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Novak, A.

Publications and source records attributed to Novak, A..

16 recordsLinked to original sources

Hub labeling makes exact distance calculation practical for large human pangenome graphs

Pangenome graphs are now a standard reference representation, and tools built on them depend on fast minimum-distance queries between graph positions, for example, when clustering seed matches during read alignment. The baseline distance index, previously used by the vg toolkit, answers such queries based on a decomposition of the graph into nested subgraphs called "snarls". Within each snarl, the index uses a dense all-pairs distance matrix. Because such a matrix is quadratic in a snarl's number of children, the large, complex snarls that often occur in many pangenome graphs dominate the overall index size. This means whole-genome graphs can be impractical to index. To mitigate this, the baseline distance index does not fully index the largest snarls, and thus cannot itself answer certain queries involving them. We replace the dense matrix with a hub labeling for snarls above a size limit. Because hub labeling and associated methods are defined for directed graphs, we introduce a directed representation of a snarl's bidirected internal "net graph", whose shortest path lengths are the net graph's minimum walk lengths. Hub labels are built using pruned landmark labeling, ordered by a contraction hierarchy. On four human chromosome pangenomes, our hub-labeling-based distance index indexes every snarl in 0.9 GB to 3.0 GB at a snarl limit of 500, an estimated 28x to 119x less space than dense distance matrices would need for those snarls with more than 500 children, and supports the 3-18.6% of node-to-node queries that the previous index cannot answer by itself. On the queries both indexes can answer, query time distributions broadly overlap. We explore the snarl limit parameter space and are able to build indexes under 50 GB for four human whole-genome pangenomes in under 6 hours, where we estimate dense matrices could have required multiple terabytes.

bioinformatics↗

Obesity-induced changes in ultrastructure and calcium release of female rat cardiomyocytes are partially reversed by aerobic exercise

Obesity is associated with an elevated risk of pathological cardiac hypertrophy, whereas exercise confers cardioprotective effects; however, the cellular mechanisms underlying these opposing influences remain incompletely defined, particularly in females. We investigated how obesity and exercise affect cardiomyocyte ultrastructure, Ca{superscript 2} release, and contractility in female Zucker Diabetic Fatty rats and their lean littermates. Animals were assigned at 12 weeks to sedentary or aerobic exercise-trained groups and maintained on a standard diet. By 18 weeks, obese rats exhibited increased body mass and myocardial hypertrophy in the absence of diabetes. Sedentary obese animals showed a reduced fraction of compact dyads and diminished stimulated and caffeine-induced Ca{superscript 2} release, while contractility remained preserved. In lean rats, exercise increased dyad density but reduced Ca{superscript 2} release, whereas in obese rats, exercise enhanced both dyad compactness and Ca{superscript 2} release. Across all groups, global cardiomyocyte ultrastructure and contractile function were similar. Type III ANOVA revealed a significant obesity x exercise interaction for dyadic structure and Ca{superscript 2} release. These findings demonstrate that obesity itself, independent of diabetes, triggers early dyadic remodeling and altered Ca{superscript 2} handling in female myocardium before detectable impairment of global cardiomyocyte structure or contractile function. Furthermore, exercise exerts beneficial effects on dyadic ultrastructure and Ca{superscript 2} signaling in obese animals. New & NoteworthyUsing a female rat model of obesity without diabetes, we demonstrate that obesity induces early remodeling of the dyadic system and impairs Ca{superscript 2} release in cardiac myocytes. We further show that the effects of aerobic exercise on dyadic structure and function are obesity-dependent, improving both dyad organization and Ca{superscript 2} signaling. These findings identify the dyadic microdomain as a vulnerable cellular site in obesity and a potential target for exercise-induced recovery.

physiology↗

GPR180 deficiency impairs mitochondrial function and insulin secretion in pancreatic β-cells

