Compounds derived from Humulus lupulus inhibit SARS-CoV-2 papain-like protease and virus replication.

BACKGROUND: Selected natural compounds exhibit very good antiviral properties. Especially, the medicinal plant Humulus lupulus (hop) contains several secondary plant metabolites some of which have previously shown antiviral activities. Among them, the prenylated chalcone xanthohumol (XN) demonstrated to be a potent inhibitor of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) main protease (Mpro). HYPOTHESIS/PURPOSE: Following the finding that xanthohumol (XN) is a potent inhibitor of SARS-CoV-2 Mpro, the effect of XN and its major derivatives isoxanthohumol (IXN), 6-prenylnaringenin (6-PN), and 8-prenylnaringenin (8-PN) from hops on SARS-CoV-2 papain-like protease (PLpro) were investigated. STUDY DESIGN: The modulatory effect of the hop compounds on PLpro were studied first in silico and then in vitro. In addition, the actual effect of hop compounds on the replication of SARS-CoV-2 in host cells was investigated. METHODS: In silico docking analysis was used to predict the binding affinity of hop compounds to the active site of PLpro. A recombinant PLpro was cloned, purified, characterized, and analyzed by small-angle X-ray scattering (SAXS), deISGylation assays, and kinetic analyses. Antiviral activity of hop compounds was assessed using the fluorescently labeled wildtype SARS-CoV-2 (icSARS-CoV-2-mNG) in Caco-2 host cells. RESULTS: Our in silico docking suggests that the purified hop compounds bind to the active site of SARS-CoV-2 PLpro blocking the access of its natural substrates. The hop-derived compounds inhibit SARS-CoV-2 PLpro with half maximal inhibitory concentration (IC50) values in the range of 59-162 µM. Furthermore, we demonstrate that XN and 6-PN, in particular, impede viral replication with IC50 values of 3.3 µM and 7.3 µM, respectively. CONCLUSION: In addition to the already known inhibition of Mpro by XN, our results show, for the first time, that hop-derived compounds target also SARS-CoV-2 PLpro which is a promising therapeutic target as it contributes to both viral replication and modulation of the immune system. These findings support the possibility to develop new hop-derived antiviral drugs targeting human coronaviruses.

Formation of dsRNA by-products during in vitro transcription can be reduced by using low steady-state levels of UTP.

Introduction: Exogeneous messenger ribonucleic acid (mRNA) can be used as therapeutic and preventive medication. However, during the enzymatic production process, commonly called in vitro transcription, by-products occur which can reduce the therapeutic efficacy of mRNA. One such by-product is double-stranded RNA (dsRNA). We therefore sought to limit the generation of dsRNA by-products during in vitro transcription. Materials and methods: In vitro transcription was performed with a DNA template including a poly(A)-tail-encoding region, dinucleotide or trinucleotide cap analogs for cotranscriptional capping, and relevant nucleoside triphosphates. Concentrations of UTP or modified UTP (m1ΨTP) and GTP were reduced and fed over the course of the reaction. mRNA was analyzed for dsRNA contamination, yield of the reaction, RNA integrity, and capping efficiency before translational activity was assessed. Results: Limiting the steady-state level of UTP or m1ΨTP during the enzymatic reaction reduced dsRNA formation, while not affecting mRNA yield or RNA integrity. Capping efficiency was optimized with the use of a combined GTP and UTP or m1ΨTP feed, while still reducing dsRNA formation. Lower dsRNA levels led to higher protein expression from the corresponding mRNAs. Discussion: Low steady-state concentrations of UTP and GTP, fed in combination over the course of the in vitro transcription reaction, produce mRNA with high capping and low levels of dsRNA formation, resulting in high levels of protein expression. This novel approach may render laborious purification steps to remove dsRNA unnecessary.

Quality of life of prosthetic and orthotic users in South India: a cross-sectional study.

BACKGROUND: The aim of this study was to compare QOL among people in India using lower-limb prostheses or orthoses with people without disability. A further aim was to compare subgroups and investigate whether QOL was associated with physical disability, gender, income, living area, and education. METHODS: A cross-sectional questionnaire study in which the World Health Organization Quality of Life-Bref was used to collect self-reported data. A total of 277 participants from India were included, 155 with disability and 122 without. Group comparisons were conducted using the Mann-Whitney U and the Kruskal-Wallis tests and associations were explored using regression analyses of the four QOL domains: physical health, psychological, social relationships, and environment. RESULTS: Participants with physical disability scored significantly lower than did participants without disability in three of the four QOL domains, i.e., physical health, (Median 14.29 vs 16.29; p < .001) psychological, (Median 14.67 vs. 15.33; p = .017) and environment (Median13.00 vs 14.00; p = .006). For people with disability those with no or irregular income and those not attending school having the lowest QOL scores in all four domains. Education was associated with all four QOL domains and income was associated with psychological and environment. Living in urban slums was associated with a higher risk of lower QOL in three QOL domains, i.e., physical health, psychology, and environment. CONCLUSIONS: Despite rehabilitation services, people with physical disability experienced lower QOL in terms of the physical health, psychological, and environmental domains than did people without disability. Community-based rehabilitation programmes for prosthetic and orthotic users need to increase and improve their rehabilitation services to increase income and improve access to education. Priority could be given to those who have no or irregular income, live in urban slums, and have not attended school to further improve their QOL.