Biology subjects

Wang, F.

Publications and source records attributed to Wang, F..

At least 181 records · Page 10Linked to original sources

bioRxiv2025

Fluorogenic Interacting Protein Stabilization for Orthogonal RNA Imaging

Live imaging of RNAs is crucial to interrogate their cellular functions, and approaches allowing fluorogenic RNA imaging with high orthogonality and contrast remains underexplored. We propose a fluorogenic interacting protein stabilization (FLIPS) strategy that enables orthogonal RNA imaging in live cells with fluorescence-labeled RNA interacting proteins (RIPs) stabilized by cognate RNA motifs. We show that the RNA motif stabilizes the otherwise degraded cognate RIP fused to a poly(arginine)- extended C-terminal degron, and the stabilizing effect is enhanced with circular permutation of the RIP. This design affords a generally applicable strategy for different RNA motifs and RIPs, enabling orthogonal and multi-color fluorescence-activated RNA imaging. Our strategy is broadly demonstrated for multiplexed, high-contrast RNA detection and imaging, single molecule RNA imaging and RNA dynamic translocation tracking in live cells. The versatility of our system highlights its potential for interrogating RNA biology and developing RNA-based imaging tools.

cell biology↗

bioRxiv2025

Target-based de novo design of cyclic peptide binders

Cyclic peptides have become a new focus in drug discovery due to their ability to bind challenging targets, including "undruggable" protein-protein interactions, with low toxicity. Despite their potential, general methods for de novo design of cyclic peptide ligands based on target protein structures remain limited. Here, we developed CYC_BUILDER, a reinforcement learning based fragment growing method for efficient assembly of peptide fragments and cyclization to generate diverse cyclic peptide binders for target proteins. CYC_BUILDER employs a Monte Carlo Tree Search (MCTS) framework to integrate seed fragment exploration, fragment fusion based peptide growth, structure optimization, evaluation and peptide cyclization. It supports peptide cyclization through both head-to-tail amide bond and disulfide bond formation. We first validated CYC_BUILDER on known protein-cyclic peptide complexes, demonstrating its ability to accurately re-generate cyclic peptide binders in terms of both sequences and binding poses. We then applied it to design cyclic peptide inhibitors for TNF, a key mediator in inflammation-related diseases. Among the nine experimentally tested designed peptides, four showed potent binding to TNF and inhibited its cellular activity. CYC_BUILDER provides an efficient tool for cyclic peptide drug design, offering significant potential for addressing challenging therapeutical targets.

biochemistry↗

bioRxiv2025

Fast and accurate modeling of TCR-peptide-MHC complexes using tFold-TCR

Alpha-beta T cell receptor ({beta}TCR) recognition of peptide-major histocompatibility complexes (pMHCs) is a corner- stone of the adaptive immune system. Fast and accurate modeling of TCR-pMHC structures is crucial for understanding TCR recognition of pMHCs at the molecular level, which is essential for the development of TCR-based therapeutics and vaccines. Despite significant interest, this challenge remains unresolved due to the diversity of TCR-pMHC interactions and limited structural data. Here, we present tFold-TCR, a high-throughput, end-to-end universal model for predicting three-dimensional (3D) atomic-level structures of TCR-pMHC complexes, capable of predicting TCRs of different classes and MHC structures from diverse systems. tFold-TCR leverages a specially trained, protein-protein interaction-sensitive large protein language model to extract intra- and inter-chain residue contact information and evolutionary relationships, bypassing the need for multiple sequence alignment (MSA) searches. It also features innovative structure prediction and flexible docking modules to enhance accuracy, particularly for interacting contacts. Compared to existing methods, including AlphaFold-3, tFold-TCR demonstrates a 30.7% increase in prediction success rate evaluated by DockQ and is over 25 times faster. These advancements enable large-scale structural characterization of TCRs and their interactions with pMHCs. Utilizing this capability, we constructed TCRStructDB, the largest database of TCR-pMHC structures to date, encompassing 2.2 million TCRs, 0.8 million pMHCs, and 45,000 TCR-pMHC complexes. TCRStructDB provides unprecedented insights into one of the most diverse receptor-ligand interactions in biology.

