Biology subjects

Weiler, J.

Publications and source records attributed to Weiler, J..

11 recordsLinked to original sources

bioRxiv2026

Restriction-site-based enrichment coupled to adaptive sampling enables long-read transposon-insertion sequencing

Long-read Transposon insertion sequencing is essential for linking genotypes to phenotypes, as short-read approaches struggle with complex genomic regions. Long-read sequencing demands high sequencing depth or targeted sequencing, which either requires extensive sample preparation or suffer from low net efficiency. We developed a simple, low-cost workflow combining enzymatic cleavage with adaptive sampling to drastically improve target recovery and simplify analysis. I-SceI restriction site was introduced into a mini-Tn5 transposon to generate a mutant library in Cupriavidus necator. Prior to Nanopore sequencing, gDNA was digested to introduce double-strand breaks precisely at insertion sites, followed by sequencing with adaptive sampling. The workflow was validated via a biofilm selection screen. Combining I-SceI digestion with adaptive sampling yielded an effective 11.3-fold enrichment (after normalizing for pore occupancy). High-depth sequencing covered 97.1% of all genes. Because reads consistently began at the exact insertion site, bioinformatic trimming was bypassed. The biofilm screen successfully identified enriched integration loci, uniquely mapped insertions within repetitive rRNA operons, and revealed co-selected, spontaneous secondary mutations.This method adds less than one hour to library preparation, reduces consumable costs by over 90%, and establishes a budget-friendly, simple and high-resolution long-read Tn-seq pipeline ideal for complex and multipartite genomes. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=78 SRC="FIGDIR/small/733934v1_ufig1.gif" ALT="Figure 1"> View larger version (13K): org.highwire.dtl.DTLVardef@e1533aorg.highwire.dtl.DTLVardef@11f3ebborg.highwire.dtl.DTLVardef@4f94b3org.highwire.dtl.DTLVardef@ab7d29_HPS_FORMAT_FIGEXP M_FIG C_FIG

genomics↗

bioRxiv2025

HIV Nef amplifies mechanical heterogeneity to promote immune evasion

Intracellular pathogens must evade cytotoxic immunity to establish persistent infection. Although immune escape is typically viewed through a biochemical lens, the ability of certain pathogens to alter the mechanical properties of infected cells suggests that biophysical mechanisms may also contribute to the process. Here, we show that a subset of CD4+ T cells infected with the human immunodeficiency virus (HIV) resist elimination through a soft phenotype that inhibits killing by mechanosensitive cytotoxic T lymphocytes (CTLs). This phenotype arises from the combined effects of the HIV virulence factor Nef, which remodels the actin cytoskeleton, and intrinsic heterogeneity in the basal cytoskeletal properties of infected T cells. Pharmacological or genetic perturbations that reverse Nef signaling to the cytoskeleton or that stiffen the filamentous-actin cortex sensitize infected cells to CTL-mediated lysis. Taken together, these findings define a novel, biophysical paradigm of immune evasion with implications for HIV cure strategies.

immunology↗

bioRxiv2024

Sensory expectations shape neural population dynamics in motor circuits

The neural basis of movement preparation has been extensively studied during self-initiated actions where motor cortical activity during preparation shows a lawful relationship to the parameters of the subsequent action1,2. However, movements are regularly triggered and constantly corrected based on sensory inputs caused by disturbances to the body or environment. Since such disturbances are often predictable and since preparing for disturbances would make movements better, we hypothesized that expectations about sensory inputs also influence preparatory activity in motor circuits. Here we show that when humans and monkeys are probabilistically cued about the direction of a future mechanical perturbation, they incorporate sensory expectations into their movement preparation and improve their corrective responses. Using high-density neural recordings, we establish that sensory expectations are widespread across the brain, including the motor cortical areas involved in preparing self-initiated actions. The geometry of these preparatory signals in the neural population state is simple, scaling directly with the probability of each perturbation direction. After perturbation onset, a condition-independent perturbation signal shifts the neural state leading to rapid responses that initially reflect sensory expectations. Based on neural networks coupled to a biomechanical model of the arm3, we show that this neural geometry emerges through training, but only when the incoming sensory information indicating perturbation direction coincides with - or is preceded by - a condition-independent signal indicating that a perturbation has occurred. Thus, motor circuit dynamics are shaped by future sensory inputs, providing clear empirical support for the idea that movement is governed by the sophisticated manipulation of sensory feedback4.

