Biology subjects

Ryan, T. M.

Publications and source records attributed to Ryan, T. M..

5 recordsLinked to original sources

bioRxiv2023

Reconstituting spore cortex peptidoglycan biosynthesis reveals a deacetylase that catalyzes transamidation

Some bacteria survive in nutrient-poor environments and resist killing by antimicrobials by forming spores. The cortex layer of the peptidoglycan cell wall that surrounds mature spores contains a unique modification, muramic-{delta}-lactam, that is essential for spore germination and outgrowth. Two proteins, the amidase CwlD and the deacetylase PdaA, are required for muramic-{delta}-lactam synthesis in cells, but their combined ability to generate muramic-{delta}-lactam has not been directly demonstrated. Here we report an in vitro reconstitution of cortex peptidoglycan biosynthesis, and we show that CwlD and PdaA together are sufficient for muramic-{delta}-lactam formation. Our method enables characterization of the individual reaction steps, and we show for the first time that PdaA has transamidase activity, catalyzing both the deacetylation of N-acetylmuramic acid and cyclization of the product to form muramic-{delta}-lactam. This activity is unique among peptidoglycan deacetylases and is notable because it may involve the direct ligation of a carboxylic acid with a primary amine. Our reconstitution products are nearly identical to the cortex peptidoglycan found in spores, and we expect that they will be useful substrates for future studies of enzymes that act on the spore cortex.

biochemistry↗

bioRxiv2021

Growth and development of trabecular structure in the calcaneus of Japanese macaques (Macaca fuscata) reflects locomotor behavior, life-history, and neuromuscular development.

We aim to broaden the analysis of bone structure by suggesting a new way to incorporate the interactions between behavior, neuromuscular development, and life-history. We examine the associations between these variables and age-related variation in trabecular structure in the calcaneus of Japanese macaques (Macaca fuscata). If skeletal markers linking these variables can be established, our inferences of the biology and behavior of fossil species would be significantly improved. We {micro}CT scanned the calcaneus in a cross-sectional sample of 36 juveniles aged between 0 and 7 years old and 5 adults at the Primate Research Institute, Japan. We calculated whole bone averages of standard trabecular properties and generated whole-bone morphometric maps of bone volume fraction and Youngs modulus. Trabecular structure is increasingly heterogeneous in older individuals. BV/TV decreases during the first month of life and increases afterwards, coinciding with the onset of independent locomotion. At birth, primary Youngs modulus is oriented orthogonal to the ossification center, but after locomotor onset bone structure becomes stiffest in the direction of joint surfaces and muscle attachments. Age-related variation in bone volume fraction is best predicted by an interaction between neuromaturation, body mass, and locomotor independence. Results support the common assumption that trabecular structure dynamically adapts to novel joint loading conditions during ontogeny. The timing of independent locomotion, body size, and neuromuscular development, are all correlated to age-related variation in the trabecular structure of the macaque calcaneus. The causal mechanisms behind the observed patterns cannot be directly inferred from our cross-sectional study. If the model presented in this paper holds up under longitudinal experimental conditions, trabecular structure can be used both to infer behavior from fossil morphology and to serve as a valuable proxy for neuromuscular maturation and life history events like locomotor onset and the achievement of an adult-like gait.

developmental biology↗

bioRxiv2020

Potential evolutionary body size reduction in a Malagasy primate (Propithecus verreauxi) in response to human size-selective hunting pressure

