Biology subjects

Larsen, R.

Publications and source records attributed to Larsen, R..

15 recordsLinked to original sources

bioRxiv2026

RNA Quality Control Enables Antibiotic Tolerance

The mechanisms underlying adaptation to antibiotic pressure within complex host environments remain incompletely understood. By experimentally evolving Streptococcus pneumoniae subjected to various antibiotics and immune states, we demonstrate populations adopting distinct adaptive strategies depending on specific selective context. General antibiotic stress drives convergent mutations in rny, encoding the RNA degradosome scaffold RNase Y, that exhibit broad-spectrum antibiotic tolerance and accelerated recovery. Single-cell transcriptomics revealed antibiotic-induced death is driven by transcriptional collapse, a catastrophic loss of RNA quantity and integrity. In contrast, rny mutants avert this via a bet-hedging strategy: a resilient minority maintains a baseline transcriptional profile, while a quiescent majority undergoes selective RNA degradation to preserve transcript fidelity. Upon stress removal, these populations execute a prioritized transcriptional ribosomal reboot, facilitating accelerated recovery. These findings identify RNA turnover as a tunable master regulator to survive combined pressures of antibiotics and immunity.

microbiology↗

bioRxiv2025

Multi-omic analysis reveals the unique glycan landscape of the blood-brain barrier glycocalyx

The blood-brain barrier (BBB) glycocalyx is the dense layer of glycans and glycoconjugates that coats the luminal surface of the central nervous system (CNS) vasculature. Despite being the first point of contact between the blood and brain, not much is known about the BBB glycocalyx. Here, we performed a multi-omic investigation of the BBB glycocalyx which revealed a unique glycan landscape characterized by enrichment of sialic acid, chondroitin sulfate, and hyaluronan. We found that the BBB glycocalyx was thicker than glycocalyces in the peripheral vasculature and that hyaluronan was the major contributor to its ultrastructure. Using endothelial RNA sequencing, we found potential genetic determinants for these differences, including BBB enrichment of genes involved in sialic acid addition and peripheral enrichment of Tmem2 and Hyal2, the only known cell-surface hyaluronidases. Glycocalyx degradation and increases in vascular permeability are widely associated with inflammation. However, we found that the BBB glycocalyx remains largely unchanged in neuroinflammation during the experimental autoimmune encephalomyelitis (EAE) model of multiple sclerosis and that its degradation is not sufficient to alter BBB permeability in health. Moreover, we showed that CNS endothelial sialic acid removal delays onset of EAE, indicating that BBB glycocalyx sialic acid may contribute to the progression of neuroinflammation. These findings underscore the unique and robust nature of the BBB glycocalyx and provide targets and tools for future studies into its role in health and neuroinflammation.

molecular biology↗

bioRxiv2021

Identification of New In Vivo TonB-FepA Rendezvous Sites

The TonB system of Gram-negative bacteria uses the protonmotive force of the cytoplasmic membrane to energize active transport of large or scarce nutrients across the outer membrane by means of customized beta-barrels known as TonB-dependent transporters (TBDTs). The lumen of each TBDT is occluded by an amino-terminal domain, called the cork, which must be displaced for transport of nutrients or translocation of the large protein toxins that parasitize the system. A complex of cytoplasmic membrane proteins consisting of TonB, ExbB and ExbD harnesses the protonmotive force that TonB transmits to the TBDT. The specifics of this energy transformation are a source of continuing interest. The amino terminal domain of a TBDT contains a region called the TonB box, that is essential for the reception of energy from TonB. This domain is the only identified site of in vivo interaction between the TBDT and TonB, occurring through a non-essential region centered on TonB residue Q160. Because TonB binds to TBDTs whether or not it is active or even intact, the mechanism and extent of cork movement in vivo has been challenging to discover. In this study, we used in vivo disulfide crosslinking between eight engineered Cys residues in Escherichia coli TonB and 42 Cys substitutions in the TBDT FepA, including the TonB box, to identify novel sites of interaction in vivo. The TonB Cys substitutions in the core of an essential carboxy terminal amphipathic helix (residues 199-216) were compared to TonB Q160C interactions. Functionality of the in vivo interactions was established when the presence of the inactive TonB H20A mutation inhibited them. A previously unknown functional interaction between the hydrophilic face of the amphipathic helix and the FepA TonB box was identified. Interaction of Q160C with the FepA TonB box appeared to be less functionally important. The two different parts of TonB also differed in their interactions with the FepA cork and barrel turns. While the TonB amphipathic helix Cys residues interacted only with Cys residues on the periplasmic face of the FepA cork, TonB Q160C interacted with buried Cys substitutions within the FepA cork, the first such interactions seen with any TBDT. Both sets of interactions required active TonB. Taken together, these data suggest a model where the amphipathic helix binds to the TonB box, causing the mechanically weak domain of the FepA cork to dip sufficiently into the periplasmic space for interaction with the TonB Q160 region, which is an interaction that does not occur if the TonB box is deleted. The TonB amphipathic helix also interacted with periplasmic turns between FepA {beta}-strands in vivo supporting a surveillance mechanism where TonB searched for TBDTs on the periplasmic face of the outer membrane.

