Biology subjects

Williamson, J.

Publications and source records attributed to Williamson, J..

14 recordsLinked to original sources

bioRxiv2026

Integration-coupled activation of promoterless combinatorial pathway libraries in Clostridium avoids burden during DNA assembly

Combinatorial DNA design and assembly is an efficient and pragmatic way to obtain high-performing metabolic pathway designs quickly. However, implementation may require organism-specific technical barriers to be overcome. Firstly, suitable expression control parts such as promoters and ribosome-binding sites (RBSs) which provide a suitable range of expression levels need to be identified or developed. Secondly, these need to be assembled into pathway-encoding combinatorial libraries of sufficient size, quality and diversity. For organisms with transformation frequencies too low to allow direct transformation of library assembly reactions, such as many Clostridium spp., assembly and amplification is typically carried out using Escherichia coli. However, if constructs are deleterious (or burdensome) to E. coli, which is often the case when using Clostridium genetic parts, poor libraries may be obtained. Here we develop a new approach called integration-coupled activation of promoterless sequences (ICAPS) to overcome this barrier and therefore enable combinatorial assembly in Clostridium. Libraries were designed and assembled as promoterless synthetic operons, preventing expression during DNA assembly, and expression was only activated later, when constructs were integrated into the host genome downstream of a promoter. Variation of expression levels was achieved using a range of context-resistant RBS sequences. This approach was used to produce a Clostridium acetobutylicum library with combinatorial expression variants of an introduced hexanol pathway. This proof of concept provides a generally-applicable approach to implement combinatorial metabolic pathway-encoding libraries in Clostridium spp., circumventing the excessive strength of Clostridium expression control parts in E. coli, and is applicable to other organisms.

synthetic biology↗

bioRxiv2025

Temperature and Ultraviolet Radiation Drive Divergent Visible and Near-Infrared Reflectance Patterns in Butterflies

O_LIClimate change poses extreme risks to biodiversity, threatening the ecosystems upon which humanity depends. Understanding the traits that mediate how organisms respond to climatic gradients in space and time will enable better predictions of the taxa and ecological communities most at risk from warming. C_LIO_LIIn insects, colouration influences thermoregulation, but warming responses must be traded off against other selective pressures. We hypothesise that butterfly wing reflectance in visible and near-infrared (NIR) spectra respond differently to temperature and ultraviolet radiation over space and time. We combined existing reflectance data of 97 butterfly species in both NIR and visible wavelengths, with long-term abundance monitoring data of 120 butterfly communities sampled across 1650 m altitude gradients from May to August in two time periods (2004/5 and 2017). C_LIO_LIThe visible and NIR reflectances of butterfly communities showed a non-linear relationship with altitude, with the lowest reflectance (darkest butterflies) at the coolest, highest sites. In contrast, community visible reflectance decreased through the year as temperatures warmed over spring-summer, whereas community NIR reflectance remained constant, revealing divergent responses of reflectance types to seasonal changes. Temperature had opposing effects on visible and NIR reflectance of butterfly communities, where increasing temperature reduced community visible reflectance strongly while increasing community NIR reflectance slightly. C_LIO_LIConsidering the effects on reflectance of shared evolutionary history in a Bayesian hierarchical model for individual species, lighter-coloured (more reflective) species were associated with warmer temperatures - flying later in spring-summer or at lower altitudes. However, instead of decreasing in reflectance through the year and across temperature gradients, species instead became lighter, we expect as a result of a Simpsons paradox. C_LIO_LIThese results emphasise how visible and NIR reflectance wavelength bands mediate butterflies responses to environmental gradients in distinct ways, despite being highly correlated across species. We also show that incorporating phylogeny into trait-environment models is essential; relying on traits alone would lead to incorrect inferences and predictions of taxa most at risk from warming climates. C_LI

ecology↗

bioRxiv2025

RobPicker: A Meta Learning Framework for Robust Identification of Macromolecules in Cryo-Electron Tomograms

Accurate particle picking of macromolecules of all kinds, shapes, and sizes in cryogenic electron tomograms (cryo-ET) is critical for understanding the molecular architecture of biological systems in their native state. Current deep learning methods have shown potential in identifying macromolecules from tomograms, but they are vulnerable to issues of noise in the training dataset obtained through human or automatic labeling and imbalanced distribution of macromolecule species. To address these limitations, we developed RobPicker, a meta-learning framework that effectively mitigates these issues by automatically learning deep neural networks to correct label errors and give greater emphasis to underrepresented macromolecule species. In evaluations across diverse cryo-ET datasets with noisy labels and imbalanced species distributions, RobPicker substantially outperforms state-of-the-art methods, particularly in identifying small and rare macromolecules. The efficiency and robustness of RobPicker can also be used for rapid fine-tuning of the tilt-series alignment, leading to improved tomogram reconstruction and enabling high-resolution cellular structural biology analysis.

