Biology subjects

Scott, A.

Publications and source records attributed to Scott, A..

At least 37 records · Page 2Linked to original sources

bioRxiv2024

Nemo knows: clownfishes differentiate cryptic host species across fine and broad geographic scales and reveal a potential adaptive radiation in the clownfish-hosting sea anemones.

The symbiosis between clownfishes (or anemonefishes) and their host sea anemones ranks among the most recognizable animal interactions on the planet. Found on coral reef habitats across the Indian and Pacific Oceans, 28 recognized species of clownfishes adaptively radiated from a common ancestor to live obligately with only 10 nominal species of host sea anemones. Are the host sea anemones truly less diverse than clownfishes? Did the symbiosis with clownfishes trigger a reciprocal adaptive radiation in sea anemones, or minimally, a co-evolutionary response to the mutualism? To address these questions, we combined fine- and broad-scale biogeographic sampling with multiple independent genomic datasets for the bubble-tip sea anemone, Entacmaea quadricolor--the most common clownfish host anemone throughout the Indo-West Pacific. Fine-scale sampling and restriction site associated DNA sequencing (RADseq) throughout the Japanese Archipelago revealed three highly divergent cryptic species: two of which co-occur throughout the Ryukyu Islands and can be differentiated by the clownfish species they host. Remarkably, broader biogeographic sampling and bait-capture sequencing reveals that this pattern is not simply the result of local ecological processes unique to Japan, but part of a deeper evolutionary signal where some species of E. quadricolor serve as host to the generalist clownfish species Amphiprion clarkii and others serve as host to the specialist clownfish A. frenatus. In total, we delimit at least five cryptic species in E. quadricolor that have diversified within the last five million years. The rapid diversification of E. quadricolor combined with functional ecological and phenotypic differentiation supports the hypothesis that this may represent an adaptive radiation in response to mutualism with clownfishes. Our data indicate that clownfishes are not merely settling in locally available hosts but recruiting to specialized host lineages with which they have co-evolved. These findings have important implications for understanding how the clownfish-sea anemone symbiosis has evolved and will shape future research agendas on this iconic model system.

evolutionary biology↗

bioRxiv2024

Characterization of Multicellular Niches Supporting Hematopoietic Stem Cells Within Distinct Zones

Previous studies of hematopoietic stem cells (HSCs) primarily focused on single cell-based niche models, yielding fruitful but conflicting findings1-5. Here we report our investigation on the fetal liver (FL) as the primary fetal hematopoietic site using spatial transcriptomics. Our study reveals two distinct niches: the portal-vessel (PV) niche and the sinusoidal niche. The PV niche, composing N-cadherin (N-cad)HiPdgfr+ mesenchymal stromal cells (MSCs), endothelial cells (ECs), and N-cadLoAlbumin+ hepatoblasts, maintains quiescent and multipotential FL-HSCs. Conversely, the sinusoidal niche, comprising ECs, hepatoblasts and hepatocytes, as well as potential macrophages and megakaryocytes, supports proliferative FL-HSCs biased towards myeloid lineages. Unlike prior reports on the role of Cxcl12, with its depletion from vessel-associated stromal cells leading to 80% of HSCs reduction in the adult bone marrow (BM)6,7, depletion of Cxcl12 via Cdh2CreERT (encoding N-cad) induces altered localization of HSCs from the PV to the sinusoidal niches, resulting in an increase of HSC number but with myeloid-bias. Similarly, we discovered that adult BM encompasses two niches within different zones, each composed of multi-cellular components: trabecular bone area (TBA, or metaphysis) supporting deep-quiescent HSCs, and central marrow (CM, or diaphysis) fostering heterogenous proliferative HSCs. This study transforms our understanding of niches by shifting from single cell-based to multicellular components within distinct zones, illuminating the intricate regulation of HSCs tailored to their different cycling states.

developmental biology↗

bioRxiv2024

Ventral pallidal GABAergic neurons drive consumption in male, but not female rats

