Torrigiani, F. | Search bioRxiv

bioRxiv2023

NON-TOXIC ACID-FREE GLYOXAL FIXATIVE FOR VETERINARY HISTOPATHOLOGY, IMMUNOHISTOCHEMISTRY AND MOLECULAR ANALYSIS

Formaldehyde fixation is worldwide the most used system for histopathological examination. However, its toxicity is well known, and preservation of proteins and nucleic acids is not optimal. Alternative fixatives warranting similar morphological quality of tissues and costs, but lacking toxicity and allowing better preservation of proteins and nucleic acids would therefore increase both safety of operators and quality of molecular analysis in pathology. This multi-institutional study aimed to compare the morphological, histochemical, immunohistochemical (IHC), and molecular analyses outcomes of a newly patented, non-toxic, acid-free Glyoxal (GAF) fixative with neutral buffered formaldehyde (NBF). Tissues from a total of 73 subjects were analyzed, including 13 necropsies. Gross features were preserved after GAF fixation, with no tissue hardening or discoloration. Cellular ultrastructure was also better preserved with GAF and histology and histochemistry on GAF-fixed samples showed good results when compared to NBF-fixed samples, with the exception of loss of tinctorial affinity of erythrocytes and mast cell granules. IHC analyses also showed comparable results with only slight and rare protocol adjustment. DNA and RNA yields were higher from GAF-fixed samples (P<0.05) and the tested genes (p53 and COX1) were better amplified. RNA scope showed positive results for c-KIT expression in GAF-fixed mast cell tumors. Based on these data, the non-toxic GAF fixative allows good macroscopical, histological and immunohistochemical analyses of tissue samples, including on-field application, and better molecular analyses when compared to NBF. This represents a promising possibility for teaching, diagnostic, and research in veterinary pathology.

pathology↗

bioRxiv2023

Identification of druggable host dependency factors shared by multiple SARS-CoV-2 variants of concern

The high mutation rate of SARS-CoV-2 leads to emergence of several variants, some of which are resistant to vaccines and drugs targeting viral elements. Targeting host dependency factors - cell proteins required for viral replication - would help avoid resistance. However, whether different SARS-CoV-2 variants induce conserved cell responses and exploit the same core host factors is still unclear. We compared three variants of concern and observed that the host transcriptional response was conserved, differing only in kinetics and magnitude. By CRISPR screening we identified the host genes required for infection by each variant: most of the identified genes were shared by multiple variants, both in lung and colon cells. We validated our hits with small molecules and repurposed FDA-approved drugs. All drugs were highly effective against all tested variants, including delta and omicron, new variants that emerged during the study. Mechanistically, we identified ROS production as a pivotal step in early virus propagation. Antioxidant drugs, such as N-acetyl cysteine (NAC), were effective against all variants both in human lung cells, and in a humanised mouse model. Our study supports the use of available antioxidant drugs, such as NAC, as a general and effective anti-COVID-19 approach.

microbiology↗