bioinformatics↗

bioRxiv2025

Immune and Mutational Profile of Gene-Edited Low-Immunogenic Human Primary Cholangiocyte Organoids

Primary human cells cultured in organoid format have great promise as potential regenerative cellular therapies. However, their immunogenicity and mutational profile remain unresolved, impeding effective long-term translation to the clinic. In this study we report, for the first time, the generation of human leukocyte antigen (HLA)-I and HLA-II knock-out expandable human primary cholangiocyte organoids (PCOs) using CRISPR-Cas9 as a potential universal low-immunogenic therapy for bile duct disorders. HLA-edited PCOs (ePCOs) displayed the same phenotypical and functional characteristics as parental un-edited PCOs. Despite minimal off-target edits, single-molecule DNA-sequencing demonstrated that ePCOs and PCOs acquire substantial mutations in culture at similar rates but without evident selection for cancer-driver mutations. ePCOs induced reduced T cell-mediated immunity and a donor-dependent NK cell cytotoxicity in vitro and evaded cytotoxic responses with increased graft survival in humanized mice in vivo. Our findings have important implications for assessment of safety and immunogenicity of organoid cellular therapies.

immunology↗

bioRxiv2024

Vimentin molecular linkages with nesprin-3 enhance nuclear deformation by cell geometric constraints

The nucleus is the organelle of the cell responsible for controlling protein expression, which has a direct effect on cellular biological functions. Here we show that the cytoskeletal protein vimentin plays an important role in increasing cell-generated forces transmitted to the cell nucleus, resulting in increased nuclear deformations in strongly polarized cells. Using micropatterned substrates to geometrically control cell shape in wild-type and vimentin-null cells, we show vimentin increases polarization and deformation of the cell nucleus. Loss of nesprin-3, which physically couples vimentin to the nuclear envelope, phenotypically copies the loss of vimentin, suggesting vimentin transmits forces to the cell nucleus through direct molecular linkages. Use of a fluorescence resonance energy transfer (FRET) sensor that binds to the nuclear envelope through lamin-A/C suggests vimentin increases the tension on the nuclear envelope. Our results indicate that nuclear shape and deformation can be modified by the vimentin cytoskeleton and its specific crosslinks to the cell nucleus.

biophysics↗

bioRxiv2023

Progesterone receptor activation regulates sensory sensitivity

Women develop chronic pain during their reproductive years more often than men, and estrogen and progesterone regulate this susceptibility. We tested whether brain progesterone receptor (PR) signaling regulates pain susceptibility. During the estrous cycle, animals were more sensitive to pain during the estrus stage than in the diestrus stage, suggesting a role for reproductive hormones, estrogen, and progesterone. We measured the pain threshold daily for four days in ovariectomized, estrogen-primed animals treated with progesterone. The pain threshold was lower 2 days later and stayed that way for the duration of the testing. A specific progesterone-receptor (PR) agonist, segesterone, promoted pain, and mice lacking PR in the brain (PRKO) did not experience lowered pain threshold when treated with progesterone or segesterone. PR activation increased the cold sensitivity but did not affect the heat sensitivity and had a small effect on light sensitivity. Finally, we evaluated whether PR activation altered experimental migraine. Segesterone and nitroglycerin (NTG) when administered sequentially, reduced pain threshold but not separately. These studies have uncovered a pain-regulating function of PRs. Targeting PRs may provide a novel therapeutic avenue to treat chronic pain in women.

neuroscience↗

bioRxiv2023

Sustained growth and rapid dispersal of chitin-associated marine bacteria

Many biogeochemical functions involve bacteria utilizing solid substrates. However, little is known about the coordination of bacterial growth with the kinetics of attachment to and detachment from such substrates. In this quantitative study of Vibrio sp. 1A01 growing on chitin particles, we reveal the heterogeneous nature of the exponentially growing culture, comprised of two co-existing subpopulations: a minority replicating on chitin particles and a non-replicating majority which was planktonic. This partition resulted from a high rate of cell detachment from particles. Despite high detachment, sustained exponential growth of cells on particles was enabled by the enrichment of extra-cellular chitinases excreted and left behind by detached cells. The "inheritance" of these chitinases sustains the colonizing subpopulation despite its reduced density. This simple mechanism helps to circumvent a tradeoff between growth and dispersal, allowing particle-associated marine heterotrophs to explore new habitats without compromising their fitness on the habitat they have already colonized.

microbiology↗

bioRxiv2022

A deep learning-based automated closed-loop optogenetic system for neuromodulation during seizures