Food intake is controlled by multiple converging signals: hormonal signals that provide information about energy homeostasis, but also hedonic and motivational aspects of food and food cues that can drive non-homeostatic or "hedonic "feeding. The ventral pallidum (VP) is a brain region implicated in the hedonic and motivational impact of food and foods cues, as well as consumption of rewards. Disinhibition of VP neurons has been shown to generate intense hyperphagia, or overconsumption. While VP gamma-Aminobutyric acidergic (GABA) neurons have been implicated in cue-elicited reward seeking and motivation, the role of these neurons in the hyperphagia resulting from VP activation remains unclear. Here, we used Designer Receptors Exclusively Activated by Designer Drugs (DREADDs) to activate or inhibit VP GABA neurons in sated male and female rats during chow and sucrose consumption. We found that activation of VP GABA neurons increases consumption of chow and sucrose in male rats, but not female rats. We also found that, while inhibition of VP GABA neurons tended to decrease sucrose consumption, this effect was not statistically significant. Together, these findings suggest that activation of VP GABA neurons can stimulate consumption of routine or highly palatable rewards selectively in male rats.

neuroscience↗

bioRxiv2024

Defining mononuclear phagocyte distribution and behaviour in the zebrafish heart

Mononuclear phagocytes (MNPs) are recognised as highly plastic, multifunctional cells that influence multiple physiological and pathophysiological states. In the heart, they support homeostatic functions, contribute to disease progression and play multiple roles in reparative and regenerative processes following tissue damage. Understanding the heterogeneous populations of cells that contribute to these diverse functions is crucial to facilitating beneficial, and limiting adverse, cardiac outcomes. However, characterisation of precise populations of cardiac immune cells remains incomplete in vertebrate models capable of endogenous regeneration, such as adult zebrafish. Here, we use a combination of transgenic lines to identify distinct MNPs in the zebrafish heart. We show that larval macrophage populations have different origins and a sub-population of csf1ra expressing cells are maintained on the surface of the adult heart. MNPs are differently distributed in the myocardium, exhibit different behaviours and are distinguished via expression level of csf1ra and mpeg1.1. Following injury, tissue resident macrophages rapidly proliferate potentially contributing to reduced scarring. The adult zebrafish heart contains multiple populations of MNPs that can be defined by existing tools. This new understanding of innate immune cell populations in the heart of adult zebrafish sheds light on the composition of a pro-regenerative cardiac microenvironment.

immunology↗

bioRxiv2024

Phylogenomics reveals coincident divergence between giant host sea anemones and the clownfish adaptive radiation.

The mutualism between clownfishes (or anemonefishes) and their giant host sea anemones are among the most immediately recognizable animal interactions on the planet and have attracted a great deal of popular and scientific attention [1-5]. However, our evolutionary understanding of this iconic symbiosis comes almost entirely from studies on clownfishes-- a charismatic group of 28 described species in the genus Amphiprion [2]. Adaptation to venomous sea anemones (Anthozoa: Actiniaria) provided clownfishes with novel habitat space, ultimately triggering the adaptive radiation of the group [2]. Clownfishes diverged from their free-living ancestors 25-30 MYA with their adaptive radiation to sea anemones dating to 13.2 MYA [2, 3]. Far from being mere habitat space, the host sea anemones also receive substantial benefits from hosting clownfishes, making the mutualistic and co-dependent nature of the symbiosis well established [4, 5]. Yet the evolutionary consequences of mutualism with clownfishes have remained a mystery from the host perspective. Here we use bait-capture sequencing to fully resolve the evolutionary relationships among the 10 nominal species of clownfish-hosting sea anemones for the first time (Figure 1). Using time-calibrated divergence dating analyses we calculate divergence times of less than 25 MYA for each host species, with 9 of 10 host species having divergence times within the last 13 MYA (Figure 1). The clownfish-hosting sea anemones thus diversified coincidently with clownfishes, potentially facilitating the clownfish adaptive radiation, and providing the first strong evidence for co-evolutionary patterns in this iconic partnership. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=187 SRC="FIGDIR/small/576469v1_fig1.gif" ALT="Figure 1"> View larger version (58K): org.highwire.dtl.DTLVardef@7053b4org.highwire.dtl.DTLVardef@876c65org.highwire.dtl.DTLVardef@dd6d9forg.highwire.dtl.DTLVardef@147fddd_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFigure 1.C_FLOATNO Diversification of the clownfish-hosting sea anemones was coincident with the clownfish adaptive radiation. A) Time-calibrated maximum likelihood cladogram of Order Actiniaria based on 328 ultra-conserved element and exon loci (75% data occupancy matrix). The three clades containing the clownfish hosting sea anemones are highlighted reflecting the multiple evolutionary origins of symbiosis with clownfishes within superfamily Actinioidea. B-D) Detailed time-calibrated maximum likelihood cladograms of Entacmaea quadricolor, Clade Stichodactylina, and Clade Heteractina, respectively. In all panels, the orange line denotes the beginning of the clownfish adaptive radiation 13 MYA. Sea anemone superfamilies Actinostoloidea, Metridioidea, Actiniernoidea, and Edwardsioidea are collapsed for clarity. C_FIG