molecular biology↗

bioRxiv2021

Automated identification of piglet brain tissue from MRI images using Region-Based Convolutional Neural Networks

Magnetic Resonance Imaging is an important tool for characterizing volumetric changes of the piglet brain during development. Typically, an early step of an imaging analysis pipeline is brain extraction, or skull stripping. Brain extractions are usually performed manually; however, this approach is time-intensive and can lead to variation between brain extractions when multiple raters are used. Automated brain extractions are important for reducing the time required for analyses and improving the uniformity of the extractions. Here we demonstrate the use of Mask R-CNN, a Region-based Convolutional Neural Network (R-CNN), for automated brain extractions of piglet brains. We validate our approach using Nested Cross-Validation on six sets of training/validation data drawn from 32 pigs. Visual inspection of the extractions shows acceptable accuracy, Dice coefficients are in the range of 0.95-0.97, and Hausdorff Distance values in the range of 4.1-8.3 voxels. These results demonstrate that R-CNNs provide a viable tool for skull stripping of piglet brains.

neuroscience↗

bioRxiv2021

Motion/direction-sensitive thalamic neurons project extensively to the middle layers of primary visual cortex

The motion/direction-sensitive and location-sensitive neurons are two major functional types in mouse visual thalamus that project to the primary visual cortex (V1). It has been proposed that the motion/direction-sensitive neurons mainly target the superficial layers in V1, in contrast to the location-sensitive neurons which mainly target the middle layers. Here, by imaging calcium activities of motion/direction-sensitive and location-sensitive axons in V1, we find no evidence for these cell-type specific laminar biases at population level. Furthermore, using a novel approach to reconstruct single-axon structures with identified in vivo response types, we show that, at single-axon level, the motion/direction-sensitive axons have middle layer preferences and project more densely to the middle layers than the location-sensitive axons. Overall, our results demonstrate that Motion/direction-sensitive thalamic neurons project extensively to the middle layers of V1, challenging the current view of the thalamocortical organizations in the mouse visual system.

neuroscience↗

bioRxiv2021

Connectivity and Synaptic Physiology in the Mouse and Human Cortex

To elucidate cortical microcircuit structure and synaptic properties we present a unique, extensive, and public synaptic physiology dataset and analysis platform. Through its application, we reveal principles that relate cell type to synapse properties and intralaminar circuit organization in the mouse and human cortex. The dynamics of excitatory synapses align with the postsynaptic cell subclass, whereas inhibitory synapse dynamics partly align with presynaptic cell subclass but with considerable overlap. Despite these associations, synaptic properties are heterogeneous in most subclass to subclass connections. The two main axes of heterogeneity are strength and variability. Cell subclasses divide along the variability axis, while the strength axis accounts for significant heterogeneity within the subclass. In human cortex, excitatory to excitatory synapse dynamics are distinct from those in mouse and short-term plasticity varies with depth across layers 2 and 3. With a novel connectivity analysis that enables fair comparisons between circuit elements, we find that intralaminar connection probability among cell subclasses exhibits a strong layer dependence.These and other findings combined with the analysis platform create new opportunities for the neuroscience community to advance our understanding of cortical microcircuits.