ObjectiveG protein-coupled receptor 180 (GPR180) has been implicated in systemic energy metabolism, primarily in adipose tissue and the liver. Given impaired whole-body glucose tolerance following GPR180 dysfunction, we aimed to determine whether GPR180 regulates pancreatic {beta}-cell function. We investigated whether GPR180 contributes to {beta}-cell insulin secretion by modulating metabolic processes that couple glucose sensing to mitochondrial energy production. MethodsPhenotyping of whole-body (Gpr180 -/-) and {beta} cell-specific Gpr180 (bGpr180-KO) knockout mice was combined with gain- and loss-of-function studies in MIN6 cells. Glucose-stimulated insulin secretion, pancreatic endocrine architecture and identity, transcriptomic and metabolic profiles, as well as mitochondrial function were assessed using in vivo and in vitro approaches, including metabolic challenge tests, histology, RNA sequencing, targeted metabolomics, respirometry, and transmission electron microscopy. ResultsLoss of GPR180 impaired first-phase insulin secretion and glucose tolerance without affecting insulin sensitivity. These defects were {beta}-cell-autonomous, as confirmed in the bGpr180-KO mice and in MIN6 cells. Functional studies revealed that GPR180 regulates mitochondrial substrate utilization, anaplerotic support of the TCA cycle, and ATP generation without affecting glucose uptake or mitochondrial biogenesis. In particular, Gpr180-deficient {beta} cells showed mitochondrial membrane depolarization, reduced oxygen consumption, and endoplasmic reticulum remodeling, altering the local mitochondrial microenvironment. In vivo, Gpr180 deletion in {beta} cells led to downregulation of mitochondrial gene programs in islets, along with altered endocrine cell identity. ConclusionsGPR180 is a previously unrecognized regulator of pancreatic {beta}-cell metabolic competence and identity, linking defects in insulin secretion with alterations in mitochondrial function and endocrine cell identity. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=198 SRC="FIGDIR/small/720098v1_ufig1.gif" ALT="Figure 1"> View larger version (87K): org.highwire.dtl.DTLVardef@1f24a65org.highwire.dtl.DTLVardef@1f2371eorg.highwire.dtl.DTLVardef@10096a5org.highwire.dtl.DTLVardef@1a242be_HPS_FORMAT_FIGEXP M_FIG C_FIG

physiology↗

Ultrasound Activated Nanobubbles Induce Durable Systemic Antitumor Immunity

Clinical outcomes in aggressive breast cancer vary widely, in part because the tumor microenvironment is structured to exclude immune infiltration. Low antigen load, dysfunctional antigen-presenting cells, T cell exclusion and exhaustion, and a stiff extracellular matrix that physically restricts immune cell trafficking work together to form a suppressive barrier that current immunotherapies struggle to overcome. We addressed this barrier using ultrasound (US)-activated nanobubbles (NBs), a drug-free intervention based on perfluoropropane-filled nanoparticles. The size and deformable phospholipid shell enable NBs to achieve deep tumor penetration and a uniform distribution throughout the entire tumor. Upon ultrasound activation, NBs generate localized mechanical forces that restore extracellular matrix elasticity, disrupt tumor transport barriers, and drive HMGB1 release, re-engaging endogenous antitumor immunity without pharmacological agents. In a syngeneic triple-negative breast cancer model, US-NB treatment depleted immunosuppressive myeloid cells 3-fold within 3 hours, followed by a greater than 5-fold increase in the ratio of antigen-experienced to suppressive T cells at 48 hours. US-NB drives rapid infiltration of CD4+ and CD8+ T cells within 48 hours. US-NB treatment achieved an 85% cure rate in the D2A1 model; cured animals maintained durable systemic immune memory, rejecting both local and systemic tumor rechallenge. Consistent therapeutic benefit was observed in a luminal B-like mammary tumor model (E0771), supporting activity across breast cancer subtypes. These results establish US-NB mechanical immunomodulation as a drug-free therapeutic strategy capable of generating robust and durable antitumor immunity, acting through biophysical tissue properties rather than tumor-specific molecular targets. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=90 SRC="FIGDIR/small/714247v1_ufig1.gif" ALT="Figure 1"> View larger version (56K): org.highwire.dtl.DTLVardef@8f274eorg.highwire.dtl.DTLVardef@5da575org.highwire.dtl.DTLVardef@19defborg.highwire.dtl.DTLVardef@7e630e_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

