Biology subjects

Grace, M.

Publications and source records attributed to Grace, M..

8 recordsLinked to original sources

bioRxiv2024

Machine learning driven image segmentation and shape clustering of algal microscopic images obtained from various water types

Algae and cyanobacteria are microorganisms found in almost all fresh and marine waters, where they can pose environmental and public health risks when they grow excessively and produce blooms. Accurate identification and quantification of these microorganisms are vital for ecological research, water quality monitoring, and public health safety. However, traditional methods of manually counting and morphologically identifying these microorganisms are time-consuming and prone to human error. Application of the machine learning-driven Fast Segment Anything Model (FastSAM), an image segmentation model, automates and potentially enhances the accuracy and efficiency of cell identification and enumeration from microscopic images. We assessed FastSAM for algal cell image segmentation, and three clustering evaluation metrics. Segmentation of microscopic images of algal and cyanobacterial cells in water and treated wastewater samples using the Convolutional Neural Network based FastSAM algorithm demonstrated benefits and challenges of this machine learning-driven image processing. Notably, the pre-trained algorithm segmented entire elements in all microscopic images used in this study. Depending on the shape, 50-100% similarity was observed between machine-based segmentation and manual validation of all segmented elements, with 100% of single cells being correctly segmented by FastSAM. The performance of clustering metrics varied between 57-94% with the Spectral Angle Mapper achieving the most accurate performance, 84-94%, compared to the manually chosen clustering benchmarks. Cyanobacterial and algal communities are biologically diverse and have ecological significance. The application of image clustering techniques in studying their cell shapes marks an important advancement in microbial ecology and environmental monitoring. As technology progresses, these methods will become increasingly utilised to decipher the complex roles that algae and cyanobacteria play in our ecosystems supporting mitigation and public health protection measures.

microbiology↗

bioRxiv2023

The HPV8 E6 protein targets the Hippo and Wnt signaling pathways as part of its arsenal to restrain keratinocyte differentiation

Infections with {beta}-genus HPVs cause hyperplastic cutaneous lesions. In individuals with the rare hereditary skin disease, epidermodysplasia verruciformis, such lesions can progress to cutaneous squamous cell carcinomas (cSCC). {beta}-HPV infections may also underlie cSCC development in chronically immunosuppressed individuals. Despite their prevalence and disease association, these viruses are not as well studied as the cancer-associated high-risk -genus HPVs. HPV-associated lesions are characterized by a marked expansion of dividing, basal-like, poorly differentiated viral cells that contain viral genomes. This reflects the ability of HPVs to inhibit epithelial cell differentiation which is likely driven by the need to establish and maintain long-term viral infections in basal-like epithelial cells. Remarkably, the {beta}-HPVs accomplish this by targeting different cellular effectors than the -genus HPVs. It was previously reported that the HPV8 E6 protein restrains epithelial cell differentiation by inhibiting Notch and TGF-{beta} signaling. Here we report that the HPV8 E6 protein can subvert Hippo signaling by activating TEAD transcriptional programs that inhibit the expression of keratinocyte differentiation markers. Moreover, we determined that HPV8 E6 can interfere with gene expression programs triggered by Wnt signaling by binding to the {beta}-catenin-associated transcriptional co-activator BCL9L and that this also serves to restrain the expression of epithelial differentiation markers. Hence the HPV8 E6 protein has evolved a remarkably large array of mechanisms to subvert the differentiation program of the infected epithelial cells. IMPORTANCEHuman papillomaviruses (HPVs) infect basal epithelial cells and cause a dramatic expansion of basal-like, proliferative cells. This reflects the ability of papillomaviruses to delay keratinocyte differentiation thereby maintaining aspects of the basal cell identity of persistently infected cells. This may enable papillomaviruses to establish and maintain long-term infections in squamous epithelial tissues. Previous work has revealed that the ability of {beta}-HPV8 E6 protein to inhibit NOTCH and TGF-{beta} signaling importantly contributes to this activity. Here we present evidence that HPV8 E6 also subverts Hippo and Wnt signaling and that these activities also aid in restraining keratinocyte differentiation.

microbiology↗

bioRxiv2022

A touchscreen device for behavioral testing in pigs

Pigs are becoming more common research models due to their utility in studying neurological conditions such as traumatic brain injury, Alzheimers disease, and Huntingtons Disease. However, behavioral tasks often require a large apparatus and are not automated, which may disinterest researchers in using important functional measures. To address this, we developed a touchscreen that pigs could be trained on for behavioral testing. A rack-mounted touchscreen monitor was placed in an enclosed, weighted audio rack. A pellet dispenser was operated by a radio frequency transceiver to deliver fruit-flavored sugar pellets from across the testing room. Programs were custom written in Python and executed by a Raspberry Pi. A behavioral shaping program was designed to train pigs to interact with the screen and setup responses for future tasks. Pigs rapidly learned to interact with the screen. To demonstrate efficacy in more complex behavior, two pigs were trained on a delay discounting tasks and two pigs on a color discrimination task. The device held up to repeated testing of large pigs and could be adjusted to the height of minipigs. The device can be easily recreated and constructed at a relatively low cost. Research topics ranging from brain injury to pharmacology to vision could benefit from behavioral tasks designed to specifically interrogate relevant function. More work will be needed to develop tests which are of specific relevance to these disciplines.

