Biology subjects

Holding, M. L.

Publications and source records attributed to Holding, M. L..

10 recordsLinked to original sources

bioRxiv2026

Snake Venom Fluidic Properties and Design of Venom Mimics as Rheological Surrogates

Snake venom composition and its contribution to toxic effects has been heavily researched, but there is a comparative lack of information on venoms fluidic properties and their relationship with fang morphology during the envenomation process. Understanding how venom flows through a fang can shed light on bite site dynamics and potentially explain bite symptoms. In this article we first conduct a broad comparative test of the rheological properties of venom from thirteen snake species, including multiple viperid and elapid snake species, revealing a shear-thinning non-Newtonian flow behavior in all studied species. However, we have not observed strong phylogenetic signal in venom fluidic properties, suggesting that flow properties may vary independently of evolutionary relationships between snake species. Second, we demonstrate that snake venoms fluidic properties can be modeled by other inexpensive, safe, and abundant shear-thinning surrogate fluids. We found that aqueous solutions of bovine serum albumin protein and xanthan gum are useful venom mimics, matching the rheological behavior of venoms from the studied snake species across a range of relevant shear rates. We further evaluated the performance of these snake venom mimics in a simulated venom delivery system, showing good and robust mimetic control of the flow properties as a function of applied pressure. By elucidating the fluidic properties of snake venom and providing a non-toxic, scalable surrogate fluid model to be used in further studies, we provide the biomedical, toxicology, evolutionary biology communities with a tool to study envenomation physics in an inexpensive and safe fashion. We suggest it is possible to design species-specific venom mimics that facilitate research on the biomechanics and fluid dynamics of venom delivery via snake bites, and inform the design of bioinspired puncture and injection devices.

bioengineering↗

bioRxiv2026

Human neurodevelopmental genes housed in massive, ancient gene deserts

Mammalian genomes contain a remarkable proportion of non-coding sequence relative to protein-coding sequence, including megabase-scale gene deserts. The origin of gene deserts and their function have long been mysteries, and most studies probing these questions have focused on specific deserts linked to transcription factors, like SOX9 and MYC. Here, we describe 21 human "lonely genes" located within some of the largest deserts in the genome, making them exceptionally isolated from other genes in linear sequence space. Together, these regions comprise roughly 3.4% of the human genome. Despite minimal sequence conservation, we find that the sizes and locations of these gene deserts are conserved, and they likely originated at the root of the vertebrate tree. In contrast to the deserts near transcription factors that have been the primary focus to date, we find that most genes housed within these massive deserts, as well as genes on the edges of deserts, are cell adhesion molecules. Additionally, many of the genes associated with these deserts are dysregulated in datasets involving chromatin modifiers linked to neurodevelopmental disorders. Using DNA FISH in mouse olfactory epithelium, we find that lonely genes appose the nuclear lamina. We propose that these ancient regions may play a structural role in the nucleus, and that this structure makes the genes within them uniquely difficult to express. While specific chromatin modifiers are able to access lonely genes, this dependence creates a regulatory vulnerability in brain development.

evolutionary biology↗

bioRxiv2026

Mapping the Mutational Landscape for Streptokinase Binding to Plasminogen

Group A Streptococcus (GAS) expresses streptokinase (SK), a critical virulence factor that non-enzymatically activates the hosts plasminogen (PLG), to an active form, PLGSK, resulting in the degradation of fibrin clots and subsequent bacterial dissemination. PLGSK formation contrasts with the physiologic activation of PLG to the serine protease plasmin via a proteolytic mechanism. As a potent thrombolytic, SK has been used as a therapeutic to treat heart attacks and strokes. GAS SK is highly specific for human PLG, and sequence variation between SK from different GAS strains has been linked to differences in PLG binding and disease severity. We now report the application of deep mutational scanning (DMS) to map the effects of [~]71% of single amino acid substitutions within SK from Group C Streptococcus, which shares high sequence homology with GAS on its ability to bind human PLG. We first demonstrate that SK expressed as a fusion protein to the p3 coat protein of M13 filamentous phage retains its capacity to bind human PLG. Our subsequent DMS analysis using this phage system identifies regions of SK in which amino acid substitutions are likely to increase or decrease its affinity for PLG. Our findings suggest a complex protein-protein interaction in which long-range protein dynamics influence the conformational activation of PLG to PLGSK. These data lay the foundation for linking SK variation between GAS strains to differences in virulence, mapping the determinants of GAS SKs human specificity, and potentially contributing to the development of improved therapeutics for heart attack and stroke.

