Biology subjects

Wilson, M. A.

Publications and source records attributed to Wilson, M. A..

At least 37 records · Page 2Linked to original sources

bioRxiv2024

Spike-wave discharges during low-current thalamic deep brain stimulation in mice

BackgroundDeep brain stimulation of central thalamus (CT-DBS) has potential for modulating states of consciousness, but it can also trigger spike-wave discharges (SWDs). ObjectivesTo report the probability of inducing SWDs during CT-DBS in awake mice. MethodsMice were implanted with electrodes to deliver unilateral and bilateral CT-DBS at different frequencies while recording EEG. We titrated stimulation current by gradually increasing it at each frequency until an SWD appeared. Subsequent stimulations to test arousal modulation were performed at the current one step below the current that caused an SWD during titration. ResultsIn 2.21% of the test stimulations (10 out of 12 mice), CT-DBS caused SWDs at currents lower than the titrated current, at currents as low as 20 A. ConclusionOur study found a small but significant probability of inducing SWDs even after titration and at relatively low currents. EEG should be closely monitored for SWDs when performing CT-DBS in both research and clinical settings.

neuroscience↗

bioRxiv2024

Distinct sets of molecular characteristics define tumor-rejecting neoantigens

Challenges in identifying tumor-rejecting neoantigens limit the efficacy of neoantigen vaccines to treat cancers, including cutaneous squamous cell carcinoma (cSCC). A minority of human cSCC tumors shared neoantigens, supporting the need for personalized vaccines. Using a UV-induced mouse cSCC model which recapitulated the mutational signature and driver mutations found in human disease, we found that CD8 T cells constrain cSCC. Two MHC class I neoantigens were identified that constrained cSCC growth. Compared to the wild-type peptides, one tumor-rejecting neoantigen exhibited improved MHC binding and the other had increased solvent accessibility of the mutated residue. Across known neoantigens that do not impact MHC binding, structural modeling of the peptide/MHC complexes indicated that increased solvent accessibility, which will facilitate TCR recognition of the neoantigen, distinguished tumor-rejecting from non-immunogenic neoantigens. This work reveals characteristics of tumor-rejecting neoantigens that may be of considerable importance in identifying optimal vaccine candidates in cSCC and other cancers.

immunology↗

bioRxiv2023

Imaging the voltage of neurons distributed across entire brains of larval zebrafish

Neurons interact in networks distributed throughout the brain. Although much effort has focused on whole-brain calcium imaging, recent advances in genetically encoded voltage indicators (GEVIs) raise the possibility of imaging voltage of neurons distributed across brains. To achieve this, a microscope must image at high volumetric rate and signal-to-noise ratio. We present a remote scanning light-sheet microscope capable of imaging GEVI-expressing neurons distributed throughout entire brains of larval zebrafish at a volumetric rate of 200.8 Hz. We measured voltage of [~]1/3 of the neurons of the brain, distributed throughout. We observed that neurons firing at different times during a sequence were located at different brain locations, for sequences elicited by a visual stimulus, which mapped onto locations throughout the optic tectum, as well as during stimulus-independent bursts, which mapped onto locations in the cerebellum and medulla. Whole-brain voltage imaging may open up frontiers in the fundamental operation of neural systems.

neuroscience↗

bioRxiv2023

Design and implementation of an asynchronous online course-based undergraduate research experience (CURE) in computational genomics

As genomics and information technologies advance, there is a growing demand for research scientists trained in bioinformatics methods to determine gene expression underlying cell biology in health and disease. One approach to increase the number of scientists proficient in bioinformatics is to expand access through online degree programs and remotely-accessible learning materials. Fully-online learners represent a significant and growing community of historically underrepresented students who are frequently excluded from research opportunities that require in-person attendance during standard operational hours. To address this opportunity gap, we developed an asynchronous course-based undergraduate research experience (CURE) for computational genomics specifically for fully-online biology students. We generated custom learning materials and leveraged remotely-accessible resources on a high performance computing cluster to address a novel research question: the effect of changing quality trimming parameters for RNA sequencing reads on the discovery of sex-based differential gene expression in the human placenta. Here we present the process by which the instructional team devised and distributed analysis to address this question over a 7.5-week CURE and provided students with concurrent training in biology, statistics, computer programming, and professional development integral to the successful execution of the project and future publications. Scores from identical learning assessments administered before and after completion of the CURE showed significant learning gains across biology and coding course objectives. Open-response progress reports were submitted weekly and identified self-reported adaptive coping strategies for challenges encountered throughout the course. The instruction team monitored the progress reports to identify problems that could be resolved through collaboration with instructors and peers via messaging platforms and virtual meetings. Analytics from the course messaging platform demonstrated that high posting engagement was strongly correlated to high normalized learning gains, showing that students can effectively use asynchronous communication platforms to facilitate learning. The online genomics CURE resulted in unanticipated positive outcomes, including students voluntarily extending their participation beyond the course duration, presenting their findings at research symposiums, and applying to graduate school. These outcomes underscore the effectiveness of this genomics CURE for training and recruitment purposes and demonstrate that students can be successful in online STEM-based research experiences if given channels for communication, bespoke and accessible learning materials, and the support of experts in the field. Online CUREs can provide valuable research experience to harness the potential of online STEM students towards a more skilled, diverse, and inclusive workforce for the advancement of biomedical science.

