Biology subjects

Richardson, K.

Publications and source records attributed to Richardson, K..

25 records · Page 2Linked to original sources

bioRxiv2023

A phenotypically robust model of Spinal and Bulbar Muscular Atrophy in Drosophila

Spinal and bulbar muscular atrophy (SBMA) is an X-linked disorder that affects males who inherit the androgen receptor (AR) gene with an abnormal CAG triplet repeat expansion. The resulting protein contains an elongated polyglutamine (polyQ) tract and causes motor neuron degeneration in an androgen-dependent manner. The precise molecular sequelae of SBMA are unclear. To assist with its investigation and the identification of therapeutic options, we report here a new model of SBMA in Drosophila melanogaster. We generated transgenic flies that express the full-length, human AR with a wild-type or pathogenic polyQ repeat. Each transgene is inserted into the same "safe harbor" site on the third chromosome of the fly as a single copy and in the same orientation. Expression of pathogenic AR, but not of its wild-type variant, in neurons or muscles leads to consistent, progressive defects in longevity and motility that are concomitant with polyQ-expanded AR protein aggregation and reduced complexity in neuromuscular junctions. Additional assays show adult fly eye abnormalities associated with the pathogenic AR species. The detrimental effects of pathogenic AR are accentuated by feeding flies the androgen, dihydrotestosterone. This new, robust SBMA model can be a valuable tool towards future investigations of this incurable disease.

neuroscience↗

bioRxiv2022

Notch Signaling and Fluid Shear Stress in Regulating Osteogenic Differentiation

Osteoporosis is a common bone and metabolic disease that is characterized by bone density loss and microstructural degeneration. Human bone marrow-derived mesenchymal stem cells (hMSCs) are multipotent progenitor cells with the potential to differentiate into various cell types, including osteoblasts, chondrocytes, and adipocytes, which have been utilized extensively in the field of bone tissue engineering and cell-based therapy. Although fluid shear stress plays an important role in bone osteogenic differentiation, the cellular and molecular mechanisms underlying this effect remain poorly understood. Here, an LNA/DNA nanobiosensor was exploited to monitor mRNA gene expression of hMSCs that were exposed to physiologically relevant fluid shear stress to examine the regulatory role of Notch signaling during osteogenic differentiation. First, the effects of fluid shear stress on cell viability, proliferation, morphology, and osteogenic differentiation were investigated and compared. Our results showed shear stress modulates hMSCs morphology and osteogenic differentiation depending on the applied shear and duration. By incorporating this LNA/DNA nanobiosensor and alkaline phosphatase (ALP) staining, we further investigated the role of Notch signaling in regulating osteogenic differentiation. Pharmacological treatment is applied to disrupt Notch signaling to investigate the mechanisms that govern shear stress induced osteogenic differentiation. Our experimental results provide convincing evidence supporting that physiologically relevant shear stress regulates osteogenic differentiation through Notch signaling. Inhibition of Notch signaling mediates the effects of shear stress on osteogenic differentiation, with reduced ALP enzyme activity and decreased Dll4 mRNA expression. In conclusion, our results will add new information concerning osteogenic differentiation of hMSCs under shear stress and the regulatory role of Notch signaling. Further studies may elucidate the mechanisms underlying the mechanosensitive role of Notch signaling in stem cell differentiation.

bioengineering↗

bioRxiv2022

Probing Notch1-Dll4 Signaling in Regulating Osteogenic Differentiation of Human Mesenchymal Stem Cells using Single Cell Nanobiosensor