animal behavior and cognition↗

bioRxiv2022

The extinction of threatened species by anthropogenic threats endangers the functional diversity of turtles and crocodiles

The Anthropocene is tightly associated with a drastic loss of species worldwide and the disappearance of their key ecosystem functions. The on-going reduction in ecosystem functionality is driven by global and local threats. The orders Testudines (turtles and tortoises) and Crocodilia (crocodiles, alligators, and gharials) contain numerous threatened, long-lived species for which their functional diversity and potential erosion by anthropogenic impacts remains unknown. Here, we examine 259 (69%) of the existing 375 species of Testudines and Crocodilia, quantifying their life history strategies (i.e., schedules of survival, development, and reproduction) from open-access data on their demography, ancestry, and threats. We find that the loss of functional diversity in simulated extinction scenarios of threatened species is greater than expected by chance. Moreover, the effects of unsustainable local consumption, diseases, and pollution are associated with specific functional strategies. In contrast, climate change, habitat disturbance, and global trade affect all species independent of their life history strategy. Importantly, the loss of functional diversity for threatened species by habitat disturbance is twice that for all other threats. Our findings highlight the importance of conservation programmes focused on preserving the functional diversity of life history strategies jointly with the phylogenetic representativity of these highly threatened groups.

ecology↗

bioRxiv2021

The Mus musculus papillomavirus type 1 E7 protein binds to the retinoblastoma tumor suppressor - implications for viral pathogenesis

The species specificity of papillomaviruses has been a significant roadblock for performing in vivo pathogenesis studies in common model organisms. The Mus musculus papillomavirus type 1 (MmuPV1) causes cutaneous papillomas that can progress to squamous cell carcinomas in laboratory mice. The papillomavirus E6 and E7 genes encode proteins that establish and maintain a cellular milieu that allows for viral genome synthesis and viral progeny synthesis in growth-arrested, terminally differentiated keratinocytes. The E6 and E7 proteins provide this activity by binding to and functionally reprogramming key cellular regulatory proteins. The MmuPV1 E7 protein lacks the canonical LXCXE motif that mediates the binding of multiple viral oncoproteins to the cellular retinoblastoma tumor suppressor protein, RB1. Our proteomic experiments, however, revealed that MmuPV1 E7 still interacts specifically with RB1. We show that MmuPV1 E7 interacts through its C-terminus with the C-terminal domain of RB1. Binding of MmuPV1 E7 to RB1 did not cause significant activation of E2F-regulated cellular genes. MmuPV1 E7 expression was shown to be essential for papilloma formation. Experimental infection of mice with MmuPV1 virus expressing an E7 mutant that is defective for binding to RB1 caused delayed onset, lower incidence, and smaller sizes of papillomas. Our results demonstrate that the MmuPV1 E7 gene is essential and that targeting non-canonical activities of RB1, which are independent of RB1s ability to modulate the expression of E2F-regulated genes, contribute to papillomavirus-mediated pathogenesis. ImportancePapillomavirus infections cause a variety of epithelial hyperplastic lesions, warts. While most warts are benign, some papillomaviruses cause lesions that can progress to squamous cell carcinomas and approximately 5% of all human cancers are caused by human papillomavirus (HPV) infections. The papillomavirus E6 and E7 proteins are thought to function to reprogram host epithelial cells to enable viral genome replication in terminally differentiated, normally growth-arrested cells. E6 and E7 lack enzymatic activities and function by interacting and functionally altering host cell regulatory proteins. Many cellular proteins that can interact with E6 and E7 have been identified, but the biological relevance of these interactions for viral pathogenesis has not been determined. This is because papillomaviruses are species-specific and do not infect heterologous hosts. Here we use a recently established mouse papillomavirus (MmuPV1) model to investigate the role of the E7 protein in viral pathogenesis. We show that MmuPV1 E7 is necessary for papilloma formation. The retinoblastoma tumor suppressor protein (RB1) is targeted by many papillomaviral E7 proteins, including cancer-associated HPVs. We show that MmuPV1 E7 can bind RB1 and that infection with a mutant MmuPV1 virus that expresses an RB1 binding defective E7 mutant caused smaller and fewer papillomas that arise with delayed kinetics.

molecular biology↗

bioRxiv2021

Life history mediates the trade-offs among different components of demographic resilience

Summary paragraphAccelerating rates of biodiversity loss underscore the need to understand how species achieve resilience -their ability to resist and recover from a/biotic disturbances. Yet, the factors determining the resilience of species remain poorly understood, due to disagreements on its definition and the lack of large-scale analyses. Here, we investigate how the life history of 785 natural populations of animals and plants predict their intrinsic ability to be resilient. We show that demographic resilience can be achieved through different combinations of compensation, resistance, and recovery after a disturbance. We demonstrate that these resilience components are highly correlated with life history traits related to the species pace of life and reproductive strategy. Species with longer generation times require longer recovery times post-disturbance, while those with greater reproductive capacity have greater resistance and compensation. Our findings highlight the key role of life history traits to understand species resilience, improving our ability to predict how natural populations cope with disturbance regimes.