microbiology↗

bioRxiv2025

Thermodynamics and selection of the plasminogen activator inhibitor-1 latency transition

Plasminogen activator inhibitor-1 (PAI-1), like other serine protease inhibitors (SERPINs), exists in a functionally active metastable conformation. Unlike other SERPINs, PAI-1 undergoes a relatively rapid rearrangement to a lower energy latent conformation. We employ deep mutational scanning (DMS) to simultaneously probe the effects of nearly all single amino acid substitutions (92%) in PAI-1 on its latency transition as well as its ability to inhibit its canonical protease target, urokinase-type plasminogen activator (uPA). The DMS results are interpreted in the context of variant effect predictors (EVE, AlphaMissense, and CPT) and protein stability predictors (EvoEF and FoldX), as well as the evolutionary conservation of the PAI-1 sequence space across extant vertebrate species. We demonstrate that while variant effect predictors can generally partition PAI-1 as functional or non-functional with respect to uPA inhibition, they perform poorly when attempting to discriminate the effects of PAI-1 variants on its latency transition. However, by employing protein stability predictors, we demonstrate that the PAI-1 latency transition is most likely driven by changes in the energy barrier to the latency transition. Finally, we show that PAI-1s ability to inhibit uPA, as well as its thermodynamic instability in its active conformation are both under purifying selection that limits the sequence space available to PAI-1. These findings suggest that DMS of collateral fitness effects, including PAI-1s latency transition, may be better interpreted in contexts other than variant effect predictors, including protein thermodynamics and evolution.

biochemistry↗

bioRxiv2025

Characterizing venom resistance in Monodelphis domestica yields new insights into mammalian blood physiology

Interactions between predators and prey are often characterized by strong selection pressures that shape extreme physiological adaptations. Venom resistance in large-bodied South American opossums (Clade Didelphini) is a striking example, as these marsupials prey on venomous snakes and exhibit remarkable resistance to their venom. While resistance is well documented in Didelphini, relatively little is known about venom resistance in the smaller, more diverse members of Didelphidae, which inhabit the same regions and encounter the same predators. Here, we investigate venom resistance in the small-bodied opossum, Monodelphis domestica, through multi-level physiological assays, examining responses to purified venom components and whole venom from sympatric and allopatric vipers. Our results show M. domestica resists venom-induced disruptions to blood coagulation, retains platelet function in the presence of platelet-disrupting venoms, and inhibits snake venom metalloproteinases. Unexpectedly, we find that M. domestica von Willebrand Factor (VWF) requires increased shear force to elongate, a previously unknown aspect of opossum blood physiology that may contribute to venom resistance and may have relevance to human coagulopathies. These findings expand the extent of venom resistance beyond large-bodied Didelphini, suggesting it is a widespread trait in South American marsupials and providing new insights into venom-mammal coevolution. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=140 SRC="FIGDIR/small/634112v2_ufig1.gif" ALT="Figure 1"> View larger version (67K): org.highwire.dtl.DTLVardef@1efb74forg.highwire.dtl.DTLVardef@ebcb82org.highwire.dtl.DTLVardef@7ddef2org.highwire.dtl.DTLVardef@1b81569_HPS_FORMAT_FIGEXP M_FIG C_FIG

evolutionary biology↗

bioRxiv2025

Tandem duplication of serpin genes yields functional variation and snake venom inhibitors.