Human mesenchymal stem cells (hMSCs) have great potential in cell-based therapies for tissue engineering and regenerative medicine due to their self-renewal and multipotent properties. Recent studies indicate that Notch1-Dll4 signaling is an important pathway in regulating osteogenic differentiation of hMSCs. However, the fundamental mechanisms that govern osteogenic differentiation are poorly understood due to a lack of effective tools to detect gene expression at single cell level. Here, we established a double-stranded locked nucleic acid (LNA)/DNA (LNA/DNA) nanobiosensor for gene expression analysis in single hMSC in both 2D and 3D microenvironments. We first characterized this LNA/DNA nanobiosensor and demonstrated the Dll4 mRNA expression dynamics in hMSCs during osteogenic differentiation. By incorporating this nanobiosensor with live hMSCs imaging during osteogenic induction, we performed dynamic tracking of hMSCs differentiation and Dll4 mRNA gene expression profiles of individual hMSC during osteogenic induction. Our results showed the dynamic expression profile of Dll4 during osteogenesis, indicating the heterogeneity of hMSCs during this dynamic process. We further investigated the role of Notch1-Dll4 signaling in regulating hMSCs during osteogenic differentiation. Pharmacological perturbation is applied to disrupt Notch1-Dll4 signaling to investigate the molecular mechanisms that govern osteogenic differentiation. In addition, the effects of Notch1-Dll4 signaling on hMSCs spheroids differentiation were also investigated. Our results provide convincing evidence supporting that Notch1-Dll4 signaling is involved in regulating hMSCs osteogenic differentiation. Specifically, Notch1-Dll4 signaling is active during osteogenic differentiation. Our results also showed that Dll4 is a molecular signature of differentiated hMSCs during osteogenic induction. Notch inhibition mediated osteogenic differentiation with reduced Alkaline Phosphatase (ALP) activity. Lastly, we elucidated the role of Notch1-Dll4 signaling during osteogenic differentiation in a 3D spheroid model. Our results showed that Notch1-Dll4 signaling is required and activated during osteogenic differentiation in hMSCs spheroids. Inhibition of Notch1-Dll4 signaling mediated osteogenic differentiation and enhanced hMSCs proliferation, with increased spheroid sizes. Taken together, the capability of LNA/DNA nanobiosensor to probe gene expression dynamics during osteogenesis, combined with the engineered 2D/3D microenvironment, enables us to study in detail the role of Notch1-Dll4 signaling in regulating osteogenesis in 2D and 3D microenvironment. These findings will provide new insights to improve cell-based therapies and organ repair techniques.

bioengineering↗

bioRxiv2021

Endurance exercise ameliorates phenotypes in Drosophila models of Spinocerebellar Ataxias

Endurance exercise is a potent intervention with widespread benefits proven to reduce disease incidence and impact across species. While endurance exercise supports neural plasticity, enhanced memory, and reduced neurodegeneration, less is known about the effect of chronic exercise on the progression of movement disorders such as ataxias. Here, we focused on three different types of ataxias, Spinocerebellar Ataxias Type (SCAs) 2, 3, and 6, belonging to the polyglutamine (polyQ) family of neurodegenerative disorders. In Drosophila models of these SCAs, flies progressively lose motor function. Here, we observe marked protection of speed and endurance in exercised SCA2 flies and modest protection in exercised SCA6 models, while no benefit is observed in SCA3 flies. Causative protein levels are reduced in SCA2 flies after chronic exercise, but not in SCA3 models, linking protein levels to exercise-based benefits. Additional investigations indicate that the exercise-inducible protein, Sestrin (Sesn) suppresses mobility decline and improves early death in SCA2 flies, even without exercise, coincident with disease protein level reduction and increased autophagic flux. These improvements depend on previously established functions of Sesn that reduce oxidative damage and modulate mTOR activity. Our study suggests differential responses of polyQ SCAs to exercise, highlighting the potential for more extensive application of exercise-based therapies in the prevention of polyQ neurodegeneration. Defining the mechanisms by which endurance exercise suppresses polyQ SCAs will open the door for more effective treatment for these diseases.

neuroscience↗

bioRxiv2021

Transcriptomic Insights into the Responses in Leaves Storing Lipid Organelles under Different Irradiances

