Biology subjects

Day, K.

Publications and source records attributed to Day, K..

12 recordsLinked to original sources

bioRxiv2025

Induction of synthetic apomixis in two sorghum hybrids enables seed yield and genotype preservation over multiple generations

Apomixis, a process of clonal reproduction through seed, has the potential to significantly change agriculture by enabling a clonal seed propagation system for hybrid crops. Here, we demonstrate that hybrid seed from synthetically induced apomictic sorghum hybrids can be generated and maintained across multiple seed generations. This was achieved through the combination of avoidance of meiosis and induced parthenogenesis. Avoidance of meiosis was generated by the CRISPR/Cas9 knockout of the sorghum meiosis genes Spo11, Rec8, and OsdL1 and OsdL3. Parthenogenesis was induced in the resultant diploid egg cell using a maize egg cell promoter to express the Cenchrus ASGR-BBML2 gene coding sequence. Two strategies incorporating these components were used to induce synthetic apomixis in two different sorghum hybrids. Each hybrid used Tx623 as a female parent and either Tx430 or the African landrace Macia as a male parent. Seed yields in the induced apomictic hybrids were consistent and stable for multiple generations following self-pollination but reduced relative to the sexual hybrids. Sorghum contains two copies of the Osd1 gene that function in meiotic non-reduction. CRISPR/Cas9 knockout of both OsdL1 and OsdL3 loci was sufficient to produce clonal hybrid progeny in conjunction with the other apomixis induction components, but this led to a significant reduction in seed set. By contrast, a single in-frame edit of either OsdL1 or OsdL3 significantly improved seed set of clonal hybrid progeny. Fine-tuning OsdL activity appears to be essential to optimizing fertility. As the efficiency of seed set in the induced synthetic sorghum apomicts was lower than that of the sexual hybrid control, additional improvements are required to unlock the agronomic potential of synthetically induced apomictic sorghum in the field.

plant biology↗

bioRxiv2024

In vitro efficacy and in vivo toxicity and retention of targeted nanoformulated carboplatin in a sustained release carrier for treatment of osteosarcoma

Objectiveevaluate 1) if targeting of platinum magnetic nanoclusters will promote uptake in osteosarcoma cells in vitro, 2) targeting will improve uptake and delivery in murine OSA in vivo compared to free carboplatin, 3) incorporation into a sustained release carrier (SRC) will prolong local retention in vivo. MethodsComplex stability and peptide loading was assessed. Drug release was tested at pH 7.4 and 5.5 and cellular uptake and cytotoxity determined for canine, human and mouse osteosarcoma. Subcutaneous murine osteosarcoma was induced and optimal dose and time until tumor growth were established. Tumor bearing mice were equally distributed between 8 treatment (0.5mg carboplatin/mouse) and 1 control group and sacrificed at 8 predetermined time points between 1 hour and 8 days. Blood, tumor site and organs were harvested for tissue ferron and platinum content analysis (ICP-MS). ResultsCarboplatin was preferentially released at pH5.5. Targeting increased cellular uptake for carboplatin 15.2-fold, and decreased IC50 at 24h and 48h. At 2 weeks, a SC injection of 1-1.56 live cells/mouse reliably resulted in a palpable tumor. Plasma platinum peaked prior to 6 hours while plasma ferron peaked at 24-48 hours. Intratumoral delivery did not lead to a sustained local presence while local delivery in a SRC after surgery did. ConclusionsTargeting of MNC-carboplatin is possible with an increased osteosarcoma cell uptake in vitro. In vivo metastatic uptake could not be assessed due to lack of metastases, but local delivery in a SRC yielded high local, and low systemic platinum concentrations in mice.

cancer biology↗

bioRxiv2023

Programming Probiotics: Diet-responsive gene expression and colonization control in engineered S. boulardii

