Biology subjects

Clark, J.

Publications and source records attributed to Clark, J..

50 records · Page 3Linked to original sources

bioRxiv2021

Temporal dynamics of base excision / single-strand break repair protein complex assembly and disassembly are modulated by the PARP1/NAD+/SIRT6 axis

Assembly and disassembly of DNA repair protein complexes at sites of DNA damage is essential to maintain genomic integrity. We investigated factors coordinating assembly of the base excision repair (BER) proteins, DNA polymerase {beta} (Pol{beta}) and XRCC1, to DNA lesion sites, identifying a new role for Pol{beta} in regulating XRCC1 disassembly from DNA repair complexes and conversely, demonstrating Pol{beta}s dependence on XRCC1 for complex assembly. RealPAR, a genetically-encoded probe for live cell imaging of poly(ADP-ribose) (PAR), reveals that Pol{beta} and XRCC1 require PAR for repair complex assembly and PAR degradation for disassembly. We find that BER complex assembly is further modulated by attenuation / augmentation of NAD+ biosynthesis. Finally, SIRT6 does not regulate PARP1 activation but impairs XRCC1 recruitment, leading to diminished Pol{beta} abundance at sites of DNA damage. These findings highlight coordinated yet independent roles for both PARP1 and SIRT6 and their regulation by NAD+ bioavailability to facilitate BER.

cancer biology↗

bioRxiv2021

Plasticity in novel environments induces larger changes in genetic variance than adaptive divergence

Adaptive plasticity allows populations to cope with environmental variation but is expected to fail as conditions become unfamiliar. In novel conditions, populations may instead rely on rapid adaptation to increase fitness and avoid extinction. Adaptation should be fastest when both plasticity and selection occur in directions of the multivariate phenotype that contain abundant genetic variation. However, tests of this prediction from field experiments are rare. Here, we quantify how additive genetic variance in a multivariate phenotype changes across an elevational gradient, and test whether plasticity and selection align with genetic variation. We do so using two closely related, but ecologically distinct, sister species of Sicilian daisy (Senecio, Asteraceae) adapted to high and low elevations on Mount Etna. Using a paternal half-sibling breeding design, we generated and then reciprocally planted c.19,000 seeds of both species, across an elevational gradient spanning each species native elevation, and then quantified mortality and five leaf traits of emergent seedlings. We found that genetic variance in leaf traits changed more across elevations than between species. The high-elevation species at novel lower elevations showed changes in the distribution of genetic variance among the leaf traits, which reduced the amount of genetic variance in the directions of selection and the native phenotype. By contrast, the low-elevation species mainly showed changes in the amount of genetic variance at the novel high elevation, and genetic variance was concentrated in the direction of the native phenotype. For both species, leaf trait plasticity across elevations was in a direction of the multivariate phenotype that contained a moderate amount of genetic variance. Together, these data suggest that where plasticity is adaptive, selection on genetic variance for an initially plastic response could promote adaptation. However, large environmental effects on genetic variance are likely to reduce adaptive potential in novel environments.

evolutionary biology↗

bioRxiv2021

Adaptive maternal effects in early life history traits help to maintain ecological resilience in novel environments for two contrasting Senecio species

While many organisms shift their development plastically to maintain fitness as environments change, such plasticity has limits. Population mean fitness is expected to decline when novel environments exceed the limits to plasticity, but the level of fitness costs is expected to vary among genotypes that can increase adaptive potential. We lack fundamental insights into how and when changes in early development traits increase adaptive potential in novel environments, which limits our ability to predict the response of natural populations to global change. To test whether genetic variation in development time is associated with increased adaptive potential in novel environments, we used a breeding design to generate c.20,000 seeds of two ecologically contrasting Sicilian species of daisies (Senecio, Asteraceae) adapted to high and low elevations on Mount Etna. We planted the seeds across four elevations that included the native range of each species, and a novel elevation. We tracked seedling mortality and measured development time as the number of days it took seedlings to establish. As predicted, genetic variance in survival increased at novel elevations. However, genetic variance in development time showed the opposite trend, decreasing at novel elevations. A strong negative genetic correlation between development time in the native range and survival at novel elevations suggested that genotypes with faster development in native environments survived better in novel environments. These results were consistent across the two ecologically contrasting species, suggesting that genetic variance in early development in native environments could be used to predict population responses to novel environments.