bioinformatics↗

bioRxiv2025

Why Accuracy Metrics Fall Short in Comparing Phenomic and Genomic Prediction Models

Phenomic selection is a new paradigm in plant breeding that uses high-throughput phenotyping technologies and machine learning models to predict traits of new individuals and make selections. This can allow breeders to evaluate more plants in higher throughput more accurately, resulting in faster rates of gain and reduced labor costs. However, phenomic prediction models are frequently benchmarked against genomic prediction models using cross-validation to demonstrate their usefulness to breeders. We argue that this is inappropriate for two reasons: 1) differences in the accuracy statistic measured by cross-validation do not reliably indicate differences in the accuracy parameter of the breeders equation, which we show analytically and through re-analysis of data from three representative phenomic prediction studies, and 2) phenomic and genomic selection tools influence other parameters of the breeders equation, so comparing accuracy, even if done properly, is insufficient to advocate for one approach over the other. We conclude that phenomic selection may be useful, but comparisons of accuracy between genomic prediction and phenomic prediction models are not.

genetics↗

bioRxiv2025

Niacin protects against vascular calcification via enhanced SIRT1 and SIRT6 signaling and promoted VSMC autophagy

BackgroundVascular calcification (VC) is a common pathologic state that often accompanies calcium-phosphorus metabolism disorder and chronic kidney diseases (CKDs) in aging individuals. Vascular smooth muscle cell (VSMC) has been widely acknowledged as one of the main cell types that involved in this process. Niacin, a lipid-lowering reagent, has been demonstrated to be beneficial in atherosclerotic disease. The correlation and mechanism between niacin and vascular calcification have not been reported so far. Methods and resultsRCS analysis based on large clinical databases indicated that diet that riches in niacin can protect against abdominal artery calcification (AAC). Our data showed that niacin treatment remarkably reduced VSMC osteogenic differentiation and senescence-associated markers under osteogenic stimulation. Moreover, niacin treatment alleviated CKD and vitamin D3-induced vascular calcification in C57BL/6J mice. Mechanistically, we for the first time demonstrated that niacin supplementation inhibited vascular calcification via maintaining both Sirtuin 1 (SIRT1) and Sirtuin 6 (SIRT6) levels. Moreover, we verified that niacin activated SIRT1 and SIRT6 promoted VSMC autophagy flux. ConclusionsThese findings may help to develop novel therapeutic strategies in the treatment and prevention of vascular calcification.

cell biology↗

bioRxiv2025

Quantifying Metagenomic Strain Associations fromMicrobiomes with Anpan

Genetic and genomic variation among microbial strains can dramatically influence their phenotypes and environmental impact, including on human health. However, inferential methods for quantifying these differences have been lacking. Strain-level metagenomic profiling data has several features that make traditional statistical methods challenging to use, including high dimensionality, extreme variation among samples, and complex phylogenetic relatedness. We present Anpan, a set of quantitative methods addressing three key challenges in microbiome strain epidemiology. First, adaptive filtering designed to interrogate microbial strain gene carriage is combined with linear models to identify strain-specific genetic elements associated with host health outcomes and other phenotypes. Second, phylogenetic generalized linear mixed models are used to characterize the association of sub-species lineages with such phenotypes. Finally, random effects models are used to identify pathways more likely to be retained or lost by outcome-associated strains. We validated our methods by simulation, showing that we achieve more accurate effect size estimation and a lower false positive rate compared to alternative methodologies. We then applied our methods to a dataset of 1,262 colorectal cancer patients, identifying functionally adaptive genes and strong phylogenetic effects associated with CRC status, sometimes complementing and sometimes extending known species-level microbiome CRC biomarkers. Anpans methods have been implemented as a publicly available R library to support microbial community strain and genetic epidemiology in a variety of contexts, environments, and phenotypes.