To increase the nutritional value of forage, transgenic ryegrass known as High Metabolizable Energy (HME) were previously generated that co-express cysteine-oleosin and diacylglycerol O-acyltransferase. HME not only accumulate lipids in the leaf but also has elevated CO2 assimilation and increased biomass. Shading is one of the most influencing factors for ryegrass growth environments particularly in swards. The aim of this study, therefore, was to determine the influence of irradiance levels on photosynthesis and gene expression in the HME leaves when compared with their corresponding non-transformant (NT). Under low light (150-250 {micro}mol m-2 s-1) and standard light (600-1000 {micro}mol m-2 s-1), the HME accumulated more lipid than NT. The previously reported elevated photosynthesis and increased biomass was observed when the HME were grown under standard light but not under low light. Under both light conditions, compared to NT, the HME had upregulated a number of transcripts involved in lipid metabolism, light capturing, photosynthesis, and sugar signalling network while downregulated genes participated in sugar and fructan biosynthesis. We further discuss how the HME differentially manipulated several genes other metabolic pathways including maintenance of redox homeostasis. Combined, the data suggests that the increased photosynthesis capacity in the HME likely corresponds to an increase of micro-lipid sink strength; these are influenced by available light energy and may be related to diffusional and biochemical activities of stomata. Overall, this work provides a clearly understanding of the changes in molecular and biochemical mechanisms underlying the carbon storing as leaf lipid sink of the HME ryegrass. One sentence summaryShading led to increase leaf lipid accumulation but limit the greater photosynthesis trait of high lipid ryegrass which may be related to limitation of biochemical activities of stomata.

molecular biology↗

bioRxiv2019

Structure and functional analysis of the Legionella chitinase ChiA reveals a novel mechanism of metal-dependent mucin degradation

Chitinases are important enzymes that contribute to the generation of carbon and nitrogen from chitin, a long chain polymer of N-acetylglucosamine that is abundant in insects, fungi, invertebrates and fish. Although mammals do not produce chitin, chitinases have been identified in bacteria that are key virulence factors in severe respiratory, gastrointestinal and urinary diseases. However, it is unclear how these enzymes are able to carry out this dual function. Legionella pneumophila is the causative agent of Legionnaires disease, an often-fatal pneumonia and its chitinase ChiA is essential for the survival of L. pneumophila in the lung. Here we report the first atomic resolution insight into the pathogenic mechanism of a bacterial chitinase. We derive an experimental model of intact ChiA and show how its N-terminal region targets ChiA to the bacterial surface after its secretion. We provide the first evidence that L. pneumophila can bind mucins on its surface but this is not dependent on chiA. This demonstrates that additional peripheral mucin binding proteins are also expressed in L. pneumophila. Finally, we show that the ChiA C-terminal chitinase domain has novel metal-dependent peptidase activity against mammalian mucins. These findings suggest that ChiA facilitates bacterial penetration of the alveolar mucosa and ChiA may be a promising target for vaccine development.

microbiology↗

bioRxiv2018

Complex interactions between local adaptation, plasticity, and sex affect vulnerability to warming in a widespread marine copepod

Predicting the response of populations to climate change requires knowledge of thermal performance. Genetic differentiation and phenotypic plasticity affect thermal performance, but the effects of sex and developmental temperatures often go uncharacterized. We used common garden experiments to test for effects of local adaptation, developmental phenotypic plasticity, and individual sex on thermal performance of the ubiquitous copepod, Acartia tonsa. Females had higher thermal tolerance than males in both populations, while the Florida population had higher thermal tolerance compared to the Connecticut population. An effect of developmental phenotypic plasticity on thermal tolerance was observed only in the Connecticut population. Ignoring sex-specific differences may result in a severe underestimation of population-level impacts of warming (i.e. - population decline due to sperm limitation). Further, despite having a higher thermal tolerance, southern populations may be more vulnerable to warming as they lack the ability to respond to increases in temperature through phenotypic plasticity.

evolutionary biology↗