The Holocene arrival of humans on Madagascar precipitated major changes to the islands biodiversity. The now-extinct, endemic "subfossil" megafauna of Madagascar were likely hunted by the islands early human inhabitants. Perhaps in part due to preferential hunting of larger prey, no surviving species on Madagascar is larger than 10 kg. Outside of Madagascar, size-selective hunting pressure has resulted in the phyletic dwarfism of many still-living species across a diversity of phyla. On Madagascar, some subfossil bones of extant lemurs are considerably larger than those of the modern members of their species, but relatively large distances between the subfossil localities and modern samples that have been compared to date makes it impossible to reject the possibility that these size differences more simply reflect pre-existing ecogeographic variation. Here, we used high-resolution 3D scan data to conduct comparative morphological analyses of subfossil and modern skeletal remains of one of the larger extant lemurs, Verreauxs sifakas (Propithecus verreauxi) from subfossil and modern sites [~]10 km adjacent: Taolambiby (bones dated to 725-560 - 1075-955 cal. years before present) and Beza Mahafaly Special Reserve, respectively. We found that the average subfossil sifaka bone (n=12) is 9% and significantly larger than that of modern sifakas (n=31 individuals; permutation test; p=0.037). When restricting the analysis to the single element and side with the largest representation in the subfossil sample (n=4 right distal femora), the average subfossil bone is 10% larger (p=0.046). While we cannot yet conclude whether this size difference reflects evolutionary change or an archaeological aggregation/taphonomic process, if this is a case of phyletic dwarfism in response to human size-selective harvesting pressures then the estimated rate of evolutionary change is slightly higher than that previously calculated for other archaeological cases of this phenomenon.

evolutionary biology↗

bioRxiv2019

Correction of z-motion artefacts to allow population imaging of synaptic activity in awake behaving mice

Functional imaging of head-fixed, awake, behaving mice using two-photon imaging of fluorescent activity reporters has become a powerful tool in the studying the function of the brain. Motion artefacts are an inevitable problem during such experiments and are routinely corrected for in x and y dimensions. However, axial (z) shifts of several microns can also occur, leading to intensity fluctuations in structures such as synapses that are small compared to the axial point-spread function of the microscope. Here we present a simple strategy to correct z-motion artefacts arising over the course of a time-series experiment in a single optical plane. Displacement in z was calculated using dye-filled blood vessels as an anatomical marker, providing high contrast images and accuracy to within [~]0.1 {micro}m. The axial profiles of ROIs corresponding to synapses were described using a Moffat function and this \"ROI-spread function\" used to correct activity traces on an ROI-by-ROI basis. We demonstrate the accuracy and utility of the procedures in simulation experiments using fluorescent beads and then apply them to correcting measurements of synaptic activity in populations of vasoactive-intestinal peptide (VIP) interneurons expressing the synaptic reporter SyGCaMP6f. Correction of z-motion artefacts had a substantial impact on the apparent correlation between synaptic activity and running speed, demonstrating the importance of correcting for these artefacts for the interpretation of in vivo imaging experiments in awake mice.\n\nSummary of Key PointsO_LIMotion artefacts associated with motor behaviour are an inevitable problem of multiphoton imaging in awake behaving animals, particularly imaging synapses.\nC_LIO_LICorrection of axial motion usually requires volumetric imaging resulting in slower rates of acquisition.\nC_LIO_LIWe describe a method that is easy to implement to correct z-motion artefacts that allows population imaging of synaptic activity while scanning a single plane in a standard multiphoton microscope.\nC_LIO_LIThe method uses a reference volume acquired in two colour channels - an activity reporter and an anatomical marker of blood vessels. The procedure estimates the z-displacement in every frame and applies an intensity correction in which the z point-spread function for each synapse is modelled as a Moffat function.\nC_LIO_LIWe demonstrate that the method allows synaptic calcium activity signals to be collected from populations of synaptic boutons in mouse primary visual cortex during locomotion.\nC_LI

neuroscience↗

bioRxiv2018

Anatomical basis and physiological role of cerebrospinal fluid transport through the murine cribriform plate

Cerebrospinal fluid (CSF) flows through the brain, transporting chemical signals and removing waste. CSF production in the brain is balanced by a constant outflow of CSF, the anatomical basis of which is poorly understood. Here we characterized the anatomy and physiological function of the CSF outflow pathway along the olfactory sensory nerves through the cribriform plate, and into the nasal epithelia. Chemical ablation of olfactory sensory nerves greatly reduced outflow of CSF through the cribriform plate. The reduction in CSF outflow did not cause an increase in intracranial pressure (ICP), consistent with an alteration in the pattern of CSF drainage or production. Our results suggest that damage to olfactory sensory neurons (such as from air pollution) could contribute to altered CSF turnover and flow, providing a potential mechanism for neurological diseases.

neuroscience↗