bioinformatics↗

bioRxiv2025

Improved Identification of Host Cell Proteins in Monoclonal Antibodies by Combining Filter Aided Sample Preparation and Native Digestion

Host cell proteins (HCPs) in biotherapeutics can present potential safety risks or compromise product stability at trace levels. Therefore, removal, testing, and characterization of HCPs are critical throughout biotherapeutic process development. While enzyme-linked immunosorbent assay (ELISA) is the gold standard for quantifying HCPs, it does not provide information on HCP identity. As a result, HCP analysis by liquid chromatography-mass spectrometry (LC-MS) has gained prominence for its ability to identifying HCPs. However, compared to ELISAs parts-per-billion sensitivity, LC-MS is limited by its dynamic range, often unable to cover magnitude difference between HCP and biotherapeutic concentrations. Thus, an effective HCP enrichment method is highly desirable. In this study, we propose a new strategy for HCP identification that combines filter-aided sample preparation (FASP) with native digestion, followed by shotgun proteomics analysis. This approach improves mAb removal compared to standard native digestion, enabling greater HCP enrichment and identification. Our method detects all spiked proteins at 1 ppm and most at 0.5 ppm, ranging from 12 to 470 kDa, demonstrating broad molecular weight coverage of HCPs. A proof-of-concept analysis using the NISTmAb standard demonstrates that our filter-aided native digestion identifies 155 more HCPs than the standard native digestion. When applying this strategy to an in-house antibody with low amounts of HCPs, we can quantify HCPs at levels as low as 0.03 ppm, demonstrating the high sensitivity in HCP characterization.

biochemistry↗

bioRxiv2024

Donor Strand Complementation and Calcium Ion Coordination Drive the Chaperone-free Polymerization of Archaeal Cannulae

Cannulae are tubular protein filaments that accumulate on the extracellular surface of the hyperthermophilic archaeon Pyrodictium abyssi during cell division. Cannulae have been postulated to act as a primitive extracellular matrix through which cells could communicate or exchange material, although their native biological function remains obscure. Here, we report cryoEM structural analyses of ex vivo cannulae and of in vitro protein assemblies derived from recombinant cannula-like proteins. Three-dimensional reconstructions of P. abyssi cannulae revealed that the structural interactions between protomers in the native and recombinant filaments were based on donor strand complementation, a form of non-covalent polymerization in which a donor {beta}-strand from one subunit is inserted into an acceptor groove in a {beta}-sheet of a neighboring subunit. Donor strand complementation in cannulae is reinforced through calcium ion coordination at the interfaces between structural subunits in the respective assemblies. While donor strand complementation occurs during the assembly of chaperone-usher pili, this process requires the participation of accessory proteins that are localized in the outer membrane. In contrast, we demonstrate that calcium ions can induce assembly of cannulae in the absence of other co-factors. Crystallographic analysis of a recombinant cannula-like protein monomer provided evidence that calcium ion binding primes the precursor for donor strand invasion through unblocking of the acceptor groove. Bioinformatic analysis suggested that structurally homologous cannula-like proteins occurred within the genomes of other hyperthermophilic archaea and were encompassed within the TasA superfamily of biomatrix proteins. CryoEM structural analyses of tubular filaments derived from in vitro assembly of a recombinant cannula-like protein from an uncultured Hyperthermus species revealed a common mode of assembly to the Pyrodictium cannulae, in which donor strand complementation and calcium ion binding stabilized longitudinal and lateral assembly in tubular 2D sheets.