genomics↗

bioRxiv2023

X-ray-driven chemistry and conformational heterogeneity in atomic resolution crystal structures of bacterial dihydrofolate reductases

Dihydrofolate reductase (DHFR) catalyzes the NADPH-dependent reduction of dihydrofolate to tetrahydrofolate. Bacterial DHFRs are targets of several important antibiotics as well as model enzymes for the role of protein conformational dynamics in enzyme catalysis. We collected 0.93 [A] resolution X-ray diffraction data from both Bacillus subtilis (Bs) and E. coli (Ec) DHFRs bound to folate and NADP+. These oxidized ternary complexes should not be able to perform chemistry, however electron density maps suggest hydride transfer is occurring in both enzymes. Comparison of low- and high-dose EcDHFR datasets show that X-rays drive partial production of tetrahydrofolate. Hydride transfer causes the nicotinamide moiety of NADP+ to move towards the folate as well as correlated shifts in nearby residues. Higher radiation dose also changes the conformational heterogeneity of Met20 in EcDHFR, supporting a solvent gating role during catalysis. BsDHFR has a different pattern of conformational heterogeneity and an unexpected disulfide bond, illustrating important differences between bacterial DHFRs. This work demonstrates that X-rays can drive hydride transfer similar to the native DHFR reaction and that X-ray photoreduction can be used to interrogate catalytically relevant enzyme dynamics in favorable cases.

biochemistry↗

bioRxiv2023

A unified open-source platform for multimodal neural recording and perturbation during naturalistic behavior

Behavioral neuroscience faces two conflicting demands: long-duration recordings from large neural populations and unimpeded animal behavior. To meet this challenge, we developed ONIX, an open-source data acquisition system with high data throughput (2GB/sec) and low closed-loop latencies (<1ms) that uses a novel 0.3 mm thin tether to minimize behavioral impact. Head position and rotation are tracked in 3D and used to drive active commutation without torque measurements. ONIX can acquire from combinations of passive electrodes, Neuropixels probes, head-mounted microscopes, cameras, 3D-trackers, and other data sources. We used ONIX to perform uninterrupted, long ([~]7 hours) neural recordings in mice as they traversed complex 3-dimensional terrain. ONIX allowed exploration with similar mobility as non-implanted animals, in contrast to conventional tethered systems which restricted movement. By combining long recordings with full mobility, our technology will enable new progress on questions that require high-quality neural recordings during ethologically grounded behaviors.

neuroscience↗

bioRxiv2023

Cucurbituril Enhances Distance Measurements of Spin-Labeled Proteins

We report complex formation between the chloroacetamide 2,6-diazaadamantane nitroxide radical (ClA-DZD) and cucurbit[7]uril (CB-7), for which the association constant in water, Ka = 1.9 x 106 M-1, is at least one order of magnitude higher than the previously studied organic radicals. The radical is highly immobilized by CB-7, as indicated by the increase of the rotational correlation time,{tau} rot, by a factor of 36, relative to that in the buffer solution. The X-ray structure of ClA-DZD@CB-7 shows the encapsulated DZD guest inside the undistorted CB-7 host, with the pendant group protruding outside. Upon addition of CB-7 to T4 Lysozyme (T4L) doubly spin-labeled with the iodoacetamide derivative of DZD, we observe the increase in{tau} rot and electron spin coherence time, Tm, along with the narrowing of inter-spin distance distributions. Sensitivity of the DEER measurements at 83 K increases by a factor 4 - 9, compared to the common spin label such as MTSL, which is not affected by CB-7. Inter-spin distances of 3-nm could be reliably measured in water/glycerol up to temperatures near the glass transition/melting temperature of the matrix at 200 K, thus bringing us closer to the goal of supramolecular recognition-enabled long-distance DEER measurements at near physiological temperatures. The X-ray structure of DZD-T4L 65 at 1.12 [A] resolution allows for unambiguous modeling of the DZD label (0.88 occupancy), indicating undisturbed structure and conformation of the protein. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=107 SRC="FIGDIR/small/554361v1_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@1171bd0org.highwire.dtl.DTLVardef@231612org.highwire.dtl.DTLVardef@17e22e5org.highwire.dtl.DTLVardef@250f1b_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2023