The tilt board task as an internally valid practice-transfer paradigm for stabilometer balance assessments

Practice-transfer paradigms are central to motor learning research, yet dynamic balance lacks standardized, internally valid practice-transfer task pairings. This study evaluated whether a mediolateral tilt board can serve as a valid transfer task for stabilometer-based balance assessment. Sixteen healthy young adults (20-35 years) completed a single session consisting of two 40-second trials on a mediolateral stabilometer and two 40-second trials on a mediolateral tilt board. Participants aimed to keep each platform horizontal during each trial. Performance outcomes were derived from platform deviation angle. Neuromuscular outcomes were derived from surface EMG of bilateral gluteus medius, vastus lateralis, and vastus medialis, including muscle synergy structure, bilateral co-activation index, RMS amplitude of muscle activation, and strategy ratios (hip-to-knee and asymmetry metrics). Between-task associations were assessed using Spearman correlations. Cross-task muscle synergy similarity was high (mean cosine similarity = 0.915 {+/-} 0.044) and close to within-task trial-to-trial similarity, indicating preserved modular coordination across devices. Performance metrics were moderately to strongly correlated between tasks (RMS deviation angle: {rho} = 0.621, p = 0.0089; time-in-balance: {rho} = 0.668, p = 0.0036). EMG-derived strategy metrics also correlated significantly across tasks, including bilateral co-activation ({rho} = 0.688, p = 0.0023), hip-to-knee ratio ({rho} = 0.765, p = 0.0003), hip asymmetry ratio ({rho} = 0.688, p=0.0023), and knee asymmetry ratio ({rho} = 0.679, p = 0.0028). In contrast, EMG RMS amplitude did not correlate across tasks ({rho} = -0.044, p = 0.873), suggesting task-specific gain of activation magnitude. Stabilometer and tilt board tasks shared a similar coordination structure and showed a high correlation in balance performance and neuromuscular strategy, supporting the tilt board as an internally valid transfer task for stabilometer-based dynamic balance paradigms. Similarity of tasks appears strongest at the level of modular control and strategy organization, with device-specific gain scaling of activation amplitude.

neuroscience↗

TSP1/TGF-β1 drives arachidonic acid metabolism to orchestrate neutrophil swarming

Neutrophil swarming has emerged as a conserved multicellular behaviour observed across tissues and pathological contexts. Yet, the molecular cues and the spatially coordinated cellular circuits that drive the process of neutrophil swarming leading to cluster formation remain poorly understood. Here, we combine spatial proteomics and lipid profiling in a model of urinary tract infection to define the epithelial-immune circuits driving neutrophil cluster formation. We identify thrombospondin-1 (TSP1)-mediated activation of transforming growth factor beta 1 (TGF-{beta}1) as key epithelial signal licensing neutrophil clustering and enhancing bacterial control. Spatial lipid analysis further reveals that TSP1/TGF-{beta}1 signalling locally activates arachidonic acid metabolism in epithelial neutrophils, with 5-lipoxygenase dependent leukotriene synthesis required for swarm formation and infection clearance. These findings uncover a spatially coordinated defence mechanism in which epithelial-derived TSP1/TGF-{beta}1 engages neutrophil lipid metabolism to orchestrate neutrophil swarming behaviour and reinforce antibacterial immunity. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=127 SRC="FIGDIR/small/688397v1_ufig1.gif" ALT="Figure 1"> View larger version (34K): org.highwire.dtl.DTLVardef@114ea92org.highwire.dtl.DTLVardef@345244org.highwire.dtl.DTLVardef@10520baorg.highwire.dtl.DTLVardef@1a7c0fe_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical abstractC_FLOATNO C_FIG

immunology↗

Astrocytic PIEZO activation by heartbeat sound promotes extracellular matrix and maturation in human brain organoids