microbiology↗

bioRxiv2024

Identification of SMARCA1 as a key regulator for Colorectal Cancer

Emerging evidence indicates that aberrations in the writing, reading, or erasure of chromatin modification codes are pivotal events in various human cancers. In this study, we conducted a systematic investigation of histone modification recognition proteins in a pair of colorectal cancer cell lines, SW480 and SW620. Using chromatin fractionation combined with data-independent acquisition mass spectrometry (DIA-MS), we developed a robust method to identify changes in histone modification recognition proteins during cancer progression. Our analysis revealed 22 proteins that were significantly upregulated and 22 proteins that were significantly downregulated in SW620 cells compared to SW480 cells. Notably, SMARCA1, a member of the ISWI family belonging to ATP-dependent chromatin remodeling complexes, was downregulated in SW620 cells compared to SW480 cells. Its high expression was strongly correlated with poor patient prognosis, aligning with the proposed role of SMARCA1 in promoting colorectal cancer (CRC) metastasis. Furthermore, reduced SMARCA1 expression altered the levels of metastasis-related matrix metalloproteinases (MMPs) in these cells. In conclusion, by systematically profiling histone modification recognition proteins in a matched pair of primary and metastatic CRC cell lines, we identified SMARCA1 as a potential driver of CRC metastasis and a promising therapeutic target for CRC patients.

cancer biology↗

bioRxiv2024

Multi-Omic Atlas reveals cytotoxic phenotype and ROS-linked metabolic quiescence as key features of CTL-resistant HIV-infected CD4+ T-cells

Cytotoxic T-lymphocytes (CTL) exert sustained pressure on reservoirs of HIV-infected cells that persist through years of antiretroviral therapy (ART). This selects for latently infected cells, but also potentially for cells that express HIV but possess intrinsic CTL resistance. We demonstrate that such resistance exists in HIV-infected CD4+ T-cells that survive rigorous CTL attack and map CTL susceptibility to cell identities and states defined by single-cell multi-omics and functional metabolic profiling. Cytotoxic CD4+ T-cells were prominently overrepresented amongst survivors, as were cells with quiescent metabolic profiles and low levels of reactive oxygen species (ROS) production. The induction of ROS production by treatment with deferoxamine sensitized these cells to CTL-mediated elimination. Reservoir-harboring cells from clinical samples share the above transcriptional features, being enriched for quiescent states. Our results provide an atlas for elucidating features of CTL resistance in HIV reservoirs, and identify oxidative stress as a therapeutic target to facilitate reservoir elimination.

immunology↗

bioRxiv2024

Fluid replacement and acute systemic inflammatory response syndrome during eight-hour extreme heat exposure in young men

Prolonged exposure to extreme heat poses significant risks, including systemic inflammatory response syndrome (SIRS), organ damage and hormonal imbalance. While fluid replacement is commonly recommended to mitigate these effects, its efficacy under uncompensable heat stress remains unclear. This study investigated the impacts of fluid replacement on thermoregulation, systemic inflammation, organ stress, cortisol levels and plasma electrolyte balance during eight-hour of extreme heat exposure in healthy young men. Twelve participants (age: 24.7{+/-}1.6 years; body surface area: 1.9{+/-}0.1 m{superscript 2}) underwent two randomized trials (dehydration: 125 mL/hour; euhydration: 375 mL/hour) in a heat chamber (40 {degrees}C, 55% RH). Biomarkers of inflammation (e.g., IL-6, IL-1{beta}), oxidative stress (e.g., MDA, SOD), organ function (ALT, BUN), cortisol, and electrolytes (sodium, potassium, chloride) were measured before and after exposure. Core temperature (Tcore) was continuously monitored. Results showed that fluid replacement significantly reduced Tcore at the end of the exposure (38.0{+/-}0.12 {degrees}C vs. 38.2{+/-}0.10 {degrees}C, p=0.046). However, it exacerbated systemic inflammation (IL-6: euhydration 19.8{+/-}4.3 pg/mL vs. dehydration 12.5{+/-}2.8 pg/mL, p<0.01) and liver stress (ALT: euhydration 45.3{+/-}6.7 U/L vs. dehydration 34.1{+/-}5.5 U/L, p=0.03). Cortisol levels decreased significantly in the euhydration group (p=0.041), potentially indicating attenuated stress resilience. Electrolyte imbalances (reduced sodium and potassium concentrations) were observed in the euhydration condition. Taken together, while fluid replacement reduced Tcore, it did not mitigate SIRS and instead exacerbated systemic inflammation, liver stress, and electrolyte imbalances, potentially through hypotonic osmotic stress. These findings underscore the need for personalized hydration strategies that balance fluid and electrolyte intake during extreme heat exposure to minimize health risks. Key pointsO_LIThis study investigated the effects of fluid replacement on acute inflammatory responses, thermoregulation, and organ stress markers in healthy young males exposed to eight hours of uncompensable heat stress. C_LIO_LIFluid replacement did not mitigate acute systemic inflammatory response syndrome (SIRS) but exacerbated inflammatory and organ stress markers despite reducing core temperature. C_LIO_LICore temperatures remained significantly lower in the euhydration group, but systemic and hepatic inflammatory markers worsened, highlighting the complexity of hydration strategies under extreme heat. C_LIO_LIWe emphasize the need for individualized hydration strategies incorporating electrolyte balance and sweat rate monitoring to minimize SIRS risk. C_LIO_LIWe provide actionable insights into heat stress physiology, with implications for occupational health guidelines, public health policies, and climate change adaptation. C_LI

