Biology subjects

Delgado, R.

Publications and source records attributed to Delgado, R..

9 recordsLinked to original sources

bioRxiv2026

Scarless SARS-CoV-2 Genome Engineering and Variant Analysis

In addition to causing cold and flu-like symptoms, Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) can also cause chronic longer-term diseases. Antiviral drugs, especially used combinatorially, have the potential to reduce the severity of individual infections and prevent the development of chronic disease. One of the safest and most versatile reverse genetics systems for SARS-CoV-2 studies is a bacterial artificial chromosome (BAC)-based system harboring the WA1 strain full-length genome and attenuating deletions in the accessory open reading frame 3a and 7b proteins (ORF3a and ORF7b, respectively). Here, a scarless genome engineering technique called En Passant mutagenesis was used to change one amino acid in the viral main protease (Mpro P132) into the residue present in contemporary Omicron strains (H132), in order to more accurately study protease inhibitors and resistance mechanisms. This recombinant, attenuated viral system yields antiviral EC50 values for the active component of approved drugs including nirmatrelvir (Paxlovid) and ensitrelvir (Xocova) and, importantly, also enables a parallel assessment of drug efflux. For instance, the antiviral potency of nirmatrelvir improves 50-fold by inhibiting the P-Glycoprotein (P-Gp) transporter with ritonavir or tariquidar, whereas the potency of ensitrelvir is unaffected. This system also enables the safe isolation and characterization of viral variants with reduced sensitivity to drugs, as evidenced by Mpro M49L compromising the efficacy of ensitrelvir. Together, these systems combine to provide safe, reliable, and quantitative approaches for Mpro variant analysis and drug testing without the biosafety concerns of conducting these experiments using wildtype isolates.

microbiology↗

bioRxiv2025

Variation at the R181 residue of p53 confers loss of p53 DNA binding cooperativity with the retention of mitochondrial-associated apoptosis

The p53 tumor suppressor binds DNA cooperatively as a tetramer, mediated by salt-bridge interactions between p53 residues E180 and R181. Variants at the R181 residue are one of the most identified TP53 pathogenic variants by germline genetic testing. We show that families with TP53 p.R181H and p.R181C variants have an attenuated cancer risk phenotype compared to patients with hotspot dominant negative loss of function TP53 variants. Despite this phenotype, we find that p53 R181H and R181C variants have significantly reduced ability to bind to p53 promoter/enhancer target sequences and transactivate p53 target genes, similar to null variants. However, p53 R181H and R181C retain wild-type p53 structure and tetramerization. In addition, R181-mutant cells undergo apoptosis through wild-type p53 activity at the mitochondria. These results indicate that retention of transcription-independent p53 tumor suppressor function results in a reduced penetrance cancer risk syndrome in humans. Statement of SignificanceInherited TP53 variants cause Li-Fraumeni Syndrome (LFS), which has significant phenotypic heterogeneity in lifetime cancer risk. Our study shows that retention of transcription-independent p53 functions, despite loss of p53 transactivation activity, results in a reduced penetrance phenotype. Classification of TP53 variants should incorporate assays of transcriptional and non-transcriptional functions.

cancer biology↗

bioRxiv2024

Partial repair causes permanent defects in papillary structure and function after reversal of urinary obstruction

Urinary obstruction causes injury to the renal papilla and leads to defects in the ability to concentrate urine which predisposes to progressive kidney injury. However, the regenerative capacity of the papilla after reversal of obstruction is poorly understood. To address this, we developed a mouse model of reversible urinary obstruction which is characterized by extensive papillary injury, followed by a robust regeneration response and complete histological recovery over a 3- month period. However, these mice have a pronounced defect in urinary concentrating capacity. We now show that this is due to permanent changes in the composition, organization, and transcriptional signatures of epithelial, endothelial, and interstitial cell lineages in the papilla. There are persistent inflammatory responses that are also seen in patients with renal stone disease but are associated with cell-specific adaptive responses to the increasingly hypoxic environment of the papilla after reversal of obstruction. Taken together, our analysis of a new model of reversible urinary obstruction reveals that partial repair leads to permanent changes in the structure and function of all of the major cellular compartments in the papilla that include both shared and distinct responses to different types of renal papillary injury in humans and mice. SummaryPartial repair after reversal of urinary obstruction leads to permanent changes in structure and function of all major cellular compartments in the renal papilla O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=145 SRC="FIGDIR/small/612436v1_ufig1.gif" ALT="Figure 1"> View larger version (57K): org.highwire.dtl.DTLVardef@1438af5org.highwire.dtl.DTLVardef@157ff60org.highwire.dtl.DTLVardef@3f09c0org.highwire.dtl.DTLVardef@9950b1_HPS_FORMAT_FIGEXP M_FIG C_FIG

pathology↗

bioRxiv2024

Re-programming of GM-CSF-dependent alveolar macrophages through GSK3 activity modulation

