SARS-CoV-2 receptor binding domain (RBD) recognizes the angiotensin converting enzyme 2 (ACE2) receptor in host cells that enables infection. Due to its antigenic specificity, RBD production is important for development of serological assays. Here we have established a system for stable RBD production in HEK 293T mammalian cells that simultaneously express the recombinant fluorescent protein dTomato, which enables kinetic monitoring of RBD expression by fluorescence microscopy. In addition, we have validated the use of this recombinant RBD in an ELISA assay for the detection of anti-RBD antibodies in serum samples of COVID-19 convalescent patients. Recombinant RBD generated using this approach can be useful for generation of antibody-based therapeutics against SARS-CoV-2, as well serological assays aimed to test antibody responses to this relevant virus.

Coffee plants and beans are prone to fungal contamination that pose health risks to consumers by producing mycotoxins like ochratoxin A (OTA). Thus, the present study aimed to analyze the mycobiota of Costa Rican coffee beans, focusing on potentially ochratoxigenic species and their in vitro susceptibility patterns to antifungal agents. Fungal isolates were obtained from cherry, green, and roasted coffee beans from Costa Rica; they were identified by morphology, MALDI-TOF technology, and sequencing. The isolation frequency (FR) was 33.10% for all the samples analyzed, 49.51% for the cherry fruits, 37.67% for the green coffee beans, and 17.33% for the roasted beans. The cherry beans were mainly contaminated with Geotrichum klebahni (46.34% FR and 90.91% relative density (RD)), while the green and roasted coffee beans were mainly infected with Aspergillus spp. (22.00% FR and 55.23% RD and 13.83% FR and 77.57% RD, respectively). A total of 46.67% of A. westerdijkiae and 20.00% of A. ochraceus produced fluorescence in YES broth related with ochratoxin production. The isolates of the Aspergillus section Circumdati were susceptible to the azole antifungals. Costa Rican coffee beans could be contaminated with mycotoxigenic fungi during storage.

Neutralization assays have become an important tool since the beginning of the COVID-19 pandemic for testing vaccine responses and therapeutic antibodies as well as for monitoring humoral immunity to SARS-CoV-2 in epidemiological studies. The spike glycoprotein (S) present on the viral surface contains a receptor binding domain (RBD) that recognizes the angiotensin-converting enzyme 2 receptor (ACE2) in host cells, allowing virus entry. The gold standard for determining SARS-CoV-2 neutralizing antibodies is the plaque reduction neutralization test (PRNT), which relies on live-virus replication performed exclusively in biosafety level 3 (BSL-3) laboratories. Here, we report the development of a surrogate live-cell imaging-based fluorescent SARS-CoV-2 neutralization assay, applicable to BSL-1 or BSL-2 laboratories, by antibody-mediated blockage of the interaction between recombinant RBD with overexpressed ACE2 receptor in a genetically modified HEK 293T stable cell line. Our approach was able to detect neutralizing antibodies both in COVID-19-positive human serum samples and polyclonal equine formulations against SARS-CoV-2. This new cell-based surrogate neutralization assay represents a virus-free fluorescence imaging alternative to the reported approaches, which can be used to detect antibody-neutralizing capabilities toward SARS-CoV-2. This assay could also be extrapolated in the future to other established and emergent viral agents.

Phospholipases A ₂ (PLA ₂ s) are a major component of snake venoms. Some of them cause severe muscle necrosis through a still unknown mechanism. Phospholipid hydrolysis is a possible explanation of their toxic action, but catalytic and toxic properties of PLA ₂ s are not directly connected. In addition, viperid venoms contain PLA ₂ -like proteins, which are very toxic even if they lack catalytic activity due to a critical mutation in position 49. Nucleolin, a main component of the nucleolus, is a disordered protein involved in many protein assembly and phase separation phenomena. In some circumstances nucleolin is exposed on the cell surface from where it is involved in the internalization of many ligands. In this work we demonstrate that Bothrops asper myotoxin II (Mt-II), a Lys49 PLA ₂ -like toxin, interacts with, and is internalized in cells by nucleolin. The internalization process is functional to the toxicity of the protein, as both an antibody and an aptamer specific for nucleolin protect cells from intoxication. We identified central RRM and the C-terminal R/F-GG domain of nucleolin as the regions involved in the interaction with Mt-II. Finally we observed that Mt-II forms, on the cell surface, amyloid-like assemblies that colocalize with nucleolin and that can be involved in the activation of the internalization process. The presence, in the three dimensional structure of Mt-II and related PLA ₂ homologues, of four exposed loops enriched in prion-like amino acid sequences reinforces this hypothesis. Phospholipases A ₂ | Lys49 myotoxins | nucleolin | amyloid-like | molecular assemblies SIGNIFICANCE: The main finding of this work, the role of nucleolin as Bothrops asper Mt-II receptor, is a remarkable step forward in understanding the mechanism of action of cytotoxic PLA ₂ s. It may suggest new strategies for anti-venom therapies and explain the anti-tumoral and anti-viral pharmacological action of snake PLA ₂ s, since nucleolin is a receptor for many growth factors and virus. The proposed internalization mechanism, via formation of molecular assemblies among Mt-II amyloid-like structures and other proteins, including nucleolin, can be of general validity. Cell surface molecular assemblies couldbepointsofselectionandconcentrationnotonlyofsnake,butalsoofmammaliansecretedPLA ₂ s, proteins involved in different pathologies, and trigger the internalization pathway only when their molarity exceeds a threshold dose.