Saccharomyces boulardii (Sb) is an emerging probiotic chassis for delivering biomolecules to the mammalian gut, offering unique advantages as the only eukaryotic probiotic. However, precise control over gene expression and gut residence time in Sb have remained challenging. To address this, we developed five ligand-responsive gene expression systems and repaired galactose metabolism in Sb, enabling inducible gene expression in this strain. Engineering these systems allowed us to construct AND logic gates, control the surface display of proteins, and turn on protein production in the mouse gut in response to a dietary sugar. Additionally, repairing galactose metabolism expanded Sbs habitat within the intestines and resulted in galactose-responsive control over gut residence time. This work opens new avenues for precise dosing of therapeutics by Sb via control over its in vivo gene expression levels and localization within the gastrointestinal tract.

bioengineering↗

bioRxiv2023

Ubiquitin-driven protein condensation promotes clathrin-mediated endocytosis

Clathrin-mediated endocytosis is an essential cellular pathway that enables signaling and recycling of transmembrane proteins and lipids. During endocytosis, dozens of cytosolic proteins come together at the plasma membrane, assembling into a highly interconnected network that drives endocytic vesicle biogenesis. Recently, multiple groups have reported that early endocytic proteins form flexible condensates, which provide a platform for efficient assembly of endocytic vesicles. Given the importance of this network in the dynamics of endocytosis, how might cells regulate its stability? Many receptors and endocytic proteins are ubiquitylated, while early endocytic proteins such as Eps15 contain ubiquitin-interacting motifs. Therefore, we examined the influence of ubiquitin on the stability of the early endocytic protein network. In vitro, we found that recruitment of small amounts of polyubiquitin dramatically increased the stability of Eps15 condensates, suggesting that ubiquitylation could nucleate endocytic assemblies. In live cell imaging experiments, a version of Eps15 that lacked the ubiquitin-interacting motif failed to rescue defects in endocytic initiation created by Eps15 knockout. Furthermore, fusion of Eps15 to a deubiquitylase enzyme destabilized nascent endocytic sites within minutes. In both in vitro and live cell settings, dynamic exchange of Eps15 proteins, a hallmark of liquid-like systems, was modulated by Eps15-Ub interactions. These results collectively suggest that ubiquitylation drives assembly of the flexible protein network responsible for catalyzing endocytic events. More broadly, this work illustrates a biophysical mechanism by which ubiquitylated transmembrane proteins at the plasma membrane could regulate the efficiency of endocytic recycling. Significance StatementThe assembly of proteins into dynamic, liquid-like condensates is an emerging principle of cellular organization. During clathrin-mediated endocytosis, a liquid-like protein network catalyzes vesicle assembly. How do cells regulate these assemblies? Here we show that ubiquitin and endocytic proteins form a dynamic, mutually-reinforcing protein network in vitro and in live cells. To probe the impact of ubiquitylation on the dynamics of endocytosis, we engineered opto-genetic control over recruitment of proteins to nascent endocytic sites. While recruitment of wildtype proteins promoted endocytosis, recruitment of deubiquitylases, enzymes capable of removing ubiquitin, resulted in disassembly of endocytic sites within minutes. These results illustrate that ubiquitylation can regulate the fate of endocytic structures, elucidating a functional connection between protein condensates, endocytosis, and ubiquitin signaling.

cell biology↗

bioRxiv2022

Mesenchymal Progenitors set the homeostatic inflammatory milieu via the TAK1-NFkB axis.

The ability of mesenchymal stromal cells to modulate inflammation is at the basis of the ongoing interest in their therapeutic potential. Yet, reliable success in clinical trials is limited, possibly due to a limited understanding of their impact on the inflammatory milieu in physiological conditions. Here we show that, at steady state, mesenchymal progenitors regulate the balance between type 1 and type 2 inflammatory milieus by acting on innate immune cells through the TAK1-NFkB pathway. Suppressing the constitutive activity of this pathway in MPs leads to skewing of the immune system toward systemic Type 2 inflammation (Th2). These changes have significant effects on diseases with an important inflammatory component, leading to a worsening of disease in a preclinical model of Th2-dependent Asthma, and a reduction of symptoms associated with Th1/Th17-dependent experimental autoimmune encephalitis.

cell biology↗

bioRxiv2021

STAT3-mediated allelic imbalance of novel genetic variant rs1047643 and B cell specific super-enhancer in association with systemic lupus erythematosus