Monocyte-derived macrophages recruited into inflamed tissues can acquire an array of functional states depending on the extracellular environment. Since the anti-inflammatory/pro-fibrotic macrophage profile is determined by MAFB, whose activity/protein levels are regulated by GSK3, we addressed the macrophage re-programming potential of GSK3 modulation. GM-CSF-dependent (GM-MO) and M-CSF-dependent monocyte-derived macrophages (M-MO) exhibited distinct levels of inactive GSK3, and inhibiting GSK3 in GM-MO led to acquisition of transcriptional, phenotypic and functional properties characteristic of M-MO (enhanced expression of IL-10 and monocyte-recruiting factors, and higher efferocytosis). These re-programming effects were also observed upon GSK3/{beta} knockdown, and through GSK3 inhibition in ex vivo isolated human alveolar macrophages (AMO). Notably, GSK3 downmodulation potentiated the transcriptional signature of Interstitial Macrophages (IMO) while suppressed the AMO-specific gene profile. Indeed, heightened levels of inactive GSK3 and MAFB-dependent proteins were observed in severe COVID-19 patients lung macrophages, highlighting the GSK3-MAFB axis as a therapeutic target for macrophage re-programming.

immunology↗

bioRxiv2022

Potent Neutralizing Activity of Polyclonal Equine Antibodies against Omicron SARS-CoV-2

Using a polyclonal approach of equine anti-SARS-CoV-2 F(ab)2 antibodies we have achieved a high level of neutralizing potency against all SARS-CoV-2 variants tested. Neutralization titers were in the range of 105-106 IU/mL including Omicron: 111,403 UI/mL, which is 2-3 orders of magnitude what is normally achieved in response to SARS-CoV-2 infection and/or vaccination. The presence of high titers of a repertoire of antibodies targeting conserved epitopes in different regions of the spike protein could plausibly account for this remarkable breadth of neutralization. These results warrant the clinical investigation of anti-SARS-CoV-2 equine polyclonal F(ab)2 antibodies as a novel therapeutic strategy against COVID-19

microbiology↗

bioRxiv2021

Endosomal proteins NPC1 and NPC2 at African swine fever virus entry/fusion

African swine fever virus (ASFV) infectious cycle starts with the viral adsorption and entry into the host cell. The virus is internalized via clathrin/dynamin mediated endocytosis and macropinocytosis. As several other viruses, ASF virion is then internalized and incorporated into the endocytic pathway. Endosomal maturation entails luminal acidification and the lowering of pH acting on the multi-layered virion structure dissolves the outer capsid. Upon decapsidation, the inner viral membrane is exposed to interact with the limiting membrane of the late endosome for fusion. Egress from endosome is related to cholesterol efflux, but it remains an intriguing process albeit essential for infection, specifically for the viral nucleic acid exit to the cytoplasm for replication. ASFV proteins E248R and E199L, with structural homology to the VACV proteins of the fusion complex, seem to have similar functions in ASFV. A direct interaction between these ASFV proteins with the cholesterol transporter protein NPC1 (Niemann-Pick C type 1) was observed, which was also shared by the E248R homologous protein L1R of VACV. Binding occurs between the transmembrane domain of E248R with the loop C of NPC1 at the same domain than EBOV binding site. These interactions suggest that these ASFV proteins are crucial for membrane fusion. CRISPR NPC1 KO Vero cells lacking NPC1 protein that were resistant to EBOV, reduced ASFV infection levels significantly. Reductions on ASFV infectivity and replication in NPC1 KO cells were accompanied by lesser viral factories of smaller size and lacking the typical cohesive morphology between endosomes and viral proteins. We observed a compensatory effect in NPC1 KO cells, elevating NPC2 levels while silencing NPC2 in Vero cells with shRNA, also reduced ASFV infection. Our findings pave the way to understand the role of these proteins at the membrane viral fusion step for several viruses. Author SummaryAfrican swine fever virus (ASFV) causes a deadly disease of pigs and wild boars that was endemic in Africa but have extended over the last years to Europe, Asia and Oceania with high socioeconomic impact. ASFV enters the cell by endocytosis and has adapted to the endosomal conditions to acquire infectivity. Viral infectivity is dependent on cholesterol traffic at the endosomes, especially at the fusion step. Fusion of the internal viral membrane with the endosomal membrane is required for the exit of the viral DNA to the cytoplasm to start replication. ASF virion internal membrane proteins E248R and E199L were found to bind the Niemann Pick C1 (NPC1) receptor at the endosome. These proteins are highly conserved among ASFV isolates and resemble proteins of the VACV entry/fusion complex. The function of NPC1 is to regulate the efflux of dietary cholesterol efflux from the endosome to the endoplasmic reticulum, which appears to be necessary for viral fusion. NPC1 knockout cells by CRISPR reduced infection affecting infectivity and early replication. Also, removing the associated endosomal protein NPC2, further declined infectivity. These results show the relevance of NPC1 receptor in the viral infection actually shared by unrelated important viral families.