neuroscience↗

bioRxiv2021

Fecal microbial transplant abates tolerance to methylone-induced hyperthermia

The microbiome-gut-brain axis has been implicated in multiple bodily systems and pathologies, and intentional manipulation of the gut-microbiome has yielded clinically significant results. Here, we examined the effects of bi-directional fecal microbial transplants (FMT) between methylone-induced hyperthermic tolerant (MHT) and methylone-naive (MN) rats. Rats treated with methylone once per week developed tolerance to methylone-induced hyperthermia by the fourth week. Once tolerant, daily bi-directional FMT between the two groups were performed for seven days prior to the next methylone treatment. The FMT abated the developed tolerance in the MHT group. When treated with methylone for the first time following FMT, recipient MN rats displayed significant tolerance to hyperthermia despite it being their initial drug treatment. Post-FMT, MHT rats displayed elevations in norepinephrine and expression of UCP1, UCP3 and TGR5 in brown adipose tissue, with reductions in expression of TGR5 and UCP3 in skeletal muscle. The pre- and post-FMT methylone tolerance phenotypes of transplant recipients are concurrent with changes in the relative abundance of several Classes of Proteobacteria, most evident for Gammaproteobacter and Alphaproteobacter. MHT recipients demonstrated a marked increase in the relative proportion of the Firmicutes Class Erysipelotrichia. These findings suggest that transplantation of gut-microbiomes can confer phenotypic responses to a drug.

pharmacology and toxicology↗

bioRxiv2020

Safety and data quality of simultaneous EEG-fMRI using multi-band fMRI imaging

PurposeSimultaneously recorded electroencephalography and functional magnetic resonance imaging (EEG-fMRI) is highly informative yet technically challenging. Until recently, there has been little information about EEG data quality and safety when used with newer multi-band (MB) fMRI sequences. Here, we measure the relative heating of a MB protocol compared with a standard single-band (SB) protocol considered to be safe. We also evaluated EEG quality recorded concurrently with the MB protocol on humans. Materials and MethodsWe compared radiofrequency (RF)-related heating at multiple electrodes and magnetic field magnitude, B1+RMS, of a MB fMRI sequence with whole-brain coverage (TR = 440 ms, MB factor = 4) against a previously recommended, safe SB sequence using a phantom outfitted with a 64-channel EEG cap. Next, 9 human subjects underwent eyes-closed resting state EEG-fMRI using the MB sequence. Additionally, in three of the subjects resting state EEG was recorded also during the SB sequence and in an fMRI-free condition to directly compare EEG data quality across scanning conditions. EEG data quality was assessed by the ability to remove gradient and cardioballistic artifacts along with a clean spectrogram. ResultsThe heating induced by the MB sequence was lower than that of the SB sequence by a factor of 0.73 {+/-} 0.38. This is consistent with an expected heating ratio of 0.64, calculated from the square of the ratio of B1+RMS values of the sequences. In the resting state EEG data, gradient and cardioballistic artifacts were successfully removed using traditional template subtraction. All subjects showed an individual alpha peak in the spectrogram with a posterior topography characteristic of eyes-closed EEG. The success of artifact rejection for the MB sequence was comparable to that in traditional SB sequences. ConclusionsOur study shows that B1+RMS is a useful indication of the relative heating of fMRI protocols. This observation indicates that simultaneous EEG-fMRI recordings using this MB sequence can be safe in terms of RF-related heating, and that EEG data recorded using this sequence is of acceptable quality after traditional artifact removal techniques.

neuroscience↗

bioRxiv2019

Complete single neuron reconstruction reveals morphological diversity in molecularly defined claustral and cortical neuron types