Mapping of allelic imbalance (AI) at heterozygous loci has the potential to establish links between genetic risk for disease and biological function. Leveraging multi-omics data for AI analysis and functional annotation, we discovered a novel functional risk variant rs1047643 at 8p23 in association with SLE. This variant displays dynamic AI of chromatin accessibility and allelic expression on FDFT1 gene in B cells with SLE. We further found a B-cell restricted super-enhancer (SE) that physically contacts with this SNP-residing locus, an interaction that also appears specifically in B cells. Quantitative analysis of open chromatin and DNA methylation profiles further demonstrated that the SE exhibits aberrant activity in B cell development with SLE. Functional studies identified that STAT3, a master factor associated with autoimmune diseases, directly regulates both the AI of risk variant and the activity of SE in cultured B cells. Our study reveals that STAT3-mediated SE activity and cis-regulatory effects of SNP rs1047643 at 8p23 locus are associated with B cell deregulation in SLE.

genetics↗

bioRxiv2021

Epigenetic Programming during thymic development sets the stage for optimal function in effector T cells via DNA demethylation

The repressive effect of DNA methylation at promoters is well-known. However, its role within conserved sequences in intragenic and intergenic regions is less clear. Using Cd4 as a model gene, here we show that DNA methylation regulates the function of stimulus-responsive regulatory elements in effector T cells. Two cis-elements orchestrate intra-and intergenic DNA demethylation of the Cd4 gene during thymic development, which in turn licenses a stimulus-responsive element, E4a, for its later function in effector cells. Deficiency in DNA demethylation leads to impaired E4a function, reduced H3K4me3 promoter levels and an inability to repel de novo DNA methylation during replication, ultimately leading to gene silencing. This physiological reduction in CD4 expression leads to a defect in Th1 polarization during cutaneous Leishmaniasis. Similar patterns of regulation were observed in a broad number of genes, highlighting an essential role for DNA demethylation during thymic development in modulating the function of stimulus-responsive elements.

immunology↗

bioRxiv2020

Frequency-dependent competition between strains imparts resilience to perturbations in a model of Plasmodium falciparum malaria transmission.

In high-transmission endemic regions, local populations of Plasmodium falciparum exhibit vast diversity of the var genes encoding its major surface antigen, with each parasite comprising multiple copies from this diverse gene pool. This strategy to evade the immune system through large combinatorial antigenic diversity is common to other hyperdiverse pathogens. It underlies a series of fundamental epidemiological characteristics, including large reservoirs of transmission from high prevalence of asymptomatics and long-lasting infections. Previous theory has shown that negative frequency-dependent selection (NFDS) mediated by the acquisition of specific immunity by hosts structures the diversity of var gene repertoires (strains), in a pattern of limiting similarity that is both non-random and non-neutral. A combination of stochastic agent-based models and network analyses has enabled the development and testing of theory in these complex adaptive systems, where assembly of local parasite diversity occurs under frequency-dependent selection and large pools of variation. We show here the application of these approaches to theory comparing the resilience of the malaria transmission system to intervention when strain diversity is assembled under (competition-based) selection vs. a form of neutrality, where immunity depends only on the number but not the genetic identity of previous infections. The transmission system is considerably more resilient under NFDS, exhibiting a lower extinction probability despite comparable prevalence during intervention. We explain this pattern on the basis of the structure of strain diversity, in particular the more pronounced fraction of highly dissimilar parasites. For simulations that survive intervention, prevalence under specific immunity is lower than under neutrality, because the recovery of diversity is considerably slower than that of prevalence and decreased var gene diversity reduces parasite transmission. A Principal Component Analysis of network features describing parasite similarity reveals that despite lower overall diversity, NFDS is quickly restored after intervention constraining strain structure and maintaining patterns of limiting similarity important to parasite persistence. Given the resulting resilience to perturbations, intervention efforts will likely require longer times than the usual practice to eliminate P. falciparum populations. We discuss implications of our findings and potential analogies for ecological communities with non-neutral assembly processes involving frequency-dependence.

