Biology subjects

Duarte, E. M.

Publications and source records attributed to Duarte, E. M..

6 recordsLinked to original sources

bioRxiv2026

Dengue virus-specific memory B cell subsets differ as a function of infection history

The four dengue virus serotypes (DENV1-4) are a major global health threat. Infection generates protective immunity over multiple exposures with different serotypes. Virus-specific memory B cells (MBCs) can contribute to lasting protection, yet their development over multiple DENV infections remains undefined. We comprehensively evaluated frequencies of nine DENV-specific B cell subsets in 58 samples from dengue cases, comparing groups with primary (1{degrees}) versus secondary (2{degrees}) DENV infection history. Longitudinal sampling from acute infection to 18 months post-symptom onset enabled assessment of DENV-specific MBC temporal dynamics. Critically, we found that DENV-specific B cell frequency differed substantially at the level of phenotypic subsets in 1{degrees} versus 2{degrees} immunity, despite no significant difference in total frequency of DENV-specific B cells. In particular, DENV-specific IgG+, IgM+, atypical, and class-switched IgD-MBCs were durable until 18 months and accumulated with multiple exposures, representing a bona fide memory compartment against DENV. Also, naive-like IgD+/IgM+ DENV-specific B cells were found. Interestingly, the peak of certain DENV-specific MBCs occurred >3-months post-symptom onset in 2{degrees} DENV immunity, suggesting potential for long-term MBC maturation. We demonstrate that DENV-specific MBC subsets differ as a function of infection history, suggesting that 2{degrees} DENV immunity does not simply generate a quantitative boost, but a qualitative reprogramming of the memory pool. Significance StatementThe four dengue virus serotypes (DENV1-4) cause the most prevalent human mosquito-borne viral disease. Dengue is a febrile disease that often results in debilitating body pain and can rapidly progress to severe disease involving shock. People are generally protected after multiple exposures to different serotypes. Memory B cells (MBCs) can contribute to lasting protection against subsequent dengue. To understand how these rare DENV-specific MBCs develop over multiple exposures, we compared samples from cases with primary versus secondary (i.e., multiple) DENV infections. We found that instead of a higher frequency of total DENV-specific MBCs, particular subsets of DENV-specific MBCs were higher and peaked later after multiple exposures. This suggests that a qualitative shift in DENV-specific MBCs may contribute to protective immunity.

immunology↗

bioRxiv2025

Dengue Virus NS1 Binds Ephrin B1 to Trigger Endothelial Dysfunction

Dengue virus (DENV), a member of the Flaviviridae family, is the most prevalent and medically important mosquito-borne viral pathogen. Infected cells secrete the viral non-structural protein 1 (NS1) into the bloodstream, where it can interact with endothelial cells to induce vascular leak. Host factors involved in NS1-mediated endothelial dysfunction are incompletely understood, leading us to investigate the host interactome of DENV NS1 in endothelial cells via a comparative mass spectrometry approach. We identified ephrin B1 (EFNB1) as a critical host factor in NS1-mediated endothelial barrier dysfunction and show that phosphorylation of EFNB1 is necessary for induction of barrier dysfunction. Further, we map the interface of the EFNB1-NS1 complex through biochemical and computational approaches, and we show that EFNB1-Fc-fusion proteins can act as decoys to block NS1-induced barrier dysfunction in vitro and in vivo. This study provides insights into the mechanism of flavivirus NS1-mediated endothelial barrier dysfunction and new avenues to target vascular leak.

microbiology↗

bioRxiv2021

Colonic Oxygen Microbubbles Augment Systemic Oxygenation and CO2 Removal in a Porcine Smoke Inhalation Model of Severe Hypoxia

Inhalation injury can lead to pulmonary complications resulting in the development of respiratory distress and severe hypoxia. Respiratory distress is one of the major causes of death in critically ill patients with a reported mortality rate of up to 45%. The present study focuses on the effect of oxygen microbubble (OMB) infusion via the colon in a porcine model of smoke inhalation-induced lung injury. Juvenile female Duroc pigs (n=6 colonic OMB, n=6 no treatment) ranging from 39-51 kg in weight were exposed to smoke under general anesthesia for 2 h. Animals developed severe hypoxia 48 h after smoke inhalation as reflected by reduction in SpO2 to 66.3 % {+/-} 13.1% and PaO2 to 45.3 {+/-} 7.6 mmHg, as well as bilateral diffuse infiltrates demonstrated on chest x-ray. Colonic OMB infusion (75 - 100 mL/kg dose) resulted in significant improvements in systemic oxygenation as demonstrated by an increase in PaO2 of 13.2 {+/-} 4.7 mmHg and SpO2 of 15.2% {+/-} 10.0% out to 2.5 h, compared to no-treatment control animals that experienced a decline in PaO2 of 8.2 {+/-} 7.9 mmHg and SpO2 of 12.9% {+/-} 18.7% over the same timeframe. Likewise, colonic OMB decreased PaCO2 and PmvCO2 by 19.7 {+/-} 7.6 mmHg and 7.6 {+/-} 6.7 mmHg, respectively, compared to controls that experienced increases in PaCO2 and PmvCO2 of 17.9 {+/-} 11.7 mmHg and 18.3 {+/-} 11.2 mmHg. We conclude that colonic OMB therapy has potential to treat patients experiencing severe hypoxemic respiratory failure. One Sentence SummaryEnteral oxygen microbubbles increase systemic oxygen and decrease carbon dioxide levels in acutely hypoxic pigs after smoke inhalation-induced respiratory failure.