evolutionary biology↗

bioRxiv2023

Modeling Cystic Fibrosis Chronic Infection Using Engineered Mucus-like Hydrogels

The airway mucus of patients with cystic fibrosis has altered properties which create a microenvironment primed for chronic infections that are difficult to treat. These complex polymicrobial airway infections and corresponding mammalian-microbe interactions are challenging to model in vitro. Here, we report the development of mucus-like hydrogels with varied compositions and viscoelastic properties reflecting differences between healthy and cystic fibrosis airway mucus. Models of cystic fibrosis and healthy airway microenvironments were created by combining the hydrogels with relevant pathogens, human bronchial epithelial cells, and an antibiotic. Notably, pathogen antibiotic resistance was not solely dependent on the altered properties of the mucus-like hydrogels but was also influenced by culture conditions including microbe species, monomicrobial or polymicrobial culture, and the presence of epithelial cells. Additionally, the cystic fibrosis airway model showed the ability to mimic features characteristic of chronic cystic fibrosis airway infections including sustained polymicrobial growth and increased antibiotic tolerance.

bioengineering↗

bioRxiv2023

Ventral pallidum projections to the ventral tegmental area reinforce but do not invigorate reward-seeking behavior

Reward-predictive cues acquire motivating and reinforcing properties that contribute to the escalation and relapse of drug use in addiction. The ventral pallidum (VP) and ventral tegmental area (VTA) are two key nodes in brain reward circuitry implicated in addiction and necessary for the performance of cue-driven behavior. Evidence suggests that VP neurons projecting to the VTA (VP[->]VTA) promote cue-induced reinstatement of drug-seeking, but the mechanisms by which these neurons do so are undefined. In addition, the role of these neurons in the pursuit of non-drug reward is not known. In the current study, we used in vivo fiber photometry and optogenetics to record from and manipulate VP[->]VTA in rats performing a discriminative stimulus task (DS task) with sucrose reward to determine the fundamental role these neurons play in invigoration and reinforcement by reward and associated discriminative cues. We find that VP[->]VTA neurons are selectively active during reward consumption, that optogenetic stimulation of these neurons paired with reward consumption biases choice, and that VP[->]VTA optogenetic stimulation is reinforcing. Critically, we found no significant encoding of cue-elicited reward-seeking vigor and acute optogenetic stimulation of these neurons paired with cue onset did not enhance the probability or vigor of reward-seeking. Our results suggest that VP[->]VTA neurons are active during the consumption of natural reward and that this activity reinforces seeking behavior.

neuroscience↗

bioRxiv2023

Potential selection and maintenance of manure-originated multi-drug resistant plasmids at sub-clinical antibiotic concentrations