Changes in an Enzyme Ensemble During Catalysis Observed by High Resolution XFEL Crystallography

Enzymes populate ensembles of structures with intrinsically different catalytic proficiencies that are difficult to experimentally characterize. We use time-resolved mix-and-inject serial crystallography (MISC) at an X-ray free electron laser (XFEL) to observe catalysis in a designed mutant (G150T) isocyanide hydratase (ICH) enzyme that enhances sampling of important minor conformations. The active site exists in a mixture of conformations and formation of the thioimidate catalytic intermediate selects for catalytically competent substates. A prior proposal for active site cysteine charge-coupled conformational changes in ICH is validated by determining structures of the enzyme over a range of pH values. A combination of large molecular dynamics simulations of the enzyme in crystallo and time-resolved electron density maps shows that ionization of the general acid Asp17 during catalysis causes additional conformational changes that propagate across the dimer interface, connecting the two active sites. These ionization-linked changes in the ICH conformational ensemble permit water to enter the active site in a location that is poised for intermediate hydrolysis. ICH exhibits a tight coupling between ionization of active site residues and catalysis-activated protein motions, exemplifying a mechanism of electrostatic control of enzyme dynamics.

biochemistry↗

bioRxiv2023

Its a Trap! Escape from an ancient, ancestral sex chromosome system and implication of Foxl2 as a primary sex determininggene in a lizard (Anguimorpha; Shinisauridae)

Although sex determination is ubiquitous in vertebrates, mechanisms of sex determination vary from environmentally-to genetically-influenced. In vertebrates, genetic sex determination is typically accomplished with sex chromosomes. Groups like mammals maintain conserved sex chromosome systems, while sex chromosomes in most vertebrate clades arent conserved across similar evolutionary timescales. One group inferred to have an evolutionarily stable mode of sex determination is Anguimorpha, a clade of charismatic taxa including: monitor lizards, Gila monsters, and crocodile lizards. The common ancestor of extant anguimorphs possessed a ZW system that has been retained across the clade. However, the sex chromosome system in the endangered, monotypic family of crocodile lizards (Shinisauridae) has remained elusive. Here, we analyze genomic data to demonstrate that Shinisaurus has replaced the ancestral anguimorph ZW system on LG7 chromosome with a novel ZW system on LG3. The linkage group LG3 corresponds to chromosome 9 in chicken, and this is the first documented use of this syntenic block as a sex chromosome in amniotes. Additionally, this [~]1Mb region harbors approximately 10 genes, including a duplication of the sex-determining transcription factor, Foxl2--critical for the determination and maintenance of sexual differentiation in vertebrates, and thus a putative primary sex determining gene for Shinisaurus.

evolutionary biology↗

bioRxiv2023

Pixel-wise programmability enables dynamic high-SNR cameras for high-speed microscopy

High-speed wide-field fluorescence microscopy has the potential to capture biological processes with exceptional spatiotemporal resolution. However, conventional cameras suffer from low signal-to-noise ratio at high frame rates, limiting their ability to detect faint fluorescent events. Here, we introduce an image sensor where each pixel has individually programmable sampling speed and phase, so that pixels can be arranged to simultaneously sample at high speed with a high signal-to-noise ratio. In high-speed voltage imaging experiments, our image sensor significantly increases the output signal-to-noise ratio compared to a low-noise scientific CMOS camera ([~]2-3 folds). This signal-to-noise ratio gain enables the detection of weak neuronal action potentials and subthreshold activities missed by the standard scientific CMOS cameras. Our camera with flexible pixel exposure configurations offers versatile sampling strategies to improve signal quality in various experimental conditions.

neuroscience↗

bioRxiv2023

Incomplete dosage balance and dosage compensation in the ZZ/ZW Gila monster (Heloderma suspectum) revealed by de novo genome assembly

Reptiles exhibit a variety of modes of sex determination, including both temperature-dependent and genetic mechanisms. Among those species with genetic sex determination, sex chromosomes of varying heterogamety (XX/XY and ZZ/ZW) have been observed with different degrees of differentiation. Karyotype studies have demonstrated that Gila monsters (Heloderma suspectum) have ZZ/ZW sex determination and this system is likely homologous to the ZZ/ZW system in the Komodo dragon (Varanus komodoensis), but little else is known about their sex chromosomes. Here, we report the assembly and analysis of the Gila monster genome. We generated a de novo draft genome assembly for a male using 10X Genomics technology. We further generated and analyzed short-read whole genome sequencing and whole transcriptome sequencing data for three males and three females. By comparing female and male genomic data, we identified four putative Z-chromosome scaffolds. These putative Z-chromosome scaffolds are homologous to Z-linked scaffolds identified in the Komodo dragon. Further, by analyzing RNAseq data, we observed evidence of incomplete dosage compensation between the Gila monster Z chromosome and autosomes and a lack of balance in Z-linked expression between the sexes. In particular, we observe lower expression of the Z in females (ZW) than males (ZZ) on a global basis, though we find evidence suggesting local gene-by-gene compensation. This pattern has been observed in most other ZZ/ZW systems studied to date and may represent a general pattern for female heterogamety in vertebrates.