physiology↗

bioRxiv2024

ANKRD5: a key component of the axoneme required for spermmotility and male fertility

Sperm motility is essential for male fertility and depends on the structural integrity of the sperm axoneme, which features a canonical "9+2" microtubule arrangement. This structure comprises nine outer doublet microtubules (DMTs) that are associated with various macromolecular complexes. Among them, the nexin-dynein regulatory complex (N-DRC) forms crossbridges between adjacent DMTs, contributing to their stabilization and enabling flagellar bending. In this study, we investigated Ankyrin repeat domain 5 (ANKRD5, also known as ANK5 or ANKEF1), a protein highly expressed in the sperm axoneme. We found that ANKRD5 interacts with DRC5/TCTE1 and DRC4/GAS8, two key components of the N-DRC, and these interactions occur independently of calcium regulation. Male Ankrd5-/- mice exhibited impaired sperm motility and infertility. Cryo-electron tomography revealed a typical "9+2" axoneme structure with intact DMTs in Ankrd5 null sperm; however, the DMTs showed pronounced morphological variability and increased structural heterogeneity. Notably, ANKRD5 deficiency did not alter ATP levels, reactive oxygen species (ROS) levels, or mitochondrial membrane potential. These findings suggest that ANKRD5 may attenuate the N-DRCs mechanical buffering-akin to a "car bumper"-between adjacent DMTs, thereby compromising axonemal stability under high mechanical stress during vigorous flagellar beating. Graphic Abstract O_FIG O_LINKSMALLFIG WIDTH=184 HEIGHT=200 SRC="FIGDIR/small/626701v3_ufig1.gif" ALT="Figure 1"> View larger version (39K): org.highwire.dtl.DTLVardef@1ca36eforg.highwire.dtl.DTLVardef@dcafe3org.highwire.dtl.DTLVardef@9f6086org.highwire.dtl.DTLVardef@17a0a0b_HPS_FORMAT_FIGEXP M_FIG C_FIG Significance StatementMale infertility affects approximately 8%-12% of men globally, with defects in sperm motility accounting for over 80% of these cases. The axoneme, which functions as the motor apparatus of the sperm, adopts a canonical "9+2" microtubule arrangement, where the nexin-dynein regulatory complex (N-DRC) plays a critical role in providing structural support between adjacent outer microtubule doublets. Elucidating the interplay between the structural organization and protein composition of the N-DRC is essential for advancing the understanding of male reproductive biology. In this study, we identify ANKRD5 as a new component of N-DRC that is essential for maintaining normal sperm motility. These findings contribute to the molecular understanding of sperm motility and highlight ANKRD5 as a potential target for the development of novel male contraceptives.