ecology↗

bioRxiv2020

A conserved amino acid in the C-terminus of HPV E7 mediates binding to PTPN14 and repression of epithelial differentiation

The human papillomavirus (HPV) E7 oncoprotein is a primary driver of HPV-mediated carcinogenesis. The E7 proteins from diverse HPV bind to the host cellular non-receptor protein tyrosine phosphatase type 14 (PTPN14) and direct it for degradation through the activity of the E7-associated host E3 ubiquitin ligase UBR4. Herein we show that a highly conserved arginine residue in the C-terminal domain of diverse HPV E7 mediates interaction with PTPN14. We found that disruption of PTPN14 binding through mutation of the C-terminal arginine did not impact the ability of several high-risk HPV E7 proteins to bind and degrade the retinoblastoma tumor suppressor or activate E2F target gene expression. HPVs infect human keratinocytes and we previously reported that both PTPN14 degradation by HPV16 E7 and PTPN14 CRISPR knockout repress keratinocyte differentiation-related genes. Now we have found that blocking PTPN14 binding through mutation of the conserved C-terminal arginine rendered both HPV16 and HPV18 E7 unable to repress differentiation-related gene expression. We then confirmed that the HPV18 E7 variant that could not bind PTPN14 was also impaired in repressing differentiation when expressed from the complete HPV18 genome. Finally, we found that the ability of HPV18 E7 to extend the lifespan of primary human keratinocytes required PTPN14 binding. CRISPR/Cas9 knockout of PTPN14 rescued keratinocyte lifespan extension in the presence of the PTPN14 binding-deficient HPV18 E7 variant. These results support the model that PTPN14 degradation by high-risk HPV E7 leads to repression of differentiation and contributes to its carcinogenic activity. IMPORTANCEHuman papillomavirus (HPV)-positive carcinomas account for nearly 5% of the global human cancer burden. The E7 oncoprotein is a primary driver of HPV-mediated carcinogenesis. HPV E7 binds and degrades the putative tumor suppressor, PTPN14. However, the impact of PTPN14 binding by E7 on cellular processes is not well defined. Here, we show that PTPN14 binding is mediated by a conserved C-terminal arginine residue of HPV E7 in vivo. Additionally, we found that PTPN14 binding contributes to the carcinogenic activity of HPV18 E7 (the second most abundant HPV type in cancers). Finally, we determined that PTPN14 binding by HPV16 E7 and HPV18 E7 represses keratinocyte differentiation. HPV-positive cancers are frequently poorly differentiated and the HPV life cycle is dependent upon the keratinocyte differentiation program. The finding that PTPN14 binding by HPV E7 impairs differentiation has significant implications for both HPV-mediated carcinogenesis and the HPV life cycle.

microbiology↗

bioRxiv2018

PTPN14 Degradation by High-Risk Human Papillomavirus E7 Limits Keratinocyte Differentiation and Contributes to HPV-Mediated Oncogenesis

High-risk human papillomavirus (HPV) E7 proteins enable oncogenic transformation of HPV-infected cells by inactivating host cellular proteins. High-risk but not low-risk HPV E7 target PTPN14 for proteolytic degradation, suggesting that PTPN14 degradation may be related to their oncogenic activity. HPV infects human keratinocytes but the role of PTPN14 in keratinocytes and the consequences of PTPN14 degradation are unknown. Using an HPV16 E7 variant that can inactivate RB1 but cannot degrade PTPN14 we found that high-risk HPV E7-mediated PTPN14 degradation impairs keratinocyte differentiation. Deletion of PTPN14 from primary human keratinocytes decreased keratinocyte differentiation gene expression. Related to oncogenic transformation, both HPV16 E7-mediated PTPN14 degradation and PTPN14 deletion promoted keratinocyte survival following detachment from a substrate. PTPN14 degradation contributed to high-risk HPV E6/E7-mediated immortalization of primary keratinocytes and HPV-positive but not HPV-negative cancers exhibit a gene expression signature consistent with PTPN14 inactivation. We find that PTPN14 degradation impairs keratinocyte differentiation and propose that this contributes to high-risk HPV E7-mediated oncogenic activity independent of RB1 inactivation.\n\nSignificance StatementHuman papillomaviruses uncouple proliferation from differentiation in order to enable virus replication in epithelial cells. HPV E7 proteins are well established to promote proliferation by binding to and inactivating retinoblastoma family proteins and other cell cycle inhibitors. However, mechanisms by which high-risk HPV oncoproteins inhibit differentiation have not been defined. This paper identifies the first mechanism by which high-risk HPV E7 inhibit keratinocyte differentiation. The inhibition of differentiation requires degradation of the cellular protein PTPN14 by high-risk HPV E7 and this degradation is related to the ability of high-risk HPV oncoproteins to immortalize keratinocytes and to cause cancer.

microbiology↗