncogenesis.

Application of Raman spectroscopy for direct analysis of Carlina acanthifolia subsp. utzka root essential oil.

Carlina genus plants e.g. Carlina acanthifolia subsp. utzka have been still used in folk medicine of many European countries and its biological activity is mostly associated with root essential oils. In the present paper, Raman spectroscopy (RS) was applied for the first time for evaluation of essential oil distribution in root of C. acnthifolia subsp. utzka and identification of root structures containing the essential oil. Furthermore, RS technique was applied to assess chemical stability of oil during drying of plant material or distillation process. Gas chromatography-mass spectrometry was used for qualitative and quantitative analysis of the essential oil. The identity of compounds was confirmed using Raman, ATR-IR and NMR spectroscopy. Carlina oxide was found to be the main component of the oil (98.96% ± 0.15). The spectroscopic study showed the high stability of essential oil and Raman distribution analysis indicated that the oil reservoirs were localized mostly in the structures of outer layer of the root while the inner part showed nearly no signal assigned to the oil. Raman spectroscopy technique enabled rapid, non-destructive direct analysis of plant material with minimal sample preparation and allowed straightforward, unambiguous identification of the essential oil in the sample.

Positive allosteric modulators of α7 nicotinic acetylcholine receptors affect neither the function of other ligand- and voltage-gated ion channels and acetylcholinesterase, nor ß-amyloid content.

The activity of positive allosteric modulators (PAMs) of α7 nicotinic acetylcholine receptors (AChRs), including 3-furan-2-yl-N-p-tolyl-acrylamide (PAM-2), 3-furan-2-yl-N-o-tolylacrylamide (PAM-3), and 3-furan-2-yl-N-phenylacrylamide (PAM-4), was tested on a variety of ligand- [i.e., human (h) α7, rat (r) α9α10, hα3-containing AChRs, mouse (m) 5-HT3AR, and several glutamate receptors (GluRs)] and voltage-gated (i.e., sodium and potassium) ion channels, as well as on acetylcholinesterase (AChE) and ß-amyloid (Aß) content. The functional results indicate that PAM-2 inhibits hα3-containing AChRs (IC50=26±6µM) with higher potency than that for NR1aNR2B and NR1aNR2A, two NMDA-sensitive GluRs. PAM-2 affects neither the activity of m5-HT3ARs, GluR5/KA2 (a kainate-sensitive GluR), nor AChE, and PAM-4 does not affect agonist-activated rα9α10 AChRs. Relevant clinical concentrations of PAM-2-4 do not inhibit Nav1.2 and Kv3.1 ion channels. These PAMs slightly enhance the activity of GluR1 and GluR2, two AMPA-sensitive GluRs. PAM-2 does not change the levels of Aß42 in an Alzheimer's disease mouse model (i.e., 5XFAD). The molecular docking and dynamics results using the hα7 model suggest that the active sites for PAM-2 include the intrasubunit (i.e., PNU-120596 locus) and intersubunit sites. These results support our previous study showing that these PAMs are selective for the α7 AChR, and clarify that the procognitive/promnesic/antidepressant activity of PAM-2 is not mediated by other targets.