Closed-loop electrical brain stimulation is becoming a popular technique proposed for use as a treatment alternative to surgical resection of brain tissue for drug-resistant seizures in epilepsy patients. Closed-loop optogenetic stimulation is an experimental alternative to electrical stimulation since it can stimulate or inhibit neurons. The closed-loop part contains an online seizure detection algorithm, which, based on the design, can detect the onset of a seizure or evaluate a running window for seizure membership. Conventional configurations of closed-loop optogenetics have several limitations, ranging from the adaptability of hardware-based implementation to inadequate, customized feature selection of seizures, among others. Here we provide a detailed description of our closed-loop components. We used a sequential, fully convolutional neural network regressor for complex feature selection of seizures against the controls from local field potential recordings. Our modular design kept the local field potential-recording headset and optical probe separate. The seizure detection and execution of light delivery are fast and can be precisely timed. This automated system is robust to noise, modular in design, flexible in use, and simple execution. When applied in vivo, the proposed work shows efficacy over the state of the arts in terms of improved seizure detection and reduction in false positives.

neuroscience↗

bioRxiv2021

Blood variation implicates respiratory limits on elevational ranges of Andean birds

The extent to which species ranges reflect intrinsic physiological tolerances is a major, unsolved question in evolutionary ecology. To date, consensus has been hindered by the limited tractability of experimental approaches across most of the tree of life. Here, we apply a macrophysiological approach to understand how hematological traits related to oxygen transport shape elevational ranges in a tropical biodiversity hotspot. Along Andean elevational gradients, we measured traits that affect blood oxygen-carrying capacity--total and cellular hemoglobin concentration and hematocrit--for 2,355 individuals of 136 bird species. We used these data to evaluate the influence of hematological traits on elevational ranges. First, we asked whether hematological plasticity is predictive of elevational range breadth. Second, we asked whether variance in hematological traits changed as a function of distance from the midpoint of the elevational range. We found that the correlation between hematological plasticity and elevational range breadth was slightly positive, consistent with a facilitative role for plasticity in elevational range expansion. We further found reduced local variation in hematological traits near elevational range limits and at high elevations, patterns consistent with intensified natural selection, reduced effective population size, or compensatory changes in other cardiohematological traits with increasing distance from species-specific optima for oxygen availability. Our findings suggest that constraints on hematological plasticity and local genetic adaptation to oxygen availability promote the evolution of the narrow elevational ranges that underpin tropical montane biodiversity.

ecology↗

bioRxiv2021

Mammals adjust diel activity across gradients of urbanization

Time is a fundamental component of ecological processes. How animal behavior changes over time has been explored through well-known ecological theories like niche partitioning and predator-prey dynamics. Yet, changes in animal behavior within the shorter 24-hour light-dark cycle have largely gone unstudied. Understanding if an animal can adjust their temporal activity to mitigate or adapt to environmental change has become a recent topic of discussion and is important for effective wildlife management and conservation. While spatial habitat is a fundamental consideration in wildlife management and conservation, temporal habitat is often ignored. We formulated a temporal resource selection model to quantify the diel behavior of eight mammal species across ten U.S. cities. We found high variability in diel activity patterns within and among species and species-specific correlations between diel activity and human population density, impervious land cover, available greenspace, vegetation cover, and mean daily temperature. We also found that some species may modulate temporal behaviors to manage both natural and anthropogenic risks. Our results highlight the complexity with which temporal activity patterns interact with local environmental characteristics, and suggest that urban mammals may use time along the 24-hour cycle to reduce risk, adapt, and therefore persist in human-dominated ecosystems.

ecology↗

bioRxiv2021

The Histone H1-like protein AlgP facilitates even spacing of polyphosphate granules in Pseudomonas aeruginosa

Synthesis of polyphosphate (polyP) is an ancient and universal stress and starvation response in bacteria. In many bacteria, polyP chains come together to form granular superstructures within cells. Some species appear to regulate polyP granule subcellular organization. Despite the critical role of polyP in starvation fitness, the composition of these structures, mechanism(s) underpinning their organization, and functional significance of such organization are poorly understood. We previously determined that granules become transiently evenly spaced on the cells long axis during nitrogen starvation in the opportunistic human pathogen Pseudomonas aeruginosa. Here, we developed a granule-enrichment protocol to screen for polyP granule-localizing proteins. We identified AlgP as a protein that associates with polyP granules. We further discovered that AlgP is required for the even spacing of polyP granules. AlgP is a DNA-binding protein with a 154 amino acid C-terminal domain enriched in KPAA repeats and variants of this repeat, with an overall sequence composition similar to the C-terminal tail of eukaryotic histone H1. Granule size, number, and spacing are significantly perturbed in the absence of AlgP, or when AlgP is truncated to remove the C-terminus. The {Delta}algP and algP{Delta}CTD mutants having fewer, larger granules. We speculate that AlgP may contribute to spacing by tethering polyP granules to the chromosome, thereby inhibiting fusion with neighboring granules. Our discovery that AlgP facilitates granule spacing allows us for the first time to directly uncouple granule biogenesis from even spacing, and will inform future efforts to explore the functional significance of granule organization on fitness during starvation. IMPORTANCEThe mechanisms underpinning polyPs pleiotropic effects on bacterial starvation physiology remain elusive. This simple polyanions lack of protein binding specificity has impeded validation of bona fide polyP-binding proteins. However, polyP forms granule superstructures with spatial specificity. Our granule enrichment protocol identified a polyP granule-associated protein in Pseudomonas aeruginosa, AlgP. AlgP was originally reported as regulator of alginate, an extracellular polysaccharide important in biofilm formation, including in cystic fibrosis (CF) chronic infections. AlgPs putative role in alginate biosynthesis has recently been called into question. We establish a distinct, previously unknown function for AlgP in modulating the subcellular organization of polyP, another polymer important for pathogenesis. In CF clinical isolates, the C-terminal repeat domain of AlgP is a hotspot for genetic rearrangements. Our finding that the C-terminus of AlgP is required for granule organization lays the groundwork for exploring the functional significance of these mutations in the evolutionary trajectory of chronic infections.