neuroscience↗

bioRxiv2024

Multi-Omic Atlas reveals cytotoxic phenotype and ROS-linked metabolic quiescence as key features of CTL-resistant HIV-infected CD4+ T-cells

Cytotoxic T-lymphocytes (CTL) exert sustained pressure on reservoirs of HIV-infected cells that persist through years of antiretroviral therapy (ART). This selects for latently infected cells, but also potentially for cells that express HIV but possess intrinsic CTL resistance. We demonstrate that such resistance exists in HIV-infected CD4+ T-cells that survive rigorous CTL attack and map CTL susceptibility to cell identities and states defined by single-cell multi-omics and functional metabolic profiling. Cytotoxic CD4+ T-cells were prominently overrepresented amongst survivors, as were cells with quiescent metabolic profiles and low levels of reactive oxygen species (ROS) production. The induction of ROS production by treatment with deferoxamine sensitized these cells to CTL-mediated elimination. Reservoir-harboring cells from clinical samples share the above transcriptional features, being enriched for quiescent states. Our results provide an atlas for elucidating features of CTL resistance in HIV reservoirs, and identify oxidative stress as a therapeutic target to facilitate reservoir elimination.

immunology↗

bioRxiv2024

The EZH2 inhibitor tazemetostat mitigates HIV immune evasion, reduces reservoir formation, and promotes durable CD8+ T-cell revitalization

Persistent HIV reservoirs in CD4 T-cells pose a barrier to curing HIV infection. We identified overexpression of enhancer of zeste homolog 2 (EZH2) in HIV-infected CD4 T- cells that survive cytotoxic T lymphocyte (CTL) exposure, suggesting a mechanism of CTL resistance. Inhibition of EZH2 with the FDA-approved drug tazemetostat increased surface expression of major histocompatibility complex class I (MHC-I) on CD4 T-cells, counterbalancing HIV Nef-mediated MHC-I downregulation. This improved CTL-mediated elimination of HIV-infected cells and suppressed viral replication in vitro. In a participant-derived xenograft mouse model, tazemetostat elevated MHC-I and the pro-apoptotic protein BIM in CD4 T-cells, facilitating CD8 T-cell-mediated reductions of HIV reservoir seeding. Additionally, tazemetostat promoted sustained skewing of CD8 T-cells toward less differentiated and exhausted phenotypes. Our findings reveal EZH2 overexpression as a novel mechanism of CTL resistance and support the clinical evaluation of tazemetostat to enhance clearance of HIV reservoirs and improve CD8+ T-cell function.

immunology↗

bioRxiv2021

Pupil-linked arousal modulates trial-by-trial motor adaptation in humans

Pupil diameter, under constant illumination, is known to reflect individuals internal states, such as surprise about observation and environmental uncertainty. Despite the growing use of pupillometry in cognitive learning studies as an additional measure for examining internal states, few studies have used pupillometry in human motor learning studies. Here we provide the first detailed characterization of pupil diameter changes in a short-term reach adaptation paradigm. We measured pupil changes in 91 human participants while they adapted to abrupt, gradual, or switching force field conditions. Sudden increases in movement error caused by the introduction/reversal of the force field resulted in strong phasic pupil dilation during movement accompanied by a transient increase in tonic pre-movement baseline pupil diameter in subsequent trials. In contrast, clear changes in pupil responses were absent when the force field was gradually introduced, indicating that error drove the changes in pupil responses. Nevertheless, we found an association between baseline pupil diameter and awareness of the gradually-introduced perturbation assessed post-experimentally. In all experiments, we also found a strong co-occurrence of larger baseline pupil diameter and slower reaction and movement time after each set break. Interestingly, error-induced pupil responses gradually became insensitive after experiencing multiple reversals. Collectively, these results suggest that tonic baseline pupil diameter reflects ones belief about environmental uncertainty, whereas phasic pupil dilation during movement reflects surprise about a sensory outcome (i.e., movement error), and both effects are modulated by novelty. Our results provide a new approach for non-verbally assessing participants internal states during motor learning.

