Biology subjects

Marques, S.

Publications and source records attributed to Marques, S..

8 recordsLinked to original sources

bioRxiv2026

Uncovering Functional Distant Mutations by Ultra-High-Throughput Screening of Dehalogenases

Conformational dynamics play a central role in enzyme function by controlling substrate access and productive binding. Yet mutations that beneficially modulate these properties are difficult to identify. Here, we used ultrahigh-throughput fluorescence-activated droplet sorting (FADS) with a bulky fluorogenic substrate derived from coumarin (COU-3) to impose steric selection pressure on the haloalkane dehalogenase LinB. Screening a focused library yielded five single substitutions located 11.5-15.5 [A] from the catalytic centre. Variant I138N showed a fourfold increase in catalytic efficiency toward COU-3 through reduced KM and increased kcat, associated with increased cap-domain flexibility and facilitated substrate entry. In contrast, variant P208S markedly reduced substrate inhibition and shifted specificity toward bulkier iodinated haloalkanes by reshaping its tunnel environment. Integrated kinetic and structural analyses revealed that screening with bulky substrates directs selection toward distal regions controlling substrate access and unproductive binding. These findings demonstrate that ultrahigh-throughput FADS can reveal dynamic mechanisms of enzyme adaptation that remain difficult to predict by rational design. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=183 SRC="FIGDIR/small/713925v1_ufig1.gif" ALT="Figure 1"> View larger version (51K): org.highwire.dtl.DTLVardef@782038org.highwire.dtl.DTLVardef@8b43f3org.highwire.dtl.DTLVardef@11a403eorg.highwire.dtl.DTLVardef@6fcaea_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2026

Host cell traversal by Plasmodium parasites is essential for sterilizing hepatic cellular immunity.

An effective vaccine capable of inducing sterile protection against Plasmodium, the causative agent of malaria, is critical to aid global eradication. Whole-organism vaccines using liver-infective sporozoites provide high levels of sterile protection against pre-erythrocytic infection. Yet, determinants of sporozoite immunogenicity remain poorly characterized. Using rodent models of vaccination, we demonstrate that the ability of Plasmodium sporozoites to actively migrate through multiple host cells prior to infecting hepatocytes is required for sterilizing immunity, regardless of the time of intrahepatic development of immunizing parasites. We further establish that host cell traversal is sufficient to trigger robust protection against Plasmodium hepatic infection. Impaired cell traversal precludes protective liver-resident memory CD8 T cell responses following vaccination, but not the production of anti-plasmodial antibodies. Our findings challenge the prevailing notion that intrahepatic parasite development is the sole determinant of whole-sporozoite vaccination-induced protection, and highlight parasite behavior traits as critical immunogenic events shaping sterilizing cellular immunity against Plasmodium liver stages.

immunology↗

bioRxiv2023

Killing them softly with EPO: a new role for erythropoietin in the homeostasis of red blood cells

The regulation of red blood cell (RBC) homeostasis is widely assumed to rely on the control of cell production by erythropoietin (EPO) and the destruction of cells at a fixed, species-specific age. In this work, we show that such a regulatory mechanism is a poor homeostatic solution to satisfy the changing needs of the body. Effective homeostatic control requires RBC lifespan to be variable and tightly regulated. We show that EPO controls RBC lifespan by determining CD47 expression in newly formed RBCs and SIRP- expression in sinusoidal macrophages. EPO also controls the initiation and intensity of anti-RBC autoimmune responses that curtail RBC lifespan in some circumstances. These mechanisms continuously modulate the rate of RBC destruction depending on oxygen availability. The finding of new homeostatic roles for EPO and autoimmunity critically challenges the current paradigm of RBC homeostasis and sets the grounds for a new approach to this field.

physiology↗

bioRxiv2022

A non-canonical sensing pathway mediates Plasmodium adaptation to AA deficiency

Eukaryotes have canonical pathways for responding to amino acid (AA) availability. Under AA-limiting conditions, the TOR complex is repressed, whereas the sensor kinase GCN2 is activated. While these pathways have been highly conserved throughout evolution, malaria parasites are a rare exception. Despite auxotrophic for most AA, Plasmodium does not have either a TOR complex nor the GCN2-downstream transcription factors. While Ile starvation has been shown to trigger eIF2 phosphorylation and a hibernatory-like response, the overall mechanisms mediating detection and response to AA fluctuation in the absence of such pathways has remained elusive. Here we show that Plasmodium parasites rely on an efficient sensing pathway to respond to AA fluctuations. A phenotypic screen of kinase knockout mutant parasites identified nek4, eIK1 and eIK2 - the last two clustering with the eukaryotic eIF2 kinases - as critical for Plasmodium to sense and respond to distinct AA-limiting conditions. Such AA-sensing pathway is temporally regulated by these kinases at distinct life cycle stages and allows parasites to actively fine-tune replication and development in response to AA availability. Collectively, our data identify a previously unknown set of heterogeneous responses to AA depletion, mediated by a complex mechanism that is critical for modulating parasite cell cycle and survival.