microbiology↗

bioRxiv2020

Probabilistic Framework for Integration of Mass Spectrum and Retention Time Information in Small Molecule Identification

MotivationIdentification of small molecules in a biological sample remains a major bottleneck in molecular biology, despite a decade of rapid development of computational approaches for predicting molecular structures using mass spectrometry (MS) data. Recently, there has been increasing interest in utilizing other information sources, such as liquid chromatography (LC) retention time (RT), to improve the MS based identifications. ResultsWe put forward a probabilistic modelling framework to integrate MS and RT data of multiple features in an LC-MS experiment. We model the MS measurements and all pairwise retention order information as a Markov random field and use efficient approximate inference for scoring and ranking potential molecular structures. Our experiments show improved identification accuracy by combining tandem mass spectrometry data (MS2) and retention orders using our approach, thereby outperforming state-of-the-art methods. Furthermore, we demonstrate the benefit of our model when only a subset of LC-MS features have MS2 measurements available besides MS1. Availability and implementationSoftware and data is freely available at https://github.com/aalto-ics-kepaco/msms_rt_score_integration. Contacteric.bach@aalto.fi

systems biology↗

bioRxiv2019

Interferon-responsive genes are targeted during the establishment of human cytomegalovirus latency

Human cytomegalovirus (HCMV) latency is an active process which remodels the latently infected cell to optimise latent carriage and reactivation. This is achieved, in part, through the expression of viral genes, including the G-protein coupled receptor US28. Here, we use an unbiased proteomic screen to assess changes in host proteins induced by US28, revealing that interferon-inducible genes are downregulated by US28. We validate that MHC Class II and two PYHIN proteins, MNDA and IFI16, are downregulated during experimental latency in primary human CD14+ monocytes. We find that IFI16 is targeted rapidly during the establishment of latency in a US28-dependent manner, but only in undifferentiated myeloid cells, a natural site of latent carriage. Finally, by overexpressing IFI16, we show that IFI16 can activate the viral major immediate early promoter and immediate early gene expression during latency via NF-{kappa}B, a function which explains why downregulation of IFI16 during latency is advantageous for the virus.\n\nImportanceHuman cytomegalovirus (HCMV) is a ubiquitous herpesvirus which infects 50-100% of humans worldwide. HCMV causes a lifelong subclinical infection in immunocompetent individuals, but is a serious cause of mortality and morbidity in the immunocompromised and in neonates. In particular, reactivation of HCMV in the transplant setting is a major cause of transplant failure and related disease. Therefore, a molecular understanding of HCMV latency and reactivation could provide insights into potential ways to target the latent viral reservoir in at-risk patient populations.

microbiology↗

bioRxiv2019

Role of Era in Assembly and Homeostasis of the Ribosomal Small Subunit

To reveal the role of the essential protein Era in the assembly of the 30S ribosomal subunit, we analyzed assembly intermediates that accumulated in Era-depleted Escherichia coli cells using quantitative mass spectrometry, cryo-electron microscopy and in-cell footprinting. Our combined approach allowed for visualization of the small subunit as it assembled and revealed that with the exception of key helices in the platform domain, all other 16S rRNA domains were able to fold even in the absence of Era. Notably, the maturing particles did not stall while waiting for the platform domain to mature and instead re-routed their folding pathway to enable concerted maturation of other structural motifs spanning multiple rRNA domains. We also found that binding of Era to the mature 30S subunit destabilized helix 44 and the decoding center preventing binding of YjeQ, another assembly factor. This work establishes Eras role in ribosome assembly and suggests new roles in maintaining ribosome homeostasis.

molecular biology↗