Heartbeat sound is one of the first rhythmic stimuli encountered by the developing human brain, yet its biological role has remained unexplored. Here we show that heartbeat sound promotes structural and functional maturation of human cortical organoids with a physiological astrocyte-to-neuron ratio. Continuous stimulation with heartbeat expanded the extracellular space (ECS), visualized by super-resolution 3D STED and 2-photon shadow imaging, and increased extracellular matrix (ECM) synthesis, detected by LC-MS/MS proteomics. Heartbeat sound promoted neuronal and astrocytic differentiation, synaptogenesis, and organoid maturation, as shown by single-cell RNA sequencing, synaptic marker immunohistochemistry, and high-density multielectrode electrophysiology. Transcriptomic analyses demonstrated astrocyte-specific expression of mechanosensitive PIEZO channels. Using Ca2+ imaging, we confirmed that low-frequency sound stimulation, including heartbeat, activates PIEZO-mediated Ca2+ responses, which in turn upregulate astrocytic ECM synthesis. Experiments with biomimetic hydrogels demonstrated that physiological matrix stiffness attenuates PIEZO activation, suggesting a mechanoprotective mechanism. Our findings demonstrate a fundamental mechanism for inducing ECM synthesis and neural differentiation through astrocytic PIEZO activation by heartbeat sound, suggesting that interoception of the internal acoustic environment is an underappreciated driver of human brain development. HighlightsO_LIHeartbeat sound activates PIEZO ion channels C_LIO_LIAstrocytic PIEZO activation promotes extracellular matrix synthesis C_LIO_LIHeartbeat sound stimulates neural differentiation and brain organoid maturation C_LI Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=103 SRC="FIGDIR/small/683816v2_ufig1.gif" ALT="Figure 1"> View larger version (38K): org.highwire.dtl.DTLVardef@1ba3159org.highwire.dtl.DTLVardef@fdd741org.highwire.dtl.DTLVardef@13ef417org.highwire.dtl.DTLVardef@b3064_HPS_FORMAT_FIGEXP M_FIG C_FIG

neuroscience↗

Determining perception thresholds of young adults to small continuous moving platform perturbations

Detecting external disturbances is vital for maintaining balance, as corrective actions are initiated to prevent falls. Quantifying peoples ability to perceive such disturbances improves our understanding of how balance is maintained. This study aims to: 1) quantify healthy young adults ability to perceive external perturbations while balancing on a stabilometer, and 2) understand the relationship between balance performance and perturbation magnitude relative to participants perception threshold. Participants (n=22; 20-35 years) completed a multiple staircase protocol. While standing on a stabilometer mounted on a moving platform, they attempted to keep it horizontal during 10-second trials with small continuous perturbations. After each trial, participants were asked whether they perceived the platform movement. Perturbation magnitudes were adjusted for the next trial based on their response. This process continued for each staircase until the termination criteria were met, at which point participants individual perception threshold was determined. Participants then performed ten 40-second trials on the stabilometer, two trials in each condition: without perturbation, perturbation at the 100%, 80%, and 50% of the individuals perception threshold, and the pilot studys minimum threshold. Balance performance was defined as time-in-balance ratio and RMS deviation angle from horizontal. Perception thresholds varied significantly between participants individuals, with an RMS acceleration ranging from 2.67 and 12.80 cm/s2. The results showed that perturbation magnitude has a significant correlation with variability in deviation angle (R=0.24, p=0.0038). The results suggest that some participants can perceive very small perturbations during a challenging balance task. Subthreshold perturbations, although very small, can influence balance performance. NEW & NOTEWORTHYTo our knowledge, this is the first study to quantitatively measure the conscious perception threshold of platform perturbations while doing a balancing task. We found that young healthy adults with little similar prior experience in tasks close to stabilameter balancing task can detect small platform perturbations while doing such challenging task. The results also showed that platform perturbations below conscious threshold can influence the balance performance on the stabilometer.