Ever since the seminal findings of Ramon y Cajal, dendritic and axonal morphology has been recognized as a defining feature of neuronal types. Yet our knowledge concerning the diversity of neuronal morphologies, in particular distal axonal projection patterns, is extremely limited. To systematically obtain single neuron full morphology on a brain-wide scale, we established a platform with five major components: sparse labeling, whole-brain imaging, reconstruction, registration, and classification. We achieved sparse, robust and consistent fluorescent labeling of a wide range of neuronal types by combining transgenic or viral Cre delivery with novel transgenic reporter lines. We acquired high-resolution whole-brain fluorescent images from a large set of sparsely labeled brains using fluorescence micro-optical sectioning tomography (fMOST). We developed a set of software tools for efficient large-volume image data processing, registration to the Allen Mouse Brain Common Coordinate Framework (CCF), and computer-assisted morphological reconstruction. We reconstructed and analyzed the complete morphologies of 1,708 neurons from the striatum, thalamus, cortex and claustrum. Finally, we classified these cells into multiple morphological and projection types and identified a set of region-specific organizational rules of long-range axonal projections at the single cell level. Specifically, different neuron types from different regions follow highly distinct rules in convergent or divergent projection, feedforward or feedback axon termination patterns, and between-cell homogeneity or heterogeneity. Major molecularly defined classes or types of neurons have correspondingly distinct morphological and projection patterns, however, we also identify further remarkably extensive morphological and projection diversity at more fine-grained levels within the major types that cannot presently be accounted for by preexisting transcriptomic subtypes. These insights reinforce the importance of full morphological characterization of brain cell types and suggest a plethora of ways different cell types and individual neurons may contribute to the function of their respective circuits.

neuroscience↗

bioRxiv2019

Simplified high-throughput methods for deep proteome analysis on the timsTOF Pro

Recent advances in mass spectrometry technology have seen remarkable increases in proteomic sequencing speed, while improvements to dynamic range have remained limited. An exemplar of this is the new timsTOF Pro instrument, which thanks to its trapped ion mobility, pushes effective fragmentation rates beyond 100Hz and provides accurate CCS values as well as impressive sensitivity. Established data dependent methodologies underutilize these advances by relying on long analytical columns and extended LC gradients to achieve comprehensive proteome coverage from biological samples. Here we describe the implementation of methods for short packed emitter columns that fully utilize instrument speed and CCS values by combining rapid generation of deep peptide libraries with enhanced matching of single shot data dependent sample analysis. Impressively, with only a 17 minute separation gradient (50 samples per day), the combination of high performance chromatography and CCS enhanced library based matching resulted in an average of 5,931 protein identifications within individual samples, and 7,244 proteins cumulatively across replicates from HeLa cell tryptic digests. Additionally, an ultra-high throughput setup utilizing 5 min gradients (180 samples per day) yielded an average of 3,666 protein identifications within individual samples and 4,659 proteins cumulatively across replicates. These workflows are simple to implement on available technology and do not require complex software solutions or custom-made consumables to achieve high throughput and deep proteome analysis from biological samples.

biochemistry↗

bioRxiv2018

A large-scale, standardized physiological survey reveals higher order coding throughout the mouse visual cortex

To understand how the brain processes sensory information to guide behavior, we must know how stimulus representations are transformed throughout the visual cortex. Here we report an open, large-scale physiological survey of neural activity in the awake mouse visual cortex: the Allen Brain Observatory Visual Coding dataset. This publicly available dataset includes cortical activity from nearly 60,000 neurons collected from 6 visual areas, 4 layers, and 12 transgenic mouse lines from 221 adult mice, in response to a systematic set of visual stimuli. Using this dataset, we reveal functional differences across these dimensions and show that visual cortical responses are sparse but correlated. Surprisingly, responses to different stimuli are largely independent, e.g. whether a neuron responds to natural scenes provides no information about whether it responds to natural movies or to gratings. We show that these phenomena cannot be explained by standard local filter-based models, but are consistent with multi-layer hierarchical computation, as found in deeper layers of standard convolutional neural networks.

neuroscience↗

bioRxiv2018

The thalamus regulates retinoic acid signaling and development of parvalbumin interneurons in postnatal mouse prefrontal cortex

