7 research outputs found
Imaging of SARS-CoV-2 infected Vero E6 cells by helium ion microscopy
No full textFrese N, Schmerer P, Wortmann M, et al. Imaging of SARS-CoV-2 infected Vero E6 cells by helium ion microscopy. Beilstein Journal of Nanotechnology. 2021;12:172-179.Helium ion microscopy (HIM) offers the opportunity to obtain direct views of biological samples such as cellular structures, virus particles, and microbial interactions. Imaging with the HIM combines sub-nanometer resolution, large depth of field, and high surface sensitivity. Due to its charge compensation capability, the HIM can image insulating biological samples without additional conductive coatings. Here, we present an exploratory HIM study of SARS-CoV-2 infected Vero E6 cells, in which several areas of interaction between cells and virus particles, as well as among virus particles, were imaged. The HIM pictures show the three-dimensional appearance of SARS-CoV-2 and the surface of Vero E6 cells at a multiplicity of infection of approximately 1 with great morphological detail. The absence of a conductive coating allows for a distinction between virus particles bound to the cell membrane and virus particles lying on top of the membrane. After prolonged imaging, it was found that ion-induced deposition of hydrocarbons from the vacuum renders the sample sufficiently conductive to allow for imaging even without charge compensation. The presented images demonstrate the potential of the HIM in bioimaging, especially for the imaging of interactions between viruses and their host organisms
Optimized protocol for Chelex-based extraction of DNA from historical skeletal remains and forensic trace samples
No full text© 2021 The Author. Published by Research Square. This is an open access article available under a Creative Commons licence.
The published version can be accessed at the following link on the publisher’s website: https://protocolexchange.researchsquare.com/article/pex-1652/v1PCR-based analysis of skeletonized human remains is a common aspect in both forensic human identification as well as Ancient DNA research. In this, both areas not merely utilize very similar methodology, but also share the same problems regarding quantity and quality of recovered DNA and presence of inhibitory substances in samples from excavated remains. To enable amplification based analysis of the remains, development of optimized DNA extraction procedures is thus a critical factor in both areas. The method paper here presents an optimized protocol for DNA extraction from ancient skeletonized remains using Chelex-100, with improved effectively in yielding amplifiable extracts from sample material excavated after centuries in a soil environment, which consequently have high inhibitor content and overall limited DNA preservation. Further studies showed that the optimized protocol can likewise be utilized for extraction of DNA from common and trace Forensic sample material
Optimized protocol for DNA extraction from ancient skeletal human remains using Chelex-100
No full text©2021 The Author. Published by AkiNik Publications. This is an open access article available under a Creative Commons licence.
The published version can be accessed at the following link on the publisher’s website: https://www.forensicpaper.com/archives/2021.v3.i1.A.35PCR-based analysis of skeletonized human remains is a common aspect in both forensic human identification as well as Ancient DNA research. In this, both areas not merely utilize very similar methodology, but also share the same problems regarding quantity and quality of recovered DNA and presence of inhibitory substances in samples from excavated remains. To enable amplification based analysis of the remains, development of optimized DNA extraction procedures is thus a critical factor in both areas. The study here presents an optimized protocol for DNA extraction from ancient skeletonized remains using Chelex-100, which proved to be effective in yielding amplifiable extracts from sample material excavated after centuries in a soil environment, which consequently have high inhibitor content and overall limited DNA preservation. Success of the optimization strategies utilized is shown in significantly improved amplification outcomes compared to the predecessor method
Amantadine Variant - Aryl Conjugates that Inhibit Multiple Amantadine Resistant M2 Mutant Influenza A Viruses
No full textOne challenge facing anti-influenza drug development is the heterogeneity of the circulating influenza A viruses, which comprise several strains with variable susceptibility to antiviral drugs. Viruses bearing the S31N mutant of the M2, such as the pandemic 2009 H1N1 and seasonal H3N2, as well as other mutants (L26F, V27A, A30T, G34E) are resistant to amantadine class of drugs. Here, we synthesized and tested many of the second generation amantadine - aryl conjugates, against the WT M2 and all the M2 amantadine resistant strains, i.e. L26F, V27A, S31N, A30T, G34E generated from WSN/33 (S31N) virus. We identified many compounds that are dual in vitro M2 WT and L26F virus inhibitors. Furthermore, few of them (21, 32, 33), having a rimantadine or diamantadine or 4-(1-adamantyl)aniline instead of amantadine in the conjugate, were in vitro inhibitors against M2 WT, L26F and S31N while one of them inhibited also the A30T virus. The electrophysiology (EP) experiments showed that these compounds blocked significantly M2 WT, L26F or even M2 V27A channels but not the M2 S31N. The observation that adamantane variants and derivatives inhibit multiple M2 mutant virus replication in cell culture, without blocking M2 channel-mediated proton current in EP is not uncommon, underlying a mechanism of antiviral activity that has not been identified