neuroscience↗

bioRxiv2020

Heritable variation in resistance to the endonuclear parasite Holospora undulata across clades of Paramecium caudatum

Resistance is a key determinant in interactions between hosts and their parasites. Understanding the amount and distribution of genetic variation in this trait can provide insights into (co)evolutionary processes and their potential to shape patterns of diversity in natural populations. Using controlled inoculation in experimental mass cultures, we investigated the quantitative genetic variation in resistance to the bacterial parasite Holospora undulata across a worldwide collection of strains of its ciliate host Paramecium caudatum. We combined the observed variation with available information on the phylogeny and biogeography of the strains. We found substantial variation in resistance among strains (with broad-sense heritability > 0.5), repeatable between laboratories and ranging from total resistance to near-complete susceptibility. Early (one week post inoculation) measurements provided higher estimates of resistance heritability than did later measurements (2-3 weeks), possibly due to diverging epidemiological dynamics in replicate cultures of the same strains. Genetic distance (based on a neutral marker) was positively correlated with the difference in resistance phenotype between strains (r = 0.45), essentially reflecting differences between highly divergent clades (haplogroups) within the host species. Haplogroup A strains, mostly European, were less resistant to the parasite (49% infection prevalence) than non-European haplogroup B strains (28%). At a smaller geographical scale (within Europe), strains that are geographically closer to the parasite origin (Southern Germany) were more susceptible to infection than those from further away. These patterns are consistent with a picture of local parasite adaptation. Our study demonstrates ample natural genetic variation in resistance on which selection can act and hints at symbiont adaptation producing signatures in geographic and lineage-specific patterns of resistance in this model system.

genetics↗

bioRxiv2020

Structure of population activity in primary motor cortex for single finger flexion and extension

How is the primary motor cortex (M1) organized to control fine finger movements? We investigated the population activity in M1 for single finger flexion and extension, using 7T functional magnetic resonance imaging (fMRI) in female and male human participants, and compared these results to the neural spiking patterns recorded in two male monkeys performing the identical task. fMRI activity patterns were distinct for movements of different fingers, but quite similar for flexion and extension of the same finger. In contrast, spiking patterns in monkeys were quite distinct for both fingers and directions, similar to what was found for muscular activity patterns. The discrepancy between fMRI and electrophysiological measurements can be explained by two (non-mutually exclusive) characteristics of the organization of finger flexion and extension movements. Given that fMRI reflects predominantly input and recurrent activity, the results can be explained by an architecture in which neural populations that control flexion or extension of the same finger produce distinct outputs, but interact tightly with each other and receive similar inputs. Additionally, neurons tuned to different movement directions for the same finger (or combination of fingers) may cluster closely together, while neurons that control different finger combinations may be more spatially separated. When measuring this organization with fMRI at a coarse spatial scale, the activity patterns for flexion and extension of the same finger would appear very similar. Overall, we suggest that the discrepancy between fMRI and electrophysiological measurements provides new insights into the general organization of fine finger movements in M1. Significance statementThe primary motor cortex (M1) is important for producing individuated finger movements. Recent evidence shows that movements that commonly co-occur are associated with more similar activity patterns in M1. Flexion and extension of the same finger, which never co-occur, should therefore be associated with distinct representations. However, using carefully controlled experiments and multivariate analyses, we demonstrate that human fMRI activity patterns for flexion or extension of the same finger are highly similar. In contrast, spiking patterns measured in monkey M1 are clearly distinct. This suggests that populations controlling opposite movements of the same finger, while producing distinct outputs, may cluster together and share inputs and local processing. These results provide testable hypotheses about the organization of hand control in M1.