developmental biology↗

bioRxiv2024

Drug Response Modeling across Cancers: Proteomics vs. Transcriptomics

Cancer cell lines are the most common in-vitro models for the evaluation of anti-cancer drug sensitivities. Past studies have been conducted to decipher and characterize the pharmacogenomic feature of cell lines based on other omics data, such as genomic mutation data and whole-genome RNA sequencing (RNA-seq) profiles. In particular, proteomic data is also an essential component for the characterization of tumours. However, different from RNA-seq datasets rich in numerous transcriptome profiles of cancer cell lines and cell viability assay of drug responses, the pharmacogenomic protein quantifications are relatively scarce. With the availability of the recently enriched proteomic dataset ProCan-DepMapSanger, we systematically evaluated the interplays among genomic mutations, transcription, and protein expressions across cancer cell lines. In general, blood cancers have higher RNA-protein correlations than those in solid cancers. The differential expression analysis on protein data helped identify more expressional and functional impact of genomic mutations of cancer genes. We also integrated the proteomic map with drug molecular chemical features to construct a bi-modal machine learning model to infer the drug sensitivities of cancer cell lines. Our results demonstrated that protein quantifications can lead to better drug response prediction performance than the model trained on transcriptome profiles. In addition, integrating protein data with drug chemical features, represented as molecular graphs and learned by Graph Neural Network, outperformed the state-of-the-art model DeepOmicNet for drug response prediction in proteomics.

bioinformatics↗

bioRxiv2024

TurboID-based proximity labeling discovers ABCF2 as an adhesion receptor for the zoonotic pathogen Pasteurella multocida

Pasteurella multocida is a zoonotic pathogen that can cause fatal infections in both animals and humans. A significant number of putative adhesive factors have been predicted to contribute to the pathogenesis of P. multocida, but their interactive proteins on host cells remain unclear. In this study, we experimentally verified the roles of three previously proposed proteins (PlpE, PtfA, Hsf-2) in the adherence of P. multocida. Through turboID-based proximity labeling screening, we identified ATP-binding cassette sub-family F member 2 (ABCF2) as a host interactive protein for PlpE/PtfA/Hsf-2. Crucial amino acid residues in PlpE, PtfA, and/or Hsf-2 that are essential for interacting with ABCF2 included Asp-123 (PlpE), Lys-88 (PtfA), Asp-136 (PtfA), Ala-464 (Hsf-2), Glu-473 (Hsf-2), and Arg-489 (Hsf-2). Knocking down or knocking out ABCF2 significantly reduced the adherence and invasion of P. multocida to host cells, while overexpression of ABCF2 markedly increased these effects. However, ABCF2 did not contribute to the adherence of other bacterial species such as Klebsiella pneumoniae and Bordetella bronchiseptica. Additionally, we demonstrated that P. multocida infection upregulated the expression of host ABCF2 by activating the p38 MAPK and NF-{kappa}B signaling pathways. Furthermore, we showed that ABCF2 was involved in the P. multocida-induced p53-dependent apoptotic signaling pathway. To the best of our knowledge, this is the first identification of ABCF2 as a host factor contributing to the adherence of P. multocida and only the second report of ABCF2s involvement in bacterial pathogenesis. ImportanceP. multocida can cause fatal infections in both animals and humans, yet the mechanisms related to its pathogenesis remain to be fully explored. In this study, we identified ABCF2 as a crucial host interactive protein for three adhesive proteins encoded by P. multocida and experimentally verified its role in the adherence and invasion of P. multocida. Furthermore, we elucidated how P. multocida modulates ABCF2 during its infection and revealed p53-dependent apoptosis as a downstream effect of ABCF2 during P. multocida infection. Given the absence of reports on ABCF2 contributing to the pathogenesis of P. multocida and only one previous report on the involvement of ABCF2 in bacterial pathogenesis prior to this study, this research could be valuable for comprehending the interactions between bacteria and hosts during bacterial infections.