Tandem duplication of genes can play a critical role in the evolution of functional novelty, but our understanding is limited concerning gene duplications role in coevolution between species. Much is known about the evolution and function of tandemly duplicated snake venom genes, however the potential of gene duplication to fuel venom resistance within prey species is poorly understood. In this study, we characterize patterns of gene duplication of the SERPINA subfamily of genes across in vertebrates and experimentally characterize functional variation in the SERPINA3-like paralogs of a wild rodent. We find the hallmarks of rapid birth-death evolution of SERPINA1-like and SERPINA3-like genes within and between rodent lineages. Next, we recombinantly expressed the 2 paralogous duplicates of SERPINA1 and 12 paralogous duplicates of SERPINA3 found in the genome of the big-eared woodrat (Neotoma macrotis), a species known to be resistant to protease-rich rattlesnake venoms. We found that two SERPINA3 paralogs inhibit snake venom serine proteases, indicating that these proteins have potential as resistance factors in SERPIN-mediated venom resistance. In addition, functional variation is apparent among paralogs, including neofunctionalization to inhibit both chymotrypsin-like and and trypsin-like proteases simultaneously for one venom-inhibiting paralog. Our results provide further evidence that the rapid evolution of SERPINA1 and SERPINA3 gene copy number across rodents has adaptive potential by producing functionally-diverse inhibitors.

evolutionary biology↗

bioRxiv2024

High-throughput amino acid-level characterization of the interactions of plasminogen activator inhibitor-1 with variably divergent proteases

While members of large paralogous protein families share structural features, their functional niches often diverge significantly. Serine protease inhibitors (SERPINs), whose members typically function as covalent inhibitors of serine proteases, are one such family. Plasminogen activator inhibitor-1 (PAI-1) is a prototypic SERPIN, which canonically inhibits tissue-and urokinase-type plasminogen activators (tPA and uPA) to regulate fibrinolysis. PAI-1 has been shown to also inhibit other serine proteases, including coagulation factor XIIa (FXIIa) and transmembrane serine protease 2 (TMPRSS2). The structural determinants of PAI-1 inhibitory function toward these non-canonical protease targets, and the biological significance of these functions, are unknown. We applied deep mutational scanning (DMS) to assess the effects of [~]80% of all possible single amino acid substitutions in PAI-1 on its ability to inhibit three putative serine protease targets (uPA, FXIIa, and TMPRSS2). Selection with each target protease generated a unique PAI-1 mutational landscape, with the determinants of protease specificity distributed throughout PAI-1s primary sequence. Next, we conducted a comparative analysis of extant orthologous sequences, demonstrating that key residues modulating PAI-1 inhibition of uPA and FXIIa, but not TMPRSS2, are maintained by purifying selection. PAI-1s activity toward FXIIa may reflect how protease evolutionary relationships predict SERPIN functional divergence, which we support via a cophylogenetic analysis of all secreted SERPINs and their cognate serine proteases. This work provides insight into the functional diversification of SERPINs and lays the framework for extending these studies to other proteases and their regulators.

evolutionary biology↗

bioRxiv2024

Characterizing population structure and documenting rapid loss of genetic diversity in Chiricahua Leopard Frogs (Lithobates chiricahuensis) with high throughput microsatellite genotyping