neuroscience↗

bioRxiv2020

Spinal stretch reflexes support efficient control of reaching

Efficiently controlling the movement of our hand requires coordinating the motion of multiple joints of the arm. Although it is widely assumed that this type of efficient control is implemented by processing that occurs in the cerebral cortex and brain stem, recent work has shown that spinal circuits can generate efficient motor output that supports keeping the hand in a static location. Here, we show that a spinal pathway can also efficiently control the hand during reaching. In our first experiment we applied multi-joint mechanical perturbations to participants elbow and wrist as they began reaching towards a target. We found that spinal stretch reflexes evoked in elbow muscles were not proportional to how much the elbow muscles were stretched but instead were efficiently scaled to the hands distance from the target. In our second experiment we applied the same elbow and wrist perturbations but had participants change how they grasped the manipulandum, diametrically altering how the same wrist perturbation moved the hand relative to the reach target. We found that changing the arms orientation diametrically altered how spinal reflexes in the elbow muscles were evoked, and in such a way that were again efficiently scaled to the hands distance from the target. These findings demonstrate that spinal circuits can help efficiently control the hand during dynamic reaching actions, and show that efficient and flexible motor control is not exclusively dependent on processing that occurs within supraspinal regions of the nervous system.

neuroscience↗

bioRxiv2018

Spinal stretch reflexes exploit musculoskeletal redundancy to support postural hand control

Motor behaviour is most efficiently controlled by only correcting disturbances or deviations that influence task success. It is currently thought that such sophisticated control is computed within a transcortical feedback pathway. Here we show that even the fastest spinal feedback pathway can produce corrective responses that adhere to this control scheme. We first applied small mechanical perturbations that flexed the elbow joint - stretching the triceps muscle - and simultaneously flexed or extended the wrist joint, displacing the hand various distances away from a central target. We then changed the arms orientation and applied the same joint perturbations, which reversed the mapping between joint motion and hand displacement. In all cases, we found that the triceps spinal stretch reflex was tuned to the hands displacement relative to the target, and not how the triceps muscle was stretched. Our findings reveal that the fastest spinal feedback pathway is capable of integrating and modulating feedback from multiple muscles to produce efficient corrective responses, forcing a re-evaluation of the how the nervous system derives the sophisticated control laws that support natural motor behaviour.

neuroscience↗

bioRxiv2017

Rapid feedback responses are flexibly coordinated across arm muscles to support goal-directed reaching

A transcortical pathway helps support goal-directed reaching by processing somatosensory information to produce rapid feedback responses across multiple joints and muscles. Here we tested whether such feedback responses can account for changes in arm configuration and for arbitrary visuomotor transformations - two manipulations that alter how muscles at the elbow and wrist need to be coordinated to achieve task success. Participants used a planar three degree-of-freedom exoskeleton robot to move a cursor to a target following a mechanical perturbation that flexed the elbow. In our first experiment, the cursor was mapped to the veridical position of the robot handle, but participants grasped the handle with two different hand orientations (thumb pointing upward or thumb point downward). We found that large rapid feedback responses were evoked in wrist extensor muscles when wrist extension helped move the cursor to the target (i.e., thumb upward), and in wrist flexor muscles when wrist flexion helped move the cursor to the target (i.e., thumb downward). In our second experiment, participants grasped the robot handle with their thumb pointing upward, but the cursors movement was either veridical, or was mirrored such that flexing the wrist moved the cursor as if the participant extended their wrist, and vice versa. After extensive practice, we found that rapid feedback responses were appropriately tuned to the wrist muscles that supported moving the cursor to the target when the cursor was mapped to the mirrored movement of the wrist, but were not tuned to the appropriate wrist muscles when the cursor was remapped to the wrists veridical movement.

neuroscience↗