microbiology↗

bioRxiv2024

Cardiac Reprogramming with Drug Resistance Alleviates Doxorubicin-induced Cardiotoxicity in Mice and Pigs

Doxorubicin (Dox)-induced cardiotoxicity represents a significant clinical adverse effect associated with cancer chemotherapy treatment. The systematic toxicity associated with the currently available drug dexrazoxane has limited its clinical utility. Inspired by cancer drug resistance mechanisms, we propose a novel strategy termed Transient overexpression of p-glycoprotein (P-gp) for Cardiac reprogramming (TopCare) to induce cardiac drug resistance as a treatment for cardiotoxicity. This approach involves reprogramming cardiomyocytes by delivering lipid nanoparticles (LNPs)-based mRNA therapeutics to induce temporary P-gp overexpression, which in turn reduces intracellular Dox levels and suppresses cytotoxic effects. Our results demonstrated that LNPs with P-gp mRNAs (P-gp LNPs) enhance P-gp expression in H9c2 cells, significantly lowering the cytotoxicity of both Dox and paclitaxel (PTX). In a mouse model with Dox-induced cardiotoxicity, it was observed that P-gp LNPs effectively promoted P-gp overexpression in cardiomyocytes, with a time-dependent decline in P-gp protein levels. This TopCare strategy resulted in improved survival rates, restored cardiac function, and reduced myocardial fibrosis and structural cardiac alterations. Furthermore, studies in large animals have shown that intrapericardial (iPC) injection of P-gp LNPs effectively mitigates adverse effects and restores cardiac function in pig models of Dox-induced cardiotoxicity. The significant cardioprotective effects achieved through cardiac drug resistance highlight the safety, efficacy, and clinical potential of the TopCare strategy for alleviating doxorubicin-induced cardiotoxicity.

bioengineering↗

bioRxiv2024

Metabolic Adaptability and Nutrient Scavenging in Toxoplasma gondii: Insights from Ingestion Pathway-Deficient Mutants

The obligate intracellular parasite Toxoplasma gondii replicates within a specialized compartment called the parasitophorous vacuole (PV). Recent work showed that despite living within a PV, Toxoplasma endocytoses proteins from the cytosol of infected host cells via a so-called ingestion pathway. The ingestion pathway is initiated by dense granule protein GRA14, which binds host ESCRT machinery to bud vesicles into the lumen of the PV. The protein-containing vesicles are internalized by the parasite and trafficked to the Plant Vacuole-like compartment (PLVAC), where cathepsin protease L (CPL) degrades the cargo and the chloroquine resistance transporter (CRT) exports the resulting peptides and amino acids to the parasite cytosol. However, although the ingestion pathway was proposed to be a conduit for nutrients, there is limited evidence for this hypothesis. We reasoned that if Toxoplasma uses the ingestion pathway to acquire nutrients, then parasites lacking GRA14, CPL, or CRT should rely more on biosynthetic pathways or alternative scavenging pathways. To explore this, we conducted a genome-wide CRISPR screen in wild-type (WT) parasites and {Delta}gra14, {Delta}cpl, and {Delta}crt mutants to identify genes that become more fitness conferring in ingestion-deficient parasites. Our screen revealed a significant overlap of genes that become more fitness conferring in the ingestion mutants compared to WT. Pathway analysis indicated that {Delta}cpl and {Delta}crt mutants relied more on pyrimidine biosynthesis, fatty acid biosynthesis, TCA cycle, and lysine degradation. Bulk metabolomic analysis showed reduced levels of glycolytic intermediates and amino acids in the ingestion mutants compared to WT, highlighting the pathways potential role in host resource scavenging. Interestingly, ingestion mutants showed an exacerbated growth defect when grown in amino acid-depleted media, suggesting a role for the Toxoplasma ingestion pathway during nutrient scarcity. ImportanceToxoplasma gondii is an obligate intracellular pathogen that infects virtually any nucleated cell in most warm-blooded animals. Infections are asymptomatic in most cases but people with weakened immunity can experience severe disease. For the parasite to replicate within the host, it must efficiently acquire essential nutrients, especially as it is unable to make several key metabolites. Understanding the mechanisms by which Toxoplasma scavenges nutrients from the host is crucial for identifying potential therapeutic targets. Our study highlights the function of the ingestion pathway in sustaining parasite metabolites and contributes to parasite replication under amino acid limiting conditions. This work advances our understanding of the metabolic adaptability of Toxoplasma.