neuroscience↗

Rational redesign of antigen binding domain improves in vivo efficacy of CD22-CAR T cells

Chimeric antigen receptor (CAR) T cells targeted to CD19 are an effective therapy for B-lineage malignancies. However, about half of patients relapse and this therapeutic, often with antigen-negative disease, warranting the targeting of other antigens. CD22 represents another promising target, with highly restricted but ubiquitous expression across the B-lineage. However, despite promising preclinical work by several groups with CD22-targeted CAR T cells targeting of this antigen in the clinic has proven difficult, with many patients relapsing with CD22Lo leukemia, contrasting to complete loss of CD19 expression post CD19-CAR. While prior work has demonstrated that a CAR with so-called "tonic" antigen-independent signaling properties has proven to be highly efficacious, tonic signaling has been shown be detrimental to long-term T cell function. Here, we demonstrate a balance between binding affinity and antigen-independent tonic signaling (as determined by length of flexible linker) in determining CAR function. We show that maximal CAR function in the settings CD22Lo and WT leukemia is maintained by boosting binding affinity without shortening flexible linker to induce tonic signaling, establishing rational modification of antigen binding domain as an important approach for modulating the function of cellular therapeutics. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=179 HEIGHT=200 SRC="FIGDIR/small/643183v1_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@1d543e3org.highwire.dtl.DTLVardef@5bf226org.highwire.dtl.DTLVardef@76cd2forg.highwire.dtl.DTLVardef@198fd37_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

A split-GAL4 driver line resource for Drosophila CNS cell types

Techniques that enable precise manipulations of subsets of neurons in the fly central nervous system have greatly facilitated our understanding of the neural basis of behavior. Split-GAL4 driver lines allow specific targeting of cell types in Drosophila melanogaster and other species. We describe here a collection of 3060 lines targeting a range of cell types in the adult Drosophila central nervous system and 1373 lines characterized in third-instar larvae. These tools enable functional, transcriptomic, and proteomic studies based on precise anatomical targeting. NeuronBridge and other search tools relate light microscopy images of these split-GAL4 lines to connectomes reconstructed from electron microscopy images. The collections are the result of screening over 77,000 split hemidriver combinations. Previously published and new lines are included, all validated for driver expression and curated for optimal cell type specificity across diverse cell types. In addition to images and fly stocks for these well-characterized lines, we make available 300,000 new 3D images of other split-GAL4 lines.

neuroscience↗

Pangenome Graph Construction from Genome Alignment with Minigraph-Cactus

Reference genomes provide mapping targets and coordinate systems but introduce biases when samples under study diverge sufficiently from them. Pangenome references seek to address this by storing a representative set of diverse haplotypes and their alignment, usually as a graph. Alternate alleles determined by variant callers can be used to construct pangenome graphs, but thanks to advances in long-read sequencing, high-quality phased assemblies are becoming widely available. Constructing a pangenome graph directly from assemblies, as opposed to variant calls, leverages the graphs ability to consistently represent variation at different scales and reduces biases introduced by reference-based variant calls. Pangenome construction in this way is equivalent to multiple genome alignment. Here we present the Minigraph-Cactus pangenome pipeline, a method to create pangenomes directly from whole-genome alignments, and demonstrate its ability to scale to 90 human haplotypes from the Human Pangenome Reference Consortium (HPRC). This tool was designed to build graphs containing all forms of genetic variation while still being practical for use with current mapping and genotyping tools. We show that this graph is useful both for studying variation within the input haplotypes, but also as a basis for achieving state of the art performance in short and long read mapping, small variant calling and structural variant genotyping. We further measure the effect of the quality and completeness of reference genomes used for analysis within the pangenomes, and show that using the CHM13 reference from the Telomere-to-Telomere Consortium improves the accuracy of our methods, even after projecting back to GRCh38. We also demonstrate that our method can apply to nonhuman data by showing improved mapping and variant detection sensitivity with a Drosophila melanogaster pangenome.

