ABSTRACT
BackgroundThe SARS-CoV-2 pandemic necessitated rapid and global responses across all areas of healthcare, including an unprecedented interest in serological immunoassays to detect antibodies to the virus. The dynamics of the immune response to SARS-CoV-2 is still not well understood and requires further investigation into the longevity of humoral immune response that is evoked due to SARS-CoV-2 infection. MethodsWe measured SARS-CoV-2 antibody levels in plasma samples from 880 people in Northern Ireland using Roche Elecsys Anti-SARS-CoV-2 IgG/IgA/IgM, Abbott SARS-CoV-2 IgG and EuroImmun IgG SARS-CoV-2 ELISA immunoassays to analyse immune dynamics over time. We undertook a laboratory evaluation for the UK-RTC AbC-19 rapid lateral flow immunoassay (LFIA), for the target condition of SARS-CoV-2 Spike protein IgG antibodies using a reference standard system to establish a characterised panel of 330 positive and 488 negative SARS-CoV-2 IgG samples. ResultsWe detected persistence of SARS-CoV-2 IgG up to 140 days (20 weeks) post infection, across all three laboratory-controlled immunoassays. On the known positive cohort, the UK-RTC AbC-19 lateral flow immunoassay showed a sensitivity of 97.58% (95.28%-98.95%) and on known negatives, showed specificity of 99.59% (98.53 %-99.95%). ConclusionsThrough comprehensive analysis of a cohort of pre-pandemic and pandemic individuals, we show detectable levels of IgG antibodies, lasting up to 140 days, providing insight to antibody levels at later time points post infection. We show good laboratory validation performance metrics for the AbC-19 rapid test for SARS-CoV-2 Spike protein IgG antibody detection in a laboratory based setting.
ABSTRACT
CRISPR/Cas9 holds immense potential to treat a range of genetic disorders. Allele-specific gene disruption induced by non-homologous end-joining (NHEJ) DNA repair offers a potential treatment option for autosomal dominant disease. Here, we successfully delivered a plasmid encoding S. pyogenes Cas9 and sgRNA to the corneal epithelium by intrastromal injection and acheived long-term knockdown of a corneal epithelial reporter gene, demonstrating gene disruption via NHEJ in vivo. In addition, we used TGFBI corneal dystrophies as a model of autosomal dominant disease to assess the use of CRISPR/Cas9 in two allele-specific systems, comparing cleavage using a SNP-derived PAM to a guide specific approach. In vitro, cleavage via a SNP-derived PAM was found to confer stringent allele-specific cleavage, while a guide-specific approach lacked the ability to distinguish between the wild-type and mutant alleles. The failings of the guide-specific approach highlights the necessity for meticulous guide design and assessment, as various degrees of allele-specificity are achieved depending on the guide sequence employed. A major concern for the use of CRISPR/Cas9 is its tendency to cleave DNA non-specifically at "off-target" sites. Confirmation that S. pyogenes Cas9 lacks the specificity to discriminate between alleles differing by a single base-pair regardless of the position in the guide is demonstrated.
Subject(s)
Clustered Regularly Interspaced Short Palindromic Repeats/genetics , Gene Editing/methods , Alleles , Animals , CRISPR-Cas Systems/genetics , CRISPR-Cas Systems/physiology , Corneal Dystrophies, Hereditary/genetics , Corneal Dystrophies, Hereditary/therapy , DNA End-Joining Repair/genetics , Mice , Mice, Mutant Strains , Mutation/genetics , Streptococcus pyogenes/enzymologyABSTRACT
<p><b>BACKGROUND</b>Histone deacetylase inhibitors (HDACIs) have been reported to induce apoptosis in cancer cells. The effects of trichostatin A (TSA) on gastric cancer cells have not been well characterized. This study was aimed to explore the effects and mechanisms of TSA on human gastric cancer SGC-7901 cells.</p><p><b>METHODS</b>The cells were treated with TSA and analyzed by cell proliferation assay, Western blot, TUNEL assay, flow cytometry by fluorescein isothiocyanate (FITC) conjugated with Annexin V and PI staining, immunofluorescence analysis, analysis of subcellular fractionation, gene chips and real time polymerase chain reaction (PCR).</p><p><b>RESULTS</b>TSA could inhibit cell growth and induced apoptosis in gastric cancer SGC-7901 cells through the regulation of apoptosis-related genes, such as Bcl-2, Bax and survivin. Further study indicated that the pan-caspase inhibitor z-VAD-fmk did not inhibit the apoptosis induced by TSA, and we did not observe the cleavage of poly ADP ribose polymerase (PARP) after TSA treatment too. In addition, apoptosis inducing factor (AIF) and EndoG were found to translocate from mitochondria to nucleus in the immunofluorescence assay and the Western analysis of subcellular fractionation confirmed the result of immunofluorescence assay.</p><p><b>CONCLUSIONS</b>The apoptosis induced by TSA in gastric cancer SGC-7901 cells involves a caspase-independent pathway.</p>