Abnormal development of GABAergic interneurons in the prefrontal cortex (PFC) is implicated in a number of psychiatric disorders. Yet, developmental mechanisms for these neurons are poorly understood. Here we show that the retinoic acid-degrading enzyme CYP26B1 is temporally expressed specifically in postnatal frontal cortex in mice, and its genetic deletion results in an increased density of parvalbumin (PV)-expressing interneurons in medial PFC during postnatal development. Furthermore, initiation of Cyp26b1 expression in neonatal PFC depends on the connections between the thalamus and the neocortex. Thus, the thalamus has a postnatal role in regulating PV neuron development in PFC by inducing Cyp26b1 and thereby restricting retinoic acid signaling. Prenatally, the lack of thalamic input causes an aberrant radial distribution of medial ganglionic eminence-derived interneurons throughout the cortex. Therefore, the thalamus controls PV neuron development in PFC both by region-specific and cortex-wide mechanisms.

neuroscience↗

bioRxiv2017

Shared and distinct transcriptomic cell types across neocortical areas

Neocortex contains a multitude of cell types segregated into layers and functionally distinct regions. To investigate the diversity of cell types across the mouse neocortex, we analyzed 12,714 cells from the primary visual cortex (VISp), and 9,035 cells from the anterior lateral motor cortex (ALM) by deep single-cell RNA-sequencing (scRNA-seq), identifying 116 transcriptomic cell types. These two regions represent distant poles of the neocortex and perform distinct functions. We define 50 inhibitory transcriptomic cell types, all of which are shared across both cortical regions. In contrast, 49 of 52 excitatory transcriptomic types were found in either VISp or ALM, with only three present in both. By combining single cell RNA-seq and retrograde labeling, we demonstrate correspondence between excitatory transcriptomic types and their region-specific long-range target specificity. This study establishes a combined transcriptomic and projectional taxonomy of cortical cell types from functionally distinct regions of the mouse cortex.

neuroscience↗

bioRxiv2017

A suite of transgenic driver and reporter mouse lines with enhanced brain cell type targeting and functionality

Modern genetic approaches are powerful in providing access to diverse types of neurons within the mammalian brain and greatly facilitating the study of their function. We here report a large set of driver and reporter transgenic mouse lines, including 23 new driver lines targeting a variety of cortical and subcortical cell populations and 26 new reporter lines expressing an array of molecular tools. In particular, we describe the TIGRE2.0 transgenic platform and introduce Cre-dependent reporter lines that enable optical physiology, optogenetics, and sparse labeling of genetically-defined cell populations. TIGRE2.0 reporters broke the barrier in transgene expression level of single-copy targeted-insertion transgenesis in a wide range of neuronal types, along with additional advantage of a simplified breeding strategy compared to our first-generation TIGRE lines. These novel transgenic lines greatly expand the repertoire of high-precision genetic tools available to effectively identify, monitor, and manipulate distinct cell types in the mouse brain.

neuroscience↗

bioRxiv2017

Aberrant Cortical Activity In Multiple GCaMP6-Expressing Transgenic Mouse Lines

Transgenic mouse lines are invaluable tools for neuroscience but as with any technique, care must be taken to ensure that the tool itself does not unduly affect the system under study. Here we report aberrant electrical activity, similar to interictal spikes, and accompanying fluorescence events in some genotypes of transgenic mice expressing GCaMP6 genetically-encoded calcium sensors. These epileptiform events have been observed particularly, but not exclusively, in mice with Emx1-Cre and Ai93 transgenes, across multiple laboratories. The events occur at >0.1 Hz, are very large in amplitude (>1.0 mV local field potentials, >10% df/f widefield imaging signals), and typically cover large regions of cortex. Many properties of neuronal responses and behavior seem normal despite these events, though rare subjects exhibit overt generalized seizures. The underlying mechanisms of this phenomenon remain unclear, but we speculate about possible causes on the basis of diverse observations. We encourage researchers to be aware of these activity patterns while interpreting neuronal recordings from affected mouse lines and when considering which lines to study.

neuroscience↗