evolutionary biology↗

bioRxiv2021

Biodegradable and Biocompatible Graphene-based Scaffolds for Functional Neural Tissue Engineering: A Strategy Approach Using Dental Pulp Stem Cells and Biomaterials

Neural tissue engineering aims to restore function of nervous system tissues using biocompatible cell-seeded scaffolds. Graphene-based scaffolds combined with stem cells deserve special attention to enhance tissue regeneration in a controlled manner. However, it is believed that minor changes in scaffold biomaterial com-position, internal porous structure, and physicochemical properties can impact cellular growth and adhesion. The current work aims to investigate in vitro biological effects of 3D graphene oxide (GO)/sodium alginate (GOSA) and reduced GOSA (RGOSA) scaffolds on dental pulp stem cells (DPSCs) in terms of cell viability and cytotoxicity. Herein, the effects of the 3D scaffolds, coating conditions, and serum supplementation on DPSCs functions are explored extensively. Biodegradation analysis revealed that addition of GO enhanced the degradation rate of composite scaffolds. Compared to the 2D surface, the cell viability of 3D scaffolds was higher (p <0.0001), highlighting the optimal initial cell adhesion to the scaffold surface and cell migration through pores. Moreover, the cytotoxicity study indicated that the incorporation of graphene supported higher DPSCs viability. It is also shown that when the mean pore size of scaffold increases, DPSCs activity decreases. In terms of coating conditions, poly-l-lysine (PLL) was the most robust coating reagent that improved cell-scaffold adherence and DPSCs metabolism activity. The cytotoxicity of GO-based scaffolds showed that DPSCs can be seeded in serum-free media without cytotoxic effects. This is critical for human translation as cellular transplants are typically serum-free. These findings suggest that proposed 3D GO-based scaffolds have favourable effects on the biological responses of DPSCs.

bioengineering↗

bioRxiv2020

Artificial light at night shifts the circadian system but still leads to physiological disruption in a wild bird

Globally increasing levels of artificial light at night (ALAN) are associated with shifts in circadian rhythms of behaviour in many wild species. However, it is still unclear whether changes in behavioural timing are underlined by parallel shifts in the molecular clock, and whether such internal shifts may differ between different tissues and physiological pathways, which could highlight circadian disruption. We tackled these questions in a comprehensive study that integrated behavioural, gene expression and metabolomic analyses. We exposed captive male great tits (Parus major) to three ALAN intensities or to dark nights, recorded their activity rhythms and obtained mid-day and midnight samples of brain, liver, spleen and blood. ALAN advanced wake-up time, and this shift was paralleled by an advance in the expression of the clock gene BMAL1 in all tissues, suggesting close links of brain and peripheral clock gene expression with activity rhythms. However, several metabolic and immune genes were desynchronised the shifted BMAL1 expression, suggesting circadian disruption of behaviour and physiology. This result was reinforced by untargeted metabolomic profiling, which showed that only 9.7% of the 755 analysed metabolites followed the behavioural shift. We suggest circadian as a key mediator of the health impacts of ALAN on wild animals.

zoology↗

bioRxiv2020

Hidden genetic variation in plasticity increases the potential to adapt to novel environments