bioengineering↗

bioRxiv2021

Arabidopsis ROOT UV-B SENSITIVE 1 and 2 Interact with Aminotransferases to Regulate Vitamin B6 Homeostasis

Pyridoxal-5-phosphate (PLP), the enzymatic cofactor form of Vitamin B6 (vitB6), is a versatile compound that has essential roles in metabolism. Cellular PLP homeostasic regulation is currently not well understood. Here we report that in Arabidopsis, biosynthesized PLP is sequestered by specific aminotransferases (ATs), and that the proteins ROOT UV-B SENSITIVE 1 (RUS1) and RUS2 function with ATs to regulate PLP homeostasis. The stunted growth phenotypes of rus1 and rus2 mutants were previously shown to be rescuable by exogenously supplied vitB6. Specific residue changes near the PLP-binding pocket in ASPARTATE AMINOTRANSFERASE2 (ASP2) also rescued rus1 and rus2 phenotypes. In this study, saturated suppressor screens identified 14 additional suppressor of rus (sor) alleles in four aminotransferase genes (ASP1, ASP2, ASP3, or ALANIN AMINOTRANSFERASE1 (AAT1)), which suppressed the rus phenotypes to varying degrees. Each of the sor mutations altered an amino acid in the PLP-binding pocket of the protein, and sor proteins were found to have reduced levels of PLP conjugation. Genetic data revealed that the availability of PLP normally requires both RUS1 and RUS2, and that increasing the number of sor mutants additively enhanced the suppression of rus phenotypes. Biochemical results showed that RUS1 and RUS2 physically interacted with ATs. Our studies suggest a mechanism in which RUS1, RUS2 and specific ATs work together to regulate PLP homeostasis in Arabidopsis.

genetics↗

bioRxiv2020

Sensitive, highly multiplexed sequencing of microhaplotypes from the Plasmodium falciparum heterozygome

BackgroundTargeted next generation sequencing offers the potential for consistent, deep coverage of information rich genomic regions to characterize polyclonal Plasmodium falciparum infections. However, methods to identify and sequence these genomic regions are currently limited. MethodsA bioinformatic pipeline and multiplex methods were developed to identify and simultaneously sequence 100 targets and applied to dried blood spot (DBS) controls and field isolates from Mozambique. For comparison, WGS data were generated for the same controls. ResultsUsing publicly available genomes, 4465 high diversity genomic regions suited for targeted sequencing were identified, representing the P. falciparum heterozygome. For this study, 93 microhaplotypes with high diversity (median HE = 0.7) were selected along with 7 drug resistance loci. The sequencing method achieved very high coverage (median 99%), specificity (99.8%) and sensitivity (90% for haplotypes with 5% within sample frequency in DBS with 100 parasites/{micro}L). In silico analyses revealed that microhaplotypes provided much higher resolution to discriminate related from unrelated polyclonal infections than biallelic SNP barcodes. DiscussionThe bioinformatic and laboratory methods outlined here provide a flexible tool for efficient, low-cost, high throughput interrogation of the P. falciparum genome, and can be tailored to simultaneously address multiple questions of interest in various epidemiological settings.

molecular biology↗

bioRxiv2019

Optimization of whole-genome sequencing of Plasmodium falciparum from low-density dried blood spot samples

BackgroundWhole-genome sequencing (WGS) is becoming increasingly useful to study the biology, epidemiology, and ecology of malaria parasites. Despite ease of sampling, DNA extracted from dried blood spots (DBS) has a high ratio of human DNA compared to parasite DNA, which poses a challenge for downstream genetic analyses. We evaluated the effects of multiple methods for DNA extraction, digestion of methylated DNA, and amplification on the quality and fidelity of WGS data recovered from DBS. ResultsAt 100 parasites/L, Chelex-Tween-McrBC samples had higher coverage (5X depth = 93% genome) than QIAamp extracted samples (5X depth = 76% genome). The two evaluated sWGA primer sets showed minor differences in overall genome coverage and SNP concordance, with a newly proposed combination of 20 primers showing a modest improvement in coverage over those previously published. ConclusionsOverall, Tween-Chelex extracted samples that were treated with McrBC digestion and are amplified using 6A10AD sWGA conditions had minimal dropout rate, higher percentages of coverage at higher depth, and more accurate SNP concordance than QiaAMP extracted samples. These findings extend the results of previously reported methods, making whole genome sequencing accessible to a larger number of low density samples that are commonly encountered in cross-sectional surveys.

molecular biology↗