ecology↗

bioRxiv2020

A scalable method for identifying recombinants from unaligned sequences

Recombination is a fundamental process in molecular evolution, and the identification of recombinant sequences is of major interest for biologists. However, current methods for detecting recombinants only work for aligned sequences, often require a reference panel, and do not scale well to large datasets. Thus they are not suitable for the analyses of highly diverse genes, such as the var genes of the malaria parasite Plasmodium falciparum, which are known to diversify primarily through recombination. We introduce an algorithm to detect recombinant sequences from an unaligned dataset. Our approach can effectively handle thousands of sequences without the need of an alignment or a reference panel, offering a general tool suitable for the analysis of many different types of sequences. We demonstrate the effectiveness of our algorithm through extensive numerical simulations; in particular, it maintains its accuracy in the presence of insertions and deletions. We apply our algorithm to a dataset of 17,335 DBL types in var genes from Ghana, enabling the comparison between recombinant and non-recombinant types for the first time. We observe that sequences belonging to the same ups type or DBL subclass recombine amongst themselves more frequently, and that non-recombinant DBL types are more conserved than recombinant ones. Author summaryRecombination is a fundamental process in molecular evolution where two genes exchange genetic material, diversifying the genes. It is important to properly model this process when reconstructing evolutionary history, and to do so we need to be able to identify recombinant genes. In this manuscript, we develop a method for this which can be applied to scenarios where current methods often fail, such as where genes are very diverse. We specifically focus on detecting recombinants in the var genes of the malaria parasite Plasmodium falciparum. These genes influence the length and severity of malaria infection, and therefore their study is critical to the treatment and prevention of malaria. They are also highly diverse, primarily because of recombination. Our analysis of genes from a cross-sectional study in Ghana study show fundamental relations between the patterns and prevalence of recombination in these genes and other important biological categorisations.

evolutionary biology↗

bioRxiv2020

PEA15 loss of function and defective cerebral development in the domestic cat

Cerebral cortical size and organization are critical features of neurodevelopment and human evolution, for which genetic investigation in model organisms can provide insight into developmental mechanisms and the causes of cerebral malformations. However, some abnormalities in cerebral cortical proliferation and folding are challenging to study in laboratory mice due to the absence of gyri and sulci in rodents. We report an autosomal recessive allele in domestic cats associated with impaired cerebral cortical expansion and folding, giving rise to a smooth, lissencephalic brain, and that appears to be caused by homozygosity for a frameshift in PEA15 (phosphoprotein expressed in astrocytes-15). Notably, previous studies of a Pea15 targeted mutation in mice did not reveal structural brain abnormalities. Affected cats, however, present with a non-progressive hypermetric gait and tremors, develop dissociative behavioral defects and aggression with age, and exhibit profound malformation of the cerebrum, with a 45% average decrease in overall brain weight, and reduction or absence of the ectosylvian, sylvian and anterior cingulate gyrus. Histologically, the cerebral cortical layers are disorganized, there is substantial loss of white matter in tracts such as the corona radiata and internal capsule, but the cerebellum is relatively spared. RNA-seq and immunohistochemical analysis reveal astrocytosis. Fibroblasts cultured from affected cats exhibit increased TNF-mediated apoptosis, and increased FGFb-induced proliferation, consistent with previous studies implicating PEA15 as an intracellular adapter protein, and suggesting an underlying pathophysiology in which increased death of neurons accompanied by increased proliferation of astrocytes gives rise to abnormal organization of neuronal layers and loss of white matter. Taken together, our work points to a new role for PEA15 in development of a complex cerebral cortex that is only apparent in gyrencephalic species. SummaryGyrification is the neurodevelopmental process in certain mammalian species during which the cerebral cortex expands and folds resulting in the classic wrinkled appearance of the brain. Abnormalities in this process underlie many congenital malformations of the brain. However, unlike many other human malformations, genetic insight into gyrification is not possible in laboratory mice because rodents have a lissencephalic or smooth cerebral cortex. We identified a mutation in domestic cats that likely causes failure of the cerebral cortex to expand and fold properly, and discovered that the mutation impairs production of a protein, PEA15 (phosphoprotein expressed in astrocytes-15), involved in intracellular signaling. Affected cats have profound abnormalities in brain development, with minimal changes in their superficial behavior and neurologic function. Additional studies of tissue and cultured cells from affected animals suggest a pathophysiologic mechanism in which increased death of neurons accompanied by increased cell division of astrocytes gives rise to abnormal organization of neuronal layers and loss of white matter. These results provide new insight into a developmental process that is unique to animals with gyrencephalic brains.