The goal of this study was to determine minimum selection concentrations of various antibiotics using four manure-originated multi-drug resistant plasmids in a surrogate Escherichia coli host. These plasmids carried genes conferring resistance phenotypes to several antibiotic classes including {beta}-lactams, lincosamides, phenicols, macrolides, sulfonamides and tetracyclines. The minimum selection concentrations of antibiotics tested in nutrient-rich medium were determined: 14.1-28.2 mg/L for penicillin G, 0.1 mg/L for oxytetracycline, 0.45 mg/L for chlortetracycline, 2 mg/L for lincomycin, 1 mg/L for florfenicol, 1.3-4 mg/L for azithromycin, 0.13-0.25 mg/L for tetracycline, 0.004-0.01 mg/L for cefotaxime. Penicillin G, oxytetracycline, chlortetracycline, lincomycin and florfenicol had minimum selection concentrations in nutrient-defined medium slightly changed within 3.5-fold range compared to those in nutrient-rich medium. The minimum selection concentrations of antibiotics interfering folic acid synthesis in bacteria were also determined: 63 mg/L for sulfamethoxazole, 11.2 mg/L for sulfisoxazole and 0.06 mg/L for trimethoprim. Mixing two antibiotics changed minimum selection concentrations within 3.7-fold range compared to those in single antibiotic tests. Relatively high plasmid loss rates (> 90%) were observed when culturing plasmid-bearing strains in antibiotic-free nutrient-rich and nutrient-defined media. Overall results suggested that these plasmids can be maintained at concentrations environmentally relevant in waste water treatment plants, sewage, manure and manured soil although they are not stable in antibiotic-free environments. IMPORTANCEAntibiotic resistance crisis is a grave concern in healthcare systems around the world. To combat this crisis, we sought to find out how likely manure-originated multi-drug resistant plasmids are to be selected and maintained in different environment matrices. Our study showed that these plasmids conferring resistance to {beta}-lactams, lincosamides, phenicols, macrolides, sulfonamides and tetracyclines can be selected at minimum selection concentrations which are lower than minimum inhibition concentrations of the E. coli host strain. Lincomycin, oxytetracycline, chlortetracycline, tetracycline, chloramphenicol, trimethoprim had minimum selection concentrations lower than the antibiotic concentrations in several environment matrices reported previously. Our findings suggest that despite the burden and the high rate of plasmid loss, these plasmids can still be selected, maintained and circulated well in some polluted environments.

microbiology↗

bioRxiv2023

Genetic characterization of extended-beta-lactamase (ESBL) plasmids captured from dairy manures

This study was to assess the gene diversity and characterize a large set of plasmids harboring extended {beta}-lactamase (ESBL) genes from raw and digested dairy manure. A total of eighty-four plasmids that were captured in this E. coli recipient were sequenced using Illumina MiSeq sequencing technology. Twenty-four plasmids of interest were subsequently sequenced using MinION technology in order that a hybrid assembly could be performed on short-and long-read sequences to circularize and complete these plasmids. The size of sequenced plasmids ranged between 40 and 260 kb with various incompatibility groups: IncC, IncI1, IncN, IncY, IncB/O/K/Z, IncX1, IncHI2, IncHI2A, IncFIB(K), IncFII. A variety of extended {beta}-lactamase genes were identified: blaCTXM -1, blaCTXM -14, blaCTXM -15, blaCTXM-27, blaCTXM-55, blaCTXM-61, blaPER-1, blaIMP-27. Interestingly, the blaIMP-27 gene, a novel metallo-{beta}-lactamase discovered in the last decade, was found located on an integrated region in the host chromosome. And one plasmid carrying the blaCMY-2 gene, an AmpC gene, also expressed ESBL phenotype. Four virulence factors, including cia, cib, traT and terC, were detected on some of these plasmids. In addition, six type-2 toxin-antitoxin systems were detected: MazF/E, PemK/I, HipA/B, YdcE/D, RelB/E and HigB/A. Twenty-two out of twenty-four complete plasmids carried putative prophage regions; and most of prophage hits were marked as incomplete, except that the largest plasmid pT525A and the IncY plasmid pT415A had prophage hits with higher scores. IMPORTANCEThe widespread of antibiotic resistant bacteria is largely due to the exchange of mobile genetic elements such as plasmids. Plasmids harboring extended {beta}-lactamase (ESBL) genes originated from dairy manure potentially become entrained in manured soil, which subsequently enter the human food chain. Currently there is a lack of detailed information on these plasmids in the environment, specifically in dairy manure. This study unveils the abundance and diversity of ESBL-carrying plasmids from both raw and digested manures which were captured in gfp-labelled E. coli CV601. In addition, the study provides insightful information of plasmid characteristics including incompatibility groups, ESBL genes combined with other resistance genes, mobile genetic elements (transposons, insertion sequence), toxin-antitoxin systems, virulence factors and prophage sequences.