Low-density lipoprotein receptor–related protein 1 (LRP1) as an auxiliary host factor for RNA viruses
No full textAmantadine variant - aryl conjugates that inhibit multiple M2 mutant - amantadine resistant influenza a viruses
No full textInfluenza A viruses can cause a serious future threat due to frequent mutations. Amantadine and rimantadine inhibit influenza A M2 wild-type (WT) viruses by binding and blocking M2 WT channel-mediated proton current. The resistant to the drugs amantadine and rimantadine influenza A viruses bearing the S31 N mutant in the M2 proton channel can be inhibited by amantadine - aryl conjugates, in which amantadine and an aryl group are linked through a methylene, which block M2 S31 N channel-mediated proton current. However, the M2 amantadine/rimantadine resistant viruses bearing one of the four mutations L26F, V27A, A30T, G34E in residues that line the M2 protein pore pose an additional concern for public health. Here, we designed 33 compounds based on the structure of three previously published and potent amantadine-aryl conjugates against M2 S31 N virus, by replacing amantadine with 16 amantadine variants. The compounds were tested against M2 WT and the five M2 amantadine-resistant viruses aiming at identifying inhibitors against multiple M2 mutant - amantadine resistant viruses. We identified 16 compounds that inhibited in vitro two influenza A viruses with M2 WT or L26F channels. Additionally, compounds 21 or 32 or 33, which are conjugates of the rimantadine variant with CMe2 (instead of CHMe in rimantadine) or the diamantylamine or the 4-(1-adamantyl)benzenamine with the 2-hydroxy-4-methoxyphenyl aryl group, were in vitro inhibitors against three influenza A viruses with M2 WT or L26F or S31 N, while compound 21 inhibited also in vitro the M2 G34E virus and 32 inhibited also in vitro the M2 A30T virus. For these compounds we performed a preliminary drug metabolism and pharmacokinetics study. Also, using electrophysiology, we showed that compound 21 was an efficient blocker of the M2 WT and M2 L26F channels, compound 32 blocked efficiently the M2 WT channel and compound 33 blocked the M2 WT, L26F and V27A channels. The drug metabolism and pharmacokinetics studies showed these compounds need further optimization
Amantadine Variants Activity Against Multiple Influenza A Viruses
No full textFuture pandemic influenza necessitates the development of new drugs against the current circulating, amantadine and rimantadine drugs resistant, influenza A M2 S31N viruses. The possibility of an antigenic shift to M2 S31 necessitates ranking the biological activities of amantadine variants. Several amantadine variants have been tested by different laboratories, but various M2 wild type influenza A strains have been used with different sensitivity against amantadine and the unambiguous comparison between potencies is not straightforward. Here, we compared the anti-influenza activities of 57 synthetic amantadine variants against influenza A WSN/33 viruses with amantadine-sensitive M2 WT, with a range of over three digits providing a reference set of potencies for structure-activity relationships, and amantadine-resistant M2 S31N proteins (and observed no potent compounds). 17 compounds were selected and tested against M2 L26F, V27A, A30T, G34E viruses. We tested few reference compounds using electrophysiology and explored point mutations which both showed that M2 is the target of potent antiviral potency against the M2 WT, L26F, V27A viruses. Major findings are: (a) Several amantadine variants from Kolocouris group block only M2 WT and M2 L26F-mediated proton current and the corresponding viruses replication. (b) A compound from Vazquez’s group is a triple blocker of M2 WT, L26F, V27A channels and viruses replication. (c) A compound from Vazquez’s group blocks only M2 L26 channel and virus replication. (d) Several compounds from Kolocouris group have potent activity against several influenza A M2 WT and three M2 S31N viruses, eg. the pandemic A/H1N1/California/07/2009 (H1N1pdm09) or A/H1N1/PuertoRico/08/1934 without blocking M2 S31N. The compounds and their cocktails while not to be more toxic than amantadine might be useful for re-purposing of amantadine class of drugs in the case (i) of the prevalence of M2 L26F and or M2 V27A strains (ii) of an antigenic shift of the virus to M2 WT and (iii) because they inhibited a broad panel of M2 WT and M2 S31N viruses including the H1N1pdm09). (d) We showed that the mechanism of antiviral activity against A/California/07/2009 or A/PR/08/1934 and possibly also M2 WT viruses compared to WSN/33 viruses is not due to inhibition of an early stage of virus infection or a late stage of M2 channel function during endocytosis or inhibition of HA binding to host cells or a different pH for HA fusion or a lysosomotropic effect