The use of molecular markers to assess genetic diversity has become a common component of recovery action plans for threatened and endangered species. In this study, we use an unusually large number of microsatellite markers (N=91) to characterize the genetic variation of Chiricahua Leopard Frogs (Lithobates chiricahuensis) across their range in order to understand their distribution of genetic variation, identify genetic bottlenecks, and measure genetic changes over time in a single, highly-managed population. Populations were best divided into three genetically distinct clusters, with the southeastern Arizona and New Mexico populations forming distinct genetic clusters. While there is moderate genetic variation distributed across the sampled populations, each population on its own shows relatively low allelic diversity. Most populations displayed strong genetic signals of recent genetic bottlenecks or a deficiency of heterozygous genotypes that is typically associated with frequent inbreeding. Populations that have a history of no management through translocations harbored the greatest number of unique alleles and overall allelic richness, especially in a subset of the Mexican populations. Finally, long-term cohort sampling at one specific site (the Southwestern Research Station in Portal, Arizona) allowed us to demonstrate how rapidly genetic diversity can decrease across a matter of years in a population with few founders. This work shows how microsatellite markers can provide important context for conservation agencies, but even a large suite of markers beyond what is typical may not be enough for populations that are extremely bottlenecked and have low levels of standing genetic diversity.

genetics↗

bioRxiv2022

Regulation of cellular LDL uptake by PROX1 and CHD7

An elevated level of low-density lipoprotein (LDL) in the bloodstream is a causal risk factor for atherosclerotic cardiovascular disease (ASCVD). The low-density lipoprotein receptor (LDLR) is a critical regulator of circulating LDL, and increasing LDLR activity is an effective therapeutic approach to reduce circulating LDL cholesterol levels. In this study, we characterize PROX1 and CHD7, two genes we previously identified in a genome-scale CRISPR screen as positive regulators of LDL uptake in HuH7 cells. We found that although disruption of either PROX1 or CHD7 significantly reduced LDL uptake, only PROX1 disruption significantly reduced the cellular levels of LDLR mRNA and surface-displayed LDLR protein. Consistent with a direct role for PROX1 in LDLR gene regulation, we also observed in publicly available data sets the presence of two liver-specific PROX1 binding sites near the LDLR locus, one of which colocalized with biochemical hallmarks of enhancer activity in hepatic tissue. Both PROX1 LDLR binding sites contained predicted PROX1 transcription factor binding motifs and colocalized with binding sites for HNF4, a known interactor for PROX1 and regulator of hepatic lipid metabolism and LDL uptake. In contrast to PROX1, no CHD7 binding sites were detected near the LDLR locus. Together, our results support a model in which both PROX1 and CHD7 promote cellular LDL uptake through distinct mechanisms, with PROX1 directly promoting LDLR gene expression and CHD7 functioning through an LDLR-independent pathway.

molecular biology↗

bioRxiv2021

Trio-binned genomes of the woodrats Neotoma bryanti and N. lepida reveal novel gene islands and rapid copy number evolution of xenobiotic metabolizing cytochrome p450 genes

The genomic architecture underlying the origins and maintenance of biodiversity is an increasingly accessible feature of species, due in large part to third-generation sequencing and novel analytical toolsets. Woodrats of the genus Neotoma provide a unique opportunity to study how vertebrate herbivores respond to climate change, as two sister species (N. bryanti and N. lepida) independently achieved a major dietary feat - switching to the novel and toxic food source creosote bush (Larrea tridentata) - in the aftermath of a natural warming event. To better understand the genetic mechanisms underlying this ability, we employed a trio binning sequencing approach with a N. bryanti x N. lepida F1 hybrid, resulting in phased, chromosome-level, highly complete, haploid genome assemblies for each species from one individual. Using these new assemblies, we explored the genomic architecture of three cytochrome p450 subfamilies (2A, 2B, and 3A) that play key roles in the metabolism of naturally occurring toxic dietary compounds. We found that woodrats show expansions of all three p450 gene families, including the evolution of multiple novel gene islands within the 2B and 3A subfamilies. Our assemblies demonstrate that trio binning from an F1 hybrid rodent effectively recovers parental genomes from species that diverged more than a million years ago. Turnover and novelty in detoxification gene islands in herbivores is widespread within distinct p450 subfamilies, and may have provided the crucial substrate for dietary adaptation during environmental change.

genomics↗