Ugalde, A. P. | Search bioRxiv

bioRxiv2026

FAβ-gal: an automated fluorescence-based quantification of the senescence-associated beta-galactosidase X-gal assay

Cellular senescence plays a pivotal role in aging and cancer, two major biomedical and socioeconomic challenges of our time. Therefore, its study has become crucial for the design of interventions based on its manipulation. In this sense, researchers have developed a wide variety of techniques to detect and quantify cellular senescence. Among them, the most popular is the original Senescence-Associated {beta}-galactosidase (SA-{beta}-gal) colorimetric assay, based on the use of the chromogenic substrate X-gal. This compound is cleaved by {beta}-galactosidase, producing an insoluble, blue precipitate of 5,5-dibromo-4,4-dichloro-indigo (commonly referred to as indigo). While this method remains the gold standard senescence assay, its quantification remains challenging due to the color-based readout. In this work, we describe a method, which we have named FA{beta}-gal (Fluorescence Analysis of {beta}-galactosidase), that exploits the far-red fluorescence of the {beta}-gal product indigo and allows the quantification of SA-{beta}-gal activity under any conventional wide-field fluorescence microscopy using the original X-gal assay. In addition, we developed workflows and software applications that standardize SA-{beta}-gal quantification in a semiautomatic and unbiased manner. We demonstrate that FA{beta}-gal measurements present a strong linear correlation with the percentage of senescent cells and show high sensitivity. Moreover, we show that this method is also applicable to tissue sections, underscoring the versatility of our approach. Therefore, FA{beta}-gal could be easily introduced into the routine of laboratories already using the original colorimetric assay, enhancing the accuracy, sensitivity and reproducibility of senescence detection.

cell biology↗

bioRxiv2023

Host factor PLAC8 is required for pancreas infection by SARS-CoV-2

BackgroundAlthough COVID-19 initially caused great concern about respiratory symptoms, mounting evidence shows that also the pancreas is productively infected by SARS-CoV-2. However, the severity of pancreatic SARS-CoV-2 infection and its pathophysiology are still under debate. Here we investigated the consequences of SARS-CoV-2 pancreatic infection and the role of the host factor Placenta-associated protein (PLAC8) MethodsWe analyzed plasma levels of pancreatic enzymes and inflammatory markers in a retrospective cohort study of 120 COVID-19 patients distributed in 3 severity-stratified groups. We studied the expression of SARS-CoV-2 and PLAC8 in the pancreas of deceased COVID-19 patients as well as in non-infected donors. We performed infection experiments in PLAC8 knock-out PDAC cell lines with full SARS-CoV-2 virus. ResultsWe found that analysis of circulating pancreatic enzymes aided the stratification of patients according to COVID-19 severity and predict outcomes. Interestingly, we found an association between PLAC8 expression and SARS-CoV-2 infection in postmortem analysis of COVID-19 patients. Using full SARS-CoV-2 infectious virus inoculum from Wuhan-1 and BA.1 strains, we demonstrated that PLAC8 is necessary for productive infection of PDAC cell lines. Finally, we observed an overlap between PLAC8 and SARS-CoV-2 immunoreactivities of the pancreas of deceased patients. ConclusionOur data indicate the human pancreas as a SARS-CoV-2 target with plausible signs of injury and demonstrate that the host factor PLAC8 is required for SARS-CoV-2 pancreatic infection, thus defining new target opportunities for COVID-19-associated pancreatic pathogenesis. Plain language summaryPrevious studies have shown that the pancreas is infected by SARS-CoV-2. However, none of these studies have described measurable pancreatic damage associated to COVID-19 severity and the pathogenesis of pancreatic SARS-CoV-2 infection remains largely unknown. Novel host factors have been proposed for SARS-CoV-2 infection of mainly the airway epithelium, none of them studied in the pancreas. Our study shows clinically relevant pancreatic damage associated with SARS-CoV-2 infiltration and assesses the predictive potential of circulating pancreatic enzymes to stratify patients according to COVID-19 severity and predict clinical outcomes in a cohort of 120 patients. Our data show that host factor Placenta-associated protein 8 (PLAC8) expression is linked to SARS-CoV-2 infection in postmortem analysis of COVID-19 patients and functionally demonstrated the full requirement of PLAC8 for SARS-CoV-2 pancreatic infection and viral replication. Our data confirm the human pancreas as a SARS-CoV-2 target with signs of injury unveiling the measurement of pancreatic enzymes for prognosis value and demonstrating that host factor PLAC8 is required for SARS-CoV-2 pancreatic infection defining new stratification and target opportunities for COVID-19-associated pancreatic pathogenesis.

cell biology↗