genetics↗

bioRxiv2018

Competition for hosts modulates vast antigenic diversity to generate persistent strain structure in Plasmodium falciparum

In their competition for hosts, parasites with antigens that are novel to host immunity will be at a competitive advantage. The resulting frequency-dependent selection can structure parasite populations into strains of limited genetic overlap. For Plasmodium falciparum-the causative agent of malaria-in endemic regions, the high recombination rates and associated vast diversity of its highly antigenic and multicopy var genes preclude such clear clustering; this undermines the definition of strains as specific, temporally-persisting gene variant combinations. We use temporal multilayer networks to analyze the genetic similarity of parasites in both simulated data and in an extensively and longitudinally sampled population in Ghana. When viewed over time, populations are structured into modules (i.e., groups) of parasite genomes whose var gene combinations are more similar within, than between, the modules, and whose persistence is much longer than that of the individual genomes that compose them. Comparison to neutral models that retain parasite population dynamics but lack competition reveals that the selection imposed by host immunity promotes the persistence of these modules. The modular structure is in turn associated with a slower acquisition of immunity by individual hosts. Modules thus represent dynamically generated niches in host immune space, which can be interpreted as strains. Negative frequency-dependent selection therefore shapes the organization of the var diversity into parasite genomes, leaving a persistence signature over ecological time scales. Multilayer networks extend the scope of phylodynamics analyses by allowing quantification of temporal genetic structure in organisms that generate variation via recombination or other non-bifurcating processes. A strain structure similar to the one described here should apply to other pathogens with large antigenic spaces that evolve via recombination. For malaria, the temporal modular structure should enable the formulation of tractable epidemiological models that account for parasite antigenic diversity and its influence on intervention outcomes.\n\nSignificanceMany pathogens, including the causative agent of malaria Plasmodium falciparum, use antigenic variation, obtained via recombination, as a strategy to evade the human immune system. The vast diversity and multiplicity of genes encoding antigenic variation in high transmission regions challenge the notion of the existence of distinct strains: temporally-persistent and specific combinations of genes relevant to epidemiology. We examine the role of human immune selection in generating such genetic population structure in the major blood-stage antigen of Plasmodium falciparum. We show, using simulated and empirical data, that immune selection generates and maintains modules of genomes with higher genetic similarity within, than between, these groups. Selection further promotes the persistence of these modules for much longer times than those of their constituent genomes. Simulations show that the temporal modular structure reduces the speed at which hosts acquire immunity to the parasite. We argue that in P. falciparum modules can be viewed as dynamic strains occupying different niches in human immune space; they are thus relevant to formulating transmission models that encompass the antigenic diversity of the parasite. Our analyses may prove useful to understand the interplay between temporal genetic structure and epidemiology in other pathogens of human and wildlife importance.

ecology↗

bioRxiv2017

Networks of genetic similarity reveal non-neutral processes shape strain structure in Plasmodium falciparum

Pathogens compete for hosts through patterns of cross-protection conferred by immune responses to antigens. In Plasmodium falciparum malaria, the var multigene family encoding for the major blood-stage antigen PfEMP1 has evolved enormous genetic diversity through ectopic recombination and mutation. With 50-60 var genes per genome, it is unclear whether immune selection can act as a dominant force in structuring var repertoires of local populations. The combinatorial complexity of the var system remains beyond the reach of existing strain theory, and previous evidence for non-random structure cannot demonstrate immune selection without comparison to neutral models. We develop two neutral models that encompass malaria epidemiology but exclude competitive interactions between parasites. These models, combined with networks of genetic similarity, reveal non-neutral strain structure in both simulated systems and an extensively sampled population in Ghana. The unique population structure we identify underlies the large transmission reservoir characteristic of highly endemic regions in Africa.

ecology↗