microbiology↗

bioRxiv2023

Seagrass genomes reveal a hexaploid ancestry facilitating adaptation to the marine environment

Seagrasses comprise the only submerged marine angiosperms, a feat of adaptation from three independent freshwater lineages within the Alismatales. These three parallel lineages offer the unique opportunity to study convergent versus lineage-specific adaptation to a fully marine lifestyle. Here, we present chromosome-level genome assemblies from a representative species of each of the seagrass lineages - Posidonia oceanica (Posidoniaceae), Cymodocea nodosa (Cymodoceaceae), and Thalassia testudinum (Hydrocharitaceae) - along with an improved assembly for Zostera marina (Zosteraceae). We also include a draft genome of Potamogeton acutifolius, a representative of Potamogetonaceae, the freshwater sister lineage to the Zosteraceae. Genome analysis reveals that all seagrasses share an ancient whole genome triplication (WGT) event, dating to the early evolution of the Alismatales. An additional whole genome duplication (WGD) event was uncovered for C. nodosa and P. acutifolius. Dating of ancient WGDs and more recent bursts of transposable elements correlate well with major geological and recent climatic events, supporting their role as rapid generators of genetic variation. Comparative analysis of selected gene families suggests that the transition from the submerged-freshwater to submerged-marine environment did not require revolutionary changes. Major gene losses related to, e.g., stomata, volatiles, defense, and lignification, are likely a consequence of the submerged lifestyle rather than the cause ( use it or lose it). Likewise, genes, often retained from the WGD and WGT, were co-opted for functions requiring the alignment of many small adaptations ( tweaking), e.g., osmoregulation, salinity, light capture, carbon acquisition, and temperature. Our ability to manage and conserve seagrass ecosystems depends on our understanding of the fundamental processes underpinning their resilience. These new genomes will accelerate functional studies and are expected to contribute to transformative solutions -- as continuing worldwide losses of the savannas of the sea are of major concern in times of climate change and loss of biodiversity.

evolutionary biology↗

bioRxiv2022

Ventral pallidal GABAergic neuron calcium activity encodes cue-driven reward-seeking and persists in the absence of reward delivery

Reward-seeking behavior is often initiated by environmental cues that signal reward availability. This is a necessary behavioral response; however, cue reactivity and reward-seeking behavior can become maladaptive. To better understand how cue elicited reward-seeking becomes maladaptive, it is important to understand the neural circuits involved in assigning appetitive value to rewarding cues and actions. Ventral pallidum (VP) neurons are known to contribute to cue elicited reward-seeking behavior and have heterogeneous responses in a discriminative stimulus (DS) task. The VP neuronal subtypes and output pathways that encode distinct aspects of the DS task remain unknown. Here, we used an intersectional viral approach with fiber photometry to record bulk calcium activity in VP GABAergic (VP GABA) neurons in male and female rats as they learned and performed the DS task. We found that VP GABA neurons are excited by reward-predictive cues but not neutral cues, and that this response develops over time. We also found that this cue-evoked response predicts reward-seeking behavior. Additionally, we found increased VP GABA calcium activity at the time of expected reward delivery, which occurred even on trials when reward was omitted. Together, these findings suggest that VP GABA neurons encode reward expectation and calcium activity in these neurons is predictive of the vigor of cue-elicited reward-seeking.