bioinformatics↗

Differential Expression of Alkaline Phosphatase and PHOSPHO1 in Bone from a Murine Model of Chronic Kidney Disease

Patients with advanced chronic kidney disease (CKD) often present with skeletal abnormalities; a condition known as renal osteodystrophy (ROD). While Tissue-nonspecific alkaline phosphatase (TNAP) and PHOSPHO1 are recognized to be critical for bone mineralization, their role in the etiology of ROD is unclear. To address this, ROD was induced in both wild-type and Phospho1 knockout (P1KO) mice using dietary adenine supplementation. The mice presented with hyperphosphatemia, hyperparathyroidism, and elevated levels of FGF23 and bone turnover markers. In particular, we noted that in CKD mice, bone mineral density (BMD) was increased in cortical bone (p < 0.05) but decreased in trabecular bone (p < 0.05). These changes were accompanied by decreased TNAP (p < 0.01) and increased PHOSPHO1 (p < 0.001) expression in wild-type CKD bones. In P1KO CKD mice, the cortical BMD phenotype was rescued, suggesting that the increased cortical BMD of CKD mice was driven by increased PHOSPHO1 expression. Other structural parameters were also improved in P1KO CKD mice. We further investigated the driver of the mineralization defects, by studying the effects of FGF23, PTH, and phosphate administration on PHOSPHO1 and TNAP expression by primary murine osteoblasts. We found both PHOSPHO1 and TNAP expression to be down-regulated in response to phosphate and PTH. While matrix mineralization was increased with phosphate (Pi), it decreased with PTH and FGF23 had no effect. The in vitro data suggest that the TNAP reduction in CKD-MBD is driven by the hyperphosphatemia and/or hyperparathyroidism noted in these mice, while the higher PHOSPHO1 expression may be a compensatory mechanism in an attempt to protect the bone from hypomineralization. We propose that increased PHOSPHO1 expression in ROD may contribute to the disordered skeletal mineralization characteristic of this progressive disorder.

cell biology↗

The effect of handrail cross-sectional design and age on applied handrail forces during reach-to-grasp balance reactions

Handrails have been shown to reduce the likelihood of falls. Despite common use, little is known about how handrail shape and size affect the forces that people can apply after balance loss, and how these forces and the corresponding ability to recover balance depend on age. Following rapid platform translations, 16 older adults and 16 sex-matched younger adults recovered their balance using seven handrail cross-sections varying in shape and size. Younger adults were able to withstand higher perturbations, but did not apply higher forces, than older adults. However, younger adults achieved their peak resultant force more quickly, which may reflect slower rates of force generation with older adults. Considering handrail design, the 38mm round handrails allowed participants to successfully recover from the largest perturbations and enabled the highest force generation. Conversely, tapered handrails had the poorest performance, resulting in the lowest force generation and withstood perturbation magnitudes. Our findings suggest that the handrail cross-sectional design affects the magnitude of force generation and may impact the success of recovery. Our findings can inform handrail design recommendations that support effective handrail use in demanding, balance recovery scenarios.

bioengineering↗

Does increased gait variability improve stability when faced with an expected balance perturbation during treadmill walking?

