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Plasminogen activator inhibitor-1 (PAI-1/Serpin E1) is classically known for its antifibrinolytic activity via inhibiting uPA and tPA of the fibrinolytic pathway. PAI-1 has a paradoxical role in tumor progression, and its molecular functions are poorly understood. PAI-1 is a widely accepted secretory protease inhibitor, however, a study suggested the localization of PAI-1 in the cytoplasm and the nucleus. Besides the plethora of its biological functions as a secretory protein, intracellular localization, and functions of PAI-1 remain unexplored at the molecular level. In this study, using various in silico approaches, we showed that PAI-1 possesses a nuclear export signal. Using the CRM1-specific inhibitor leptomycin B, we demonstrated that PAI-1 has a functional CRM1-dependent NES, indicating the possibility of its nuclear localization. Further, we confirm that PAI-1 is localized in the nucleus of endothelial cells using fluorescence microscopy and immunoprecipitation. Notably, we identified an unconventional distribution of PAI-1 in the PML bodies of the nucleus of normal endothelial cells, while the protein was restricted in the cytoplasm of slow-growing cells. The data showed that the localization of PAI-1 in PML bodies is highly correlated with the growth potential of endothelial cells. This conditional nucleocytoplasmic shuttling of PAI-1 during the aging of cells could impart a strong link to its age-related functions and tumor progression. Together, this study identifies the novel behavior of PAI-1 that might be linked with cell aging and may be able to unveil the elusive role of PAI-1 in tumor progression.
RESUMEN
Background: Direct whole genome sequencing (WGS) of Mycobacterium tuberculosis (Mtb) can be used as a tool to study drug resistance, mixed infections, and within-host diversity. However, WGS is challenging to obtain from clinical samples due to low number of bacilli against a high background. Methods: We prospectively collected 34 samples (sputum, n = 17; bronchoalveolar lavage, n = 13; and pus, n = 4) from patients with active tuberculosis (TB). Prior to DNA extraction, we used a ligand-mediated magnetic bead method to enrich Mtb from clinical samples and performed WGS on Illumina platform. Results: Mtb was definitively identified based on WGS from 88.2% (30/34) of the samples, of which 35.3% (12/34) were smear negative. The overall median genome coverage was 15.2% (interquartile range [IQR], 7.7%-28.2%). There was a positive correlation between load of bacilli on smears and genome coverage (P < .001). We detected 58 genes listed in the World Health Organization mutation catalogue in each positive sample (median coverage, 85% [IQR, 61%-94%]), enabling the identification of mutations missed by routine diagnostics. Mutations causing resistance to rifampicin, isoniazid, streptomycin, and ethambutol were detected in 5 of 34 (14.7%) samples, including the rpoB S441A mutation that confers resistance to rifampicin, which is not covered by Xpert MTB/RIF. Conclusions: We demonstrate the feasibility of magnetic bead-based enrichment for culture-free WGS of Mtb from clinical specimens, including smear-negative samples. This approach can also be integrated with low-cost sequencing workflows such as targeted sequencing for rapid detection of Mtb and drug resistance.
RESUMEN
The pandemic of coronavirus disease 2019 (COVID-19) caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has affected millions of people and claimed thousands of lives. Starting in China, it is arguably the most precipitous global health calamity of modern times. The entire world has rocked back to fight against the disease and the COVID-19 vaccine is the prime weapon. Even though the conventional vaccine development pipeline usually takes more than a decade, the escalating daily death rates due to COVID-19 infections have resulted in the development of fast-track strategies to bring in the vaccine under a year's time. Governments, companies, and universities have networked to pool resources and have come up with a number of vaccine candidates. Also, international consortia have emerged to address the distribution of successful candidates. Herein, we summarize these unprecedented developments in vaccine science and discuss the types of COVID-19 vaccines, their developmental strategies, and their roles as well as their limitations.