neuroscience↗

bioRxiv2022

Paracrine CSF1 signaling regulates macrophage migration dynamics towards ovarian cancer cells in a 3D microfluidic model that recapitulates in vivo infiltration patterns in patient-derived xenograft models

Ovarian cancer is the second most deadly gynecologic cancer in the United States, and tumorassociated macrophages in the ovarian cancer microenvironment are the most abundant immune cell type and are associated poor survival. Here, we utilize three-dimensional microfluidic assays to investigate the dynamics of macrophage infiltration towards ovarian cancer cells. Experimental results demonstrate that both ovarian cancer cell lines and patient-derived xenograft models promote the infiltration of macrophages into a 3D collagen type I extracellular matrix. Additionally, blocking CSF1 signaling reduced the number of recruited macrophages as well as migration speed, while macrophage recruitment was enhanced by addition of recombinant CSF1. We further demonstrated that results obtained with our microfluidic model are consistent with the recruitment of macrophages in vivo by patient-derived xenograft models, and that a xenograft model with high CSF1 expression showed an enhanced ability to recruit macrophages both in vitro and in vivo. These results highlight the role of CSF1 signaling in ovarian cancer, as well as the utility of microfluidic models in recapitulating the 3D ovarian cancer microenvironment.

bioengineering↗

bioRxiv2021

Epigenetic remodeling during monolayer cell expansion reduces therapeutic potential

Understanding how cells remember previous mechanical environments to influence their fate, or mechanical memory, informs the design of biomaterials and therapies in medicine. Current regeneration therapies require two-dimensional (2D) cell expansion processes to achieve large cell populations critical for the repair of damaged (e.g. connective and musculoskeletal) tissues. However, the influence of mechanical memory on cell fate following expansion is unknown, and mechanisms defining how physical environments influence the therapeutic potential of cells remain poorly understood. Here, we show that the organization of histone H3 trimethylated at lysine 9 (H3K9me3) and expression of tissue-identifying genes in primary cartilage cells (chondrocytes) transferred to three-dimensional (3D) hydrogels depends on the number of previous population doublings on tissue culture plastic during 2D cell expansion. Decreased levels of H3K9me3 occupying promoters of dedifferentiation genes after the 2D culture were also retained in 3D culture. Suppression of H3K9me3 during expansion of cells isolated from a murine model similarly resulted in the loss of the chondrocyte phenotype and global remodeling of nuclear architecture. In contrast, increasing levels of H3K9me3 through inhibiting H3K9 demethylases partially rescued the chondrogenic nuclear architecture and gene expression, which has important implications for tissue repair therapies, where expansion of large numbers of phenotypically-suitable cells is required. Overall, our findings indicate mechanical memory in primary cells is encoded in the chromatin architecture, which impacts cell fate and the phenotype of expanded cells. SIGNIFICANCE STATEMENTTissue regeneration procedures, such as cartilage defect repair (e.g. Matrix-induced Autologous Chondrocyte Implantation) often require cell expansion processes to achieve sufficient cells to transplant into an in vivo environment. However, the chondrocyte cell expansion on 2D stiff substrates induces epigenetic changes that persist even when the chondrocytes are transferred to a different (e.g. 3D) or in vivo environment. Treatments to alter epigenetic gene regulation may be a viable strategy to improve existing cartilage defect repair procedures and other tissue engineering procedures that involve cell expansion.

bioengineering↗

bioRxiv2021

Estrogen regulates early embryonic development of the olfactory sensory system via estrogen responsive glia