Rapid environmental change is forcing populations into novel environments where plasticity will no longer maintain fitness. When populations are exposed to novel environments, evolutionary theory predicts that genetic variation in fitness will increase and should be associated with genetic differences in plasticity. If true, then genetic variation in plasticity can increase adaptive potential in novel environments, and population persistence via rapid adaptation is more likely. To test whether genetic variation in fitness increases in novel environments and is associated with plasticity, we transplanted 8,149 clones of 314 genotypes of a Sicilian daisy (Senecio chrysanthemifolius) within and outside its native range, and quantified genetic variation in fitness, and plasticity in leaf traits and gene expression. Although mean fitness declined by 87% in the novel environment, genetic variance in fitness increased threefold and was correlated with plasticity in leaf traits. High fitness genotypes showed greater plasticity in gene expression, but lower plasticity in most leaf traits. Interestingly, genotypes with greater fitness in the novel environment had the lowest fitness at the native site. These results suggest that standing genetic variation in plasticity could help populations to persist and adapt to novel environments, despite remaining hidden in native environments.

evolutionary biology↗

bioRxiv2020

Broiler Chickens and Early Life Programming: Microbiome transplant-induced cecal bacteriome dynamics and phenotypic effects

The concept of successional trajectories describes how small differences in initial community composition can magnify through time and lead to significant differences in mature communities. For many animals, the types and sources of early-life exposures to microbes have been shown to have significant and long-lasting effects on the community structure and/or function of the microbiome. In modern commercial poultry production, chicks are reared as a single age cohort and do not directly encounter adult birds. This scenario is likely to initiate a trajectory of microbial community development that is significantly different than non-industrial settings where chicks are exposed to a much broader range of environmental and fecal inocula; however, the comparative effects of these two scenarios on microbiome development and function remain largely unknown. In this work, we performed serial transfers of cecal material through multiple generations of birds to first derive a stable source of inoculum. Subsequently, we compared microbiome development between chicks receiving this passaged cecal material, versus an environmental inoculum, to test the hypothesis that the first exposure of newly hatched chicks to microbes determines early GI microbiome structure and may have longer-lasting effects on bird health and development. Cecal microbiome dynamics and bird weights were tracked for a two-week period, with half of the birds in each treatment group exposed to a pathogen challenge at 7 days of age. We report that: i) a relatively stable community was derived after a single passage of transplanted cecal material, ii) this cecal inoculum significantly but ephemerally altered community structure relative to the environmental inoculum and PBS controls, and iii) either microbiome transplant administered at day-of-hatch appeared to have some protective effects against pathogen challenge relative to uninoculated controls. Differentially abundant taxa were identified across treatment types that may inform future studies aimed at identifying strains associated with beneficial phenotypes.

microbiology↗

bioRxiv2020

Glutathione S-transferase: A Candidate Gene for Berry Color in Muscadine Grapes (Vitis rotundifolia)

Muscadine grapes (Vitis rotundifolia Michx.) are a specialty crop cultivated in the southern United States. Muscadines (2n=40) belong to the Muscadinia subgenus of Vitis, while all other cultivated grape species belong to the subgenus Euvitis (2n=38). The berry color locus in muscadines has been mapped to a 0.8 Mbp region syntenic with chromosome 4 of V. vinifera. In this study, we identified glutathione S-transferase4 (GST4) as a likely candidate gene for anthocyanin transport within the berry color locus. PCR and KASP genotyping identified a single intragenic SNP (C/T) marker corresponding to a proline to leucine mutation within the muscadine GST4 (VrGST4) that differentiated black (CC and CT) from bronze (TT) muscadines in 65 breeding selections, 14 cultivars, and 320 progeny from two mapping populations. Anthocyanin profiling on a subset of the progeny indicated a dominant VrGST4 action, with no allele dosage effect on total anthocyanin content or composition of individual anthocyanins. Proanthocyanidin content was similar in the seeds of both black and bronze genotypes, and seeds had much higher VrGST3 expression and lower VrGST4 expression than skins. VrGST4 expression was higher in post-veraison berries of black muscadines compared to pre-veraison berries, but no changes in gene expression in pre- and post-veraison berries were observed in the bronze muscadine cultivar. VrMybA1 expression was higher in post-veraison berries of both black and bronze muscadines. These results suggest that berry pigmentation in muscadines is regulated by a mechanism distinct from the MybA gene cluster that is responsible for berry color variation in V. vinifera.