microbiology↗

bioRxiv2021

Monitoring the effects of chemical stimuli on live cells with metasurface-enhanced infrared reflection spectroscopy

Infrared spectroscopy has found wide applications in the analysis of biological materials. A more recent development is the use of engineered nanostructures - plasmonic metasurfaces - as substrates for metasurface-enhanced infrared reflection spectroscopy (MEIRS). Here, we demonstrate that strong field enhancement from plasmonic metasurfaces enables the use of MEIRS as a highly informative analytic technique for real-time monitoring of cells. By exposing live cells cultured on a plasmonic metasurface to chemical compounds, we show that MEIRS can be used as a label-free phenotypic assay for detecting multiple cellular responses to external stimuli: changes in cell morphology, adhesion, lipid composition of the cellular membrane, as well as intracellular signaling. Using a focal plane array detection system, we show that MEIRS also enables spectro-chemical imaging at the single-cell level. The described metasurface-based all-optical sensor opens the way to a scalable, high-throughput spectroscopic assay for live cells.

bioengineering↗

bioRxiv2020

DC/L-SIGN recognition of spike glycoprotein promotes SARS-CoV-2 trans-infection and can be inhibited by a glycomimetic antagonist

The efficient spread of SARS-CoV-2 resulted in a pandemic that is unique in modern history. Despite early identification of ACE2 as the receptor for viral spike protein, much remains to be understood about the molecular events behind viral dissemination. We evaluated the contribution of C-type lectin receptors (CLRS) of antigen-presenting cells, widely present in air mucosa and lung tissue. DC-SIGN, L-SIGN, Langerin and MGL bind to diverse glycans of the spike using multiple interaction areas. Using pseudovirus and cells derived from monocytes or T-lymphocytes, we demonstrate that while virus capture by the CLRs examined does not allow direct cell infection, DC/L-SIGN, among these receptors, promote virus transfer to permissive ACE2+ cells. A glycomimetic compound designed against DC-SIGN, enable inhibition of this process. Thus, we described a mechanism potentiating viral capture and spreading of infection. Early involvement of APCs opens new avenues for understanding and treating the imbalanced innate immune response observed in COVID-19 pathogenesis

immunology↗

bioRxiv2019

Glycosylation-dependent enhanced cell-binding and infectivity through DC-SIGN in the West African Ebola virus Makona

Since its discovery in Zaire in 1976, most ebolavirus outbreaks described occurred mainly in remote and poorly communicated areas of Central Africa and affected a limited number of individuals. Nonetheless, the Ebola epidemic that began in West Africa at the end of 2013 spread rapidly and reached an unprecedented scale. This epidemic was caused by the Makona variant of Zaire ebolavirus (EBOV). Monitoring of the EBOV Makona evolution throughout the epidemic identified the A82V substitution in the EBOV glycoprotein (GP) at the beginning of the epidemic, which correlated with its rapid spread. The Makona GP-V82 variant exhibits slightly higher human and primate cell infectivity. Host factors responsible for the enhanced transmission of EBOV Makona have yet to be identified. Here we show that the GP A82V substitution increases EBOV avidity for its DC-SIGN lectin receptor, which enhances cell-binding and infectivity of dendritic cells and macrophages, the primary cell subsets infected during the initial stages of the disease. Using a pseudotype lentivirus system, we identified two GP N-linked glycosylation responsible of the augmented Makona GP-V82 cell infection. Crystal structures indicated how the GP A82V substitution drives exposure of a key glycan and facilitates lectin recognition. Thus, DC-SIGN might be an important host determinant for the unique dissemination of EBOV during the 2013-2016 epidemic, which likely promoted host-to-host transmission by enhancing viral infection of dendritic cells in the skin and mucosa. This study also reveals that variations in virus envelope glycosylations can be a common pathway for virus adaptation to human transmission.\n\nSynopsisThe Zaire ebolavirus (EBOV) Makona, responsible of the largest outbreak of Ebola virus disease (EVD) in West Africa from 2013-2016, displayed a glycosylation-dependent enhancement of binding and subsequent infectivity in cells expressing the DC-SIGN surface lectin.

microbiology↗