Estrogen is well-known to regulate development of sexual dimorphisms of the brain, however its role in the brain during early embryonic development prior to sex-differentiation is unclear. Using estrogen biosensor zebrafish models, we found that estrogen activity in the embryonic brain occurs specifically in a type of glia located within the OB, which we name estrogen-responsive olfactory bulb/EROB cells. With estrogen activity, EROB cells extend their ramified projections that overlay the OB outermost layer and tightly interact with olfactory sensory neurons (OSNs) at the olfactory glomeruli. Pharmacologically inhibiting estrogen activity and/or EROB cell ablation impedes olfactory glomerular development, including OSN pathfinding, topological organisation of olfactory glomeruli and inhibitory neurogenesis in the OB. Furthermore, activation of this estrogen/EROB-dependent mechanism decreases the intrinsic neuronal activity primarily in the OB, and this alteration of estrogen signalling disrupts olfaction-mediated behaviour. We propose that estrogen acts on glia to regulate development of functional OB circuits, thereby modulating the local intrinsic excitability in the OB and olfaction-mediated behaviour. Our data also suggest a possibility that the estrogen/EROB cascade may be an important site of action for environmental estrogens causative of neurodevelopmental impairments in animals and humans.

developmental biology↗

bioRxiv2021

Spatially Interacting Phosphorylation Sites and Mutations in Cancer

Advances in mass-spectrometry have generated increasingly large-scale proteomics datasets containing tens of thousands of phosphorylation sites (phosphosites) that require prioritization. We develop a bioinformatics tool called HotPho and systematically discover 3D co-clustering of phosphosites and cancer mutations on protein structures. HotPho identifies 474 such hybrid clusters containing 1,255 co-clustering phosphosites, including RET p.S904/Y928, the conserved HRAS/KRAS p.Y96, and IDH1 p.Y139/IDH2 p.Y179 that are adjacent to recurrent mutations on protein structures not found by linear proximity approaches. Hybrid clusters, enriched in histone and kinase domains, frequently include expression-associated mutations experimentally shown as activating and conferring genetic dependency. Approximately 300 co-clustering phosphosites are verified in patient samples of 5 cancer types or previously implicated in cancer, including CTNNB1 p.S29/Y30, EGFR p.S720, MAPK1 p.S142, and PTPN12 p.S275. In summary, systematic 3D clustering analysis highlights nearly 3,000 likely functional mutations and over 1,000 cancer phosphosites for downstream investigation and evaluation of potential clinical relevance.

bioinformatics↗

bioRxiv2020

COPB2 haploinsufficiency causes a coatopathy with osteoporosis and developmental delay

Coatomer complexes function in the sorting and trafficking of proteins between subcellular organelles. Pathogenic variants in coatomer subunits or associated factors have been reported in multi-systemic disorders, i.e., coatopathies, that can affect the skeletal and central nervous systems. We have identified loss-of-function variants in COPB2, a component of the coatomer complex I (COPI), in individuals presenting with osteoporosis, fractures and developmental delay of variable severity. Because the role of COPB2 in bone has not been characterized, we studied the effect of COPB2 deficiency on skeletal development in mice and zebrafish. Copb2+/- mice showed low bone mass and decreased bone strength. In zebrafish, larvae carrying a copb2 heterozygous frameshift variant showed delayed mineralization. copb2-null embryos showed endoplasmic reticulum (ER) and Golgi disorganization, and embryonic lethality. COPB2 siRNA-treated fibroblasts showed delayed collagen trafficking with retention of type I collagen in the ER and Golgi, and altered distribution of Golgi markers. Our data suggest that COPB2 haploinsufficiency leads to disruption of intracellular collagen trafficking and osteoporosis, which may improve with ascorbic acid supplementation. This work highlights the role of COPI complex as a critical regulator of bone mass and identifies a new form of coatopathy due to COPB2 deficiency.

genetics↗

bioRxiv2020

Nuclear stiffness decreases with disruption of the extracellular matrix in living tissues

Reciprocal interactions between the cell nucleus and the extracellular matrix lead to macroscale tissue phenotype changes. The extracellular environment is physically linked to the nuclear envelope and provides cues to maintain nuclear structure and cellular homeostasis regulated in part by mechanotransduction mechanisms. However, little is known about how structure and properties of the extracellular matrix in living tissues impacts nuclear mechanics, and current experimental challenges limit the ability to detect and directly measure nuclear mechanics while cells are within the native tissue environment. Here, we hypothesized that enzymatic disruption of the tissue matrix results in a softer tissue, affecting the stiffness of embedded cell and nuclear structures. We aimed to directly measure nuclear mechanics without perturbing the native tissue structure to better understand nuclear interplay with the cell and tissue microenvironments. To accomplish this, we expanded an atomic force microscopy needle-tip probe technique that probes nuclear stiffness in cultured cells to measure the nuclear envelope and cell membrane stiffness within native tissue. We validated this technique by imaging needle penetration and subsequent repair of the plasma and nuclear membranes of HeLa cells stably expressing the membrane repair protein CHMP4B-GFP. In the native tissue environment ex vivo, we found that while enzymatic degradation of viable cartilage tissues with collagenase 3 (MMP-13) and aggrecanase-1 (ADAMTS-4) decreased tissue matrix stiffness, cell and nuclear membrane stiffness is also decreased. Finally, we demonstrated the capability for cell and nucleus elastography using the AFM needle-tip technique. These results demonstrate disruption of the native tissue environment that propagates to the plasma membrane and interior nuclear envelope structures of viable cells.

biophysics↗

bioRxiv2020

Pleiotropy and Epistasis Within and Between Signaling Pathways Defines the Genetic Architecture of Fungal Virulence

Cryptococcal disease is estimated to affect nearly a quarter of a million people annually. Environmental isolates of Cryptococcus deneoformans, which make up 15 to 30% of clinical infections in temperate climates such as Europe, vary in their pathogenicity, ranging from benign to hyper-virulent. Key traits that contribute to virulence, such as the production of the pigment melanin, an extracellular polysaccharide capsule, and the ability to grow at human body temperature have been identified, yet little is known about the genetic basis of variation in such traits. Here we investigate the genetic basis of melanization, capsule size, thermal tolerance, oxidative stress resistance, and antifungal drug sensitivity using quantitative trait locus (QTL) mapping in progeny derived from a cross between two divergent C. deneoformans strains. Using a "function-valued" QTL analysis framework that exploits both time-series information and growth differences across multiple environments, we identified QTL for each of these virulence traits and drug susceptibility. For three QTL we identified the underlying genes and nucleotide differences that govern variation in virulence traits. One of these genes, RIC8, which encodes a regulator of cAMP-PKA signaling, contributes to variation in four virulence traits: melanization, capsule size, thermal tolerance, and resistance to oxidative stress. Two major effect QTL for amphotericin B resistance map to the genes SSK1 and SSK2, which encode key components of the HOG pathway, a fungal-specific signal transduction network that orchestrates cellular responses to osmotic and other stresses. We also discovered complex epistatic interactions within and between genes in the HOG and cAMP-PKA pathways that regulate antifungal drug resistance and resistance to oxidative stress. Our findings advance the understanding of virulence traits among diverse lineages of Cryptococcus, and highlight the role of genetic variation in key stress-responsive signaling pathways as a major contributor to phenotypic variation. Author summaryDifferent environmental isolates (strains) of the same microbial species can vary greatly in their ability to cause disease, ranging from avirulent to hypervirulent. What makes some strains deadly pathogens, while others are relatively benign? This study describes the characterization of key genetic differences that underlie variation in traits thought to promote virulence in Cryptococcus deneoformans, a wide-spread opportunistic fungal pathogen. Using a combination of quantitative genetic and molecular genetic approaches we dissected the genetic architecture of virulence-related cellular traits (melanin production and the production of a polysaccharide capsule), physiological responses to stress (tolerance of thermal, oxidative, and osmotic stress), and sensitivity to multiple antifungal drugs. Strikingly we find that variation in most of these traits is governed by a small number of genetic differences that modify the function of two major cell signaling networks, cyclic AMP-Protein Kinase A (cAMP-PKA) signaling and a fungal specific MAP-kinase cascade called the high osmolarity glycerol (HOG) pathway. Similar to recent studies in a number of other fungal species, our findings point to an outsize role for a small number of highly pleiotropic signaling pathways in potentiating phenotypic variation both within and between fungal species.

genetics↗