microbiology↗

bioRxiv2021

A spatiotemporally resolved single cell atlas of the Plasmodium liver stage

Malaria infection involves an obligatory, yet clinically silent liver stage1,2. Hepatocytes operate in repeating units termed lobules, exhibiting heterogeneous gene expression patterns along the lobule axis3, but the effects of hepatocyte zonation on parasite development have not been molecularly explored. Here, we combine single-cell RNA sequencing4 and single-molecule transcript imaging5 to characterize the hosts and parasites temporal expression programs in a zonally-controlled manner for the rodent malaria parasite Plasmodium berghei ANKA. We identify differences in parasite gene expression in distinct zones, and a sub-population of periportally-biased hepatocytes that harbor abortive infections associated with parasitophorous vacuole breakdown. These abortive hepatocytes up-regulate immune recruitment and key signaling programs. They exhibit reduced levels of Plasmodium transcripts, perturbed parasite mRNA localization, and may give rise to progressively lower abundance of periportal infections. Our study provides a resource for understanding the liver stage of Plasmodium infection at high spatial resolution and highlights heterogeneous behavior of both the parasite and the host hepatocyte.

genomics↗

bioRxiv2021

Attrition Rate in Infant fNIRS Research: A Meta-Analysis

Understanding the trends and predictors of attrition rate, or the proportion of collected data that is excluded from the final analyses, is important for accurate research planning, assessing data integrity, and ensuring generalizability. In this pre-registered meta-analysis, we reviewed 182 publications in infant (0-24 months) functional near-infrared spectroscopy (fNIRS) research published from 1998 to April 9, 2020 and investigated the trends and predictors of attrition. The average attrition rate was 34.23% among 272 experiments across all 182 publications. Among a subset of 136 experiments which reported the specific reasons of subject exclusion, 21.50% of the attrition were infant-driven while 14.21% were signal-driven. Subject characteristics (e.g., age) and study design (e.g., fNIRS cap configuration, block/trial design, and stimulus type) predicted the total and subject-driven attrition rates, suggesting that modifying the recruitment pool or the study design can meaningfully reduce the attrition rate in infant fNIRS research. Based on the findings, we established guidelines on reporting the attrition rate for scientific transparency and made recommendations to minimize the attrition rates. We also launched an attrition rate calculator (LINK) to aid with research planning. This research can facilitate developmental cognitive neuroscientists in their quest toward increasingly rigorous and representative research. HighlightsO_LIAverage attrition rate in infant fNIRS research is 34.23% C_LIO_LI21.50% of the attrition are infant-driven (e.g., inattentiveness) while 14.21% are signal-driven (e.g., poor optical contact) C_LIO_LISubject characteristics (e.g., age) and study design (e.g., fNIRS cap configuration, block/trial design, and stimulus type) predict the total and infant-driven attrition rates C_LIO_LIModifying the recruitment pool or the study design can meaningfully reduce the attrition rate in infant fNIRS research C_LI

neuroscience↗

bioRxiv2019

Improved transcriptome annotation and read-through transcript identification with TIF-Seq2

Eukaryotic transcriptomes are complex involving thousands of overlapping transcripts. The interleaved nature of the transcriptome limits our ability to identify regulatory regions and, in some cases, can lead to misinterpretation of gene expression. To improve the understanding of the overlapping transcriptome, we have developed an optimized method, TIF-Seq2, able to sequence simultaneously the 5 and 3 ends of individual RNA molecules at single-nucleotide resolution. We investigated the transcriptome of a well characterized human cell line (K562) and identify thousands of unannotated transcript isoforms. By focusing on transcripts which are challenging to be investigated with RNA-seq, we accurately defined boundaries of lowly expressed unannotated and read-though transcripts putatively encoding fusion genes. We validated our results by targeted long-read sequencing and standard RNA-Seq for chronic myeloid leukaemia patient samples. Taking the advantage of TIF-Seq2, we explore transcription regulation among the overlapping units and investigate their crosstalk. We show that most overlapping upstream transcripts use poly(A) sites within the first 2 kb of the downstream transcription unit. Our work shows that, by paring the 5 and 3 end of each RNA, TIF-Seq2 can improve the annotation of complex genomes, facilitates accurate assignment of promoters to genes and easily identify transcriptionally fused genes. Key pointsO_LIStudy of TSS-PAS co-occurrence allows dissecting complex overlapping transcription units. C_LIO_LIPartially overlapping transcription units in human commonly use PAS within the first 2Kb. C_LIO_LITIF-Seq2 facilitates the identification of lowly expressed and transcriptionally fused genes. C_LI

genomics↗

bioRxiv2017

Single-cell transcriptomic profiling of progenitors of the oligodendrocyte lineage reveals transcriptional convergence during development

Pdgfra+ oligodendrocyte precursor cells (OPCs) arise in distinct specification waves during embryogenesis in the central nervous system (CNS). It is unclear whether there is a correlation between these waves and different transcriptional oligodendrocyte (OL) states at adult stages. Here we present a bulk and single-cell transcriptomics resource providing insights on how transitions between these states occur. We show that E13.5 Pdgfra+ populations are not OPCs, exhibiting instead hallmarks of neural progenitors. A subset of these progenitors, which we refer as pre-OPCs, rewires their transcriptional landscape, converging into indistinguishable OPC states at E17.5 and post-natal stages. P7 brain and spinal cord OPCs present similar transcriptional profiles at the single-cell level, indicating that OPC states are not region-specific. Postnatal OPC progeny of E13.5 Pdgfra+ have electrophysiological and transcriptional profiles similar to OPCs derived from subsequent specification waves. In addition, lineage tracing indicates that a subset of E13.5 Pdgfra+ cells also originate cells of the pericyte lineage. In summary, our results indicate that embryonic Pdgfra+ cells are diverse and give rise at post-natal stages to distinct cell lineages, including OPCs with convergent transcriptional profiles in different CNS regions.

neuroscience↗