BackgroundCurrently, there is uncertainty as to whether movement variability is errorful or exploratory. Research questionThis study aimed to determine if gait variability represents exploration to improve stability. We hypothesized that 1) spatiotemporal gait features will be more variable prior to an expected perturbation than during unperturbed walking, and 2) increased spatiotemporal gait variability pre-perturbation will correlate with improved stability post-perturbation. MethodsSixteen healthy young adults completed 15 treadmill walking trials within a motion simulator under two conditions: unperturbed and expecting a perturbation. Participants were instructed not to expect a perturbation for unperturbed trials, and to expect a single transient medio-lateral balance perturbation for perturbed trials. Kinematic data were collected during the trials. Twenty steps were recorded post-perturbation. Unperturbed and pre-perturbation gait variabilities were defined by the short- and long-term variabilities of step length, width, and time, using 100 steps from pre-perturbation and unperturbed trials. Paired t-tests identified between-condition differences in variabilities. Stability was defined as the number of steps to centre of mass restabilization post-perturbation. Multiple regression analyses determined the effect of pre-perturbation variability on stability. ResultsLong-term step width variability was significantly higher pre-perturbation compared to unperturbed walking (mean difference=0.28cm, p=0.0073), with no significant differences between conditions for step length or time variabilities. There was no significant relationship between pre-perturbation variability and post-perturbation restabilization. SignificanceIncreased pre-perturbation step width variability was neither beneficial nor detrimental to stability. However, the increased variability in medio-lateral foot placement suggests that participants adopted an exploratory strategy in anticipation of a perturbation.

neuroscience↗

Genotyping structural variants in pangenome graphs using the vg toolkit

Structural variants (SVs) remain challenging to represent and study relative to point mutations despite their demonstrated importance. We show that variation graphs, as implemented in the vg toolkit, provide an effective means for leveraging SV catalogs for short-read SV genotyping experiments. We benchmarked vg against state-of-the-art SV genotypers using three sequence-resolved SV catalogs generated by recent long-read sequencing studies. In addition, we use assemblies from 12 yeast strains to show that graphs constructed directly from aligned de novo assemblies improve genotyping compared to graphs built from intermediate SV catalogs in the VCF format.

bioinformatics↗

Role Of The Membrane Cholesterol-Glycosphingolipid Complex As A Transistor To Regulate GSL Receptor Function And Signaling Of Both Lipids

Cholesterol and glycosphingolipids (GSL) are the major species that accumulate in plasma membrane lipid rafts. These complexes imbue the membrane with increased order, which in turn, plays a central role in the transmembrane signaling foci lipid rafts provide. In addition, both GSL and cholesterol binding can mediate (separate) signal pathways. We have shown that cholesterol and GSLs however, form a complex in which the GSL sugar is reoriented from a membrane perpendicular to parallel format, becoming largely unavailable for exogenous ligand binding. Similarly, the steroid hydroxyl is masked, restricting access of cholesterol ligands. This was observed in model and cell membranes and in human tumour frozen tissue sections. We now show the order of exogenous ligand binding plays a significant role to determine the extent of GSL or cholesterol receptor activity. Ligand binding to cholesterol enhances subsequent GSL recognition and vice versa, suggesting that ligand binding to \"free\" receptor (membrane perpendicular GSL carbohydrate, nonmasked cholesterol) can result in partial dissociation of the GSL/cholesterol complex to allow additional GSL ligand and cholesterol ligand binding. Since many GSLs can complex with membrane cholesterol, the binding of a single cholesterol ligand may unmask cholesterol-complexed GSL for increased binding of both a single or multiple GSL-specific ligands. We show that multiple cholesterol-masked GSLs can be coincident in tissues. This provides a mechanism for GSL-dependent signal amplification and diversification, representing a biological transistor, regulating amplitude and potentially, diversity of GSL signaling. The process represents a new mechanism of cross-talk between GSL and cholesterol signaling. This is of clinical importance since we have found cholesterol/GSL masking applies to monoclonal anti GSL antibodies in development and in current use as antineoplastic therapeutics.

cell biology↗