evolutionary biology↗

bioRxiv2023

Concerning the eXclusion in human genomics: The choice of sex chromosome representation in the human genome drastically affects number of identified variants

Over the past 30 years, a community of scientists have pieced together every base pair of the human reference genome from telomere-to-telomere. Interestingly, most human genomics studies omit more than 5% of the genome from their analyses. Under normal circumstances, omitting any chromosome(s) from analysis of the human genome would be reason for concern--the exception being the sex chromosomes. Sex chromosomes in eutherians share an evolutionary origin as an ancestral pair of autosomes. In humans, they share three regions of high sequence identity (~98-100%), which--along with the unique transmission patterns of the sex chromosomes--introduce technical artifacts into genomic analyses. However, the human X chromosome bears numerous important genes--including more "immune response" genes than any other chromosome--which makes its exclusion irresponsible when sex differences across human diseases are widespread. To better characterize the effect that including/excluding the X chromosome may have on variants called, we conducted a pilot study on the Terra cloud platform to replicate a subset of standard genomic practices using both the CHM13 reference genome and sex chromosome complement-aware (SCC-aware) reference genome. We compared quality of variant calling, expression quantification, and allele-specific expression using these two reference genome versions across 50 human samples from the Genotype-Tissue-Expression consortium annotated as females. We found that after correction, the whole X chromosome (100%) can generate reliable variant calls--allowing for the inclusion of the whole genome in human genomics analyses as a departure from the status quo of omitting the sex chromosomes from empirical and clinical genomics studies.

genomics↗

bioRxiv2023

A lizard is never late: squamate genomics as a recent catalyst for understanding microchromosome and sex chromosome evolution

In 2011, the first high-quality genome assembly of a squamate reptile (lizard or snake) was published for the green anole. Dozens of genome assemblies were subsequently published over the next decade, yet these assemblies were largely inadequate for answering fundamental questions regarding genome evolution in squamates due to their lack of contiguity or annotation. As the "genomics age" was beginning to hit its stride in many organismal study systems, progress in squamates was largely stagnant following the publication of the green anole genome. In fact, zero high-quality (chromosome-level) squamate genomes were published between the years 2012-2017. However, since 2018, an exponential increase in high-quality genome assemblies has materialized with 24 additional high-quality genomes published for species across the squamate tree of life. As the field of squamate genomics is rapidly evolving, we provide a systematic review from an evolutionary genomics perspective. We collated a near-complete list of publicly available squamate genome assemblies from more than half-a-dozen international and third-party repositories and systematically evaluated them with regard to their overall quality, phylogenetic breadth, and usefulness for continuing to provide accurate and efficient insights into genome evolution across squamate reptiles. This review both highlights and catalogs the currently available genomic resources in squamates and their ability to address broader questions in vertebrates, specifically sex chromosome and microchromosome evolution, while addressing why squamates may have received less historical focus and has caused their progress in genomics to lag behind peer taxa.

genomics↗

bioRxiv2023

Adaptations to water stress and pastoralism in the Turkana of northwest Kenya

The Turkana people inhabit arid regions of east Africa--where temperatures are high and water is scarce--and they practice subsistence pastoralism, such that their diet is primarily composed of animal products. Working with Turkana communities, we sequenced 367 genomes and identified 8 regions putatively involved in adaptation to water stress and pastoralism. One of these regions includes a putative enhancer for STC1--a kidney-expressed gene involved in the response to dehydration and the metabolism of purine-rich foods such as red meat. We show that STC1 is induced by antidiuretic hormone in humans, is associated with urea levels in the Turkana themselves, and is under strong selection in this population (s[~]0.041). This work highlights that partnerships with subsistence-level groups can lead to new models of human physiology with biomedical relevance.

evolutionary biology↗

bioRxiv2022

Evolutionary and biomedical implications of sex differences in the primate brain transcriptome

Humans exhibit sex differences in the prevalence of many neurodevelopmental and neurodegenerative conditions. To better understand the translatability of a critical nonhuman primate model, the rhesus macaque, we generated