genetics↗

bioRxiv2020

Daphnia magna modifies its gene expression extensively in response to caloric restriction revealing a novel effect on haemoglobin isoform preference

Caloric restriction (CR) produces clear phenotypic effects within and between generations of the model crustacean Daphnia magna. We have previously established that micro RNAs and cytosine methylation change in response to CR in this organism, and we demonstrate here that CR has a dramatic effect on gene expression. Over 6000 genes were differentially expressed between CR and well-fed D. magna, with a bias towards up-regulation of genes under caloric restriction. We identified a highly expressed haemoglobin gene that responds to CR by changing isoform proportions. Specifically, a transcript containing three erythrocruorin domains was strongly down-regulated under CR in favour of transcripts containing fewer or no such domains. This change in the haemoglobin mix is similar to the response to hypoxia in Daphnia, which is mediated through the transcription factor hypoxia-inducible factor 1, and ultimately the mTOR signalling pathway. This is the first report of a role for haemoglobin in the response to CR. We also observed high absolute expression of super-oxide dismutase (SOD) in normally-fed individuals, which contrasts with observations of high SOD levels under in CR in other taxa. However, key differentially expressed genes, like SOD, were not targeted by differentially expressed micro-RNAs. Whether the link between Haemoglobin and CR is the case in other organisms, or is related to the aquatic lifestyle, remains to be tested. It suggests that one response to CR may be to simply transport less oxygen and lower respiration.

genomics↗

bioRxiv2020

Functional loss of a non-canonical BCOR-PRC1.1 complex accelerates SHH-driven medulloblastoma formation

Medulloblastoma is a childhood brain tumor arising from the developing cerebellum. In Sonic Hedgehog (SHH)-subgroup medulloblastoma, aberrant activation of SHH signaling causes increased proliferation of granule neuron progenitors (GNPs) and predisposes these cells to tumorigenesis. A second, cooperating genetic hit is often required to push these hyperplastic cells to malignancy and confer mutation-specific characteristics associated with oncogenic signaling. Somatic loss-of-function mutations of the transcriptional co-repressor BCOR are recurrent and highly enriched in SHH-medulloblastoma. To investigate BCOR as a putative tumor suppressor, we used a germline genetically engineered mouse model to delete exons 9/10 of Bcor (Bcor{Delta}E9-10) in GNPs during development. This leads to reduced expression of C-terminally truncated BCOR (BCOR{Delta}E9-10). While Bcor{Delta}E9-10 alone did not promote tumorigenesis or affect GNP differentiation, Bcor{Delta}E9-10 combined with loss of the SHH-receptor gene Ptch1 resulted in highly penetrant medulloblastomas. In Ptch1+/-;Bcor{Delta}E9-10 tumors, the growth factor gene Igf2 was aberrantly upregulated, and ectopic Igf2 overexpression was sufficient to drive tumorigenesis in Ptch1+/- GNPs. BCOR directly regulates Igf2, likely through the PRC1.1 complex; the repressive histone mark H2AK119Ub is decreased at the Igf2 promoter in Ptch1+/-;Bcor{Delta}E9-10 tumors. Overall, our data suggests that BCOR-PRC1.1 disruption leads to Igf2 overexpression, which transforms preneoplastic cells to malignant tumors.

cancer biology↗

bioRxiv2020

Ecology and evolutionary history determine plastic responses to environmental variation in two closely related species