microbiology↗

bioRxiv2024

Comparing neurocognitive mechanisms of mathematical ability and fluency in children: insights from an fNIRS study

BackgroundEarly proficiency in mathematics is a strong predictor of later academic success and life achievement, considering the practical skills that mastering the subject enables students to equip. Yet, there exists a paucity of research into the neural mechanisms supporting mathematical abilities in young children. Recent research utilises resting state functional connectivity (RSFC), a measure of the coherence of brain activity among brain regions in the absence of tasks, to understand the functional roles of these regions. MethodsWe analysed the RSFC of 45 children to investigate the intrinsic cognitive processes underpinning arithmetic processing in three regions of interest (ROIs): middle frontal gyrus, inferior parietal lobule, and precuneus. Correlations between RSFC among these regions and mathematics or math fluency scores, derived from the Wechsler Individual Achievement Test (WIAT-III), were examined. ResultsRSFC between the right precuneus and both the ipsilateral middle frontal gyrus and inferior parietal region may be associated with arithmetic processing speed and accuracy, while cross-hemispheric RSFC between the right precuneus and the left inferior parietal lobule appears to be associated with problem-solving and numeracy skills. RSFC between the right precuneus and left inferior parietal lobule differed in children performing below the 10th percentile in mathematics (out of 45 participants). ConclusionsThe results suggest that children of the same age may follow different neural development trajectories. More targeted and differentiated interventions are essential to offer additional and early support for students struggling with mathematics.

neuroscience↗

bioRxiv2024

Human heart assembloids with autologous tissue-resident macrophages recreate physiological immuno-cardiac interactions

Interactions between the developing heart and the embryonic immune system are essential for proper cardiac development and maintaining homeostasis, with disruptions linked to various diseases. While human pluripotent stem cell (hPSC)-derived organoids are valuable models for studying human organ function, they often lack critical tissue-resident immune cells. Here, we introduce an advanced human heart assembloid model, termed hHMA (human heart-macrophage assembloid), which fully integrates autologous cardiac tissue- resident macrophages (MPs) with pre-existing human heart organoids (hHOs). Through multi-omic analyses, we confirmed that these MPs are phenotypically similar to embryonic cardiac tissue-resident MPs and remain viable in the assembloids over time. The inclusion of MPs significantly impacts hHMA development, influencing cardiac cellular composition, boosting cellular communication, remodeling the extracellular matrix, promoting ventricular morphogenesis, and enhancing sarcomeric maturation. Our findings indicate that MPs contribute to homeostasis via efferocytosis, integrate into the cardiomyocyte electrical system, and support catabolic metabolism. To demonstrate the versatility of this model, we developed a platform to study cardiac arrhythmias by chronic exposure to pro-inflammatory factors linked to arrhythmogenesis in clinical settings, successfully replicating key features of inflammasome-mediated atrial fibrillation. Overall, this work introduces a robust platform for examining the role of immune cells in cardiac development, disease mechanisms, and drug discovery, bridging the gap between in vitro models and human physiology. These findings offer insights into cardiogenesis and inflammation-driven heart disease, positioning the hHMA system as an invaluable tool for future cardiovascular research and therapeutic development.

bioengineering↗