The evolution of plastic responses to external cues allows species to track the environmental variation they regularly experience. However, it remains unclear how plasticity evolves during adaptation. To test whether distinct patterns of plasticity is associated with recent adaptive divergence, we quantified plasticity for two closely related but ecologically divergent Sicilian daisy species (Senecio, Asteraceae). We sampled c.40 genotypes of each species from natural populations on and around Mt Etna and then reciprocally transplanted multiple clones of each genotype into four field sites along an elevational gradient representing each species native range, the edge of their range, and conditions outside their native range. At each elevation we quantified survival and measured leaf traits that included investment (specific leaf area), morphology, chlorophyll fluorescence, pigment content and gene expression. As evidence of adaptive divergence, both species performed better at their native site and better than the species from the other habitat. Traits and differentially expressed genes that changed with elevation in one species often showed little change in the other species, or changed in the opposite direction. Adaptive divergence is therefore associated with the evolution of distinct plastic responses to environmental variation, despite these two species sharing a recent common ancestor.

evolutionary biology↗

bioRxiv2019

Complementary Performances of Convolutional and Capsule Neural Networks on Classifying Microfluidic Images of Dividing Yeast Cells

Microfluidic-based assays have become effective high-throughput approaches to examining replicative aging of budding yeast cells. Deep learning may offer an efficient way to analyze a large number of images collected from microfluidic experiments. Here, we compare three deep learning architectures to classify microfluidic time-lapsed images of dividing yeast cells into categories that represent different stages in the yeast replicative aging process. We found that convolutional neural networks outperformed capsule networks in terms of accuracy, precision, and recall. The capsule networks had the most robust performance at detecting one specific category of cell images. An ensemble of three best-fitted single-architecture models achieves the highest overall accuracy, precision, and recall due to complementary performances. In addition, extending classification classes and augmentation of the training dataset can improve the predictions of the biological categories in our study. This work lays a useful framework for sophisticated deep-learning processing of microfluidics-based assays of yeast replicative aging.

bioinformatics↗

bioRxiv2018

PI3Kδ hyper-activation promotes the development of B cells that exacerbate Streptococcus pneumoniae infection in an antibody-independent manner

Streptococcus pneumoniae is a major cause of pneumonia and a leading cause of death world-wide. Antibody-mediated immune responses can offer protection against repeated exposure to S. pneumoniae, yet vaccines only offer partial protection. Patients with Activated PI3K{delta} Syndrome (APDS) are highly susceptible to S. pneumoniae. We generated a conditional knockin mouse model of this disease and identified a CD19+B220- B cell subset that is induced by PI3K{delta} signaling, is resident in the lungs, and which promotes increased susceptibility to S. pneumoniae during the early phase of infection via an antibody-independent mechanism. We show that an inhaled PI3K{delta} inhibitor improves survival rates following S. pneumoniae infection in wild-type mice and in mice with activated PI3K{delta}. These results suggest that a subset of B cells in the lung can promote the severity of S. pneumoniae infection, representing a novel therapeutic target.

immunology↗

bioRxiv2017

C. elegans DBL-1/BMP Regulates Lipid Accumulation via Interaction with Insulin Signaling

Metabolic homeostasis is coordinately controlled by diverse inputs, which must be understood to combat metabolic disorders. Here we introduce DBL-1, the C. elegans BMP2/4 homolog, as a significant regulator of lipid homeostasis. We used neutral lipid staining and a lipid droplet marker to demonstrate that both increases and decreases in DBL-1/BMP signaling result in reduced lipid stores and lipid droplet count. We find that lipid droplet size, however, correlates positively with the level of DBL 1/BMP signaling. Regulation of lipid accumulation in the intestine occurs through non-cell-autonomous signaling, since expression of SMA-3, a Smad signal transducer, in the epidermis (hypodermis) is sufficient to rescue the loss of lipid accumulation. Finally, genetic evidence indicates that DBL-1/BMP functions upstream of Insulin/IGF-1 Signaling (IIS) in lipid metabolism. We conclude that BMP signaling regulates lipid metabolism in C. elegans through inter-organ signaling to IIS, shedding light on a less well-studied regulatory mechanism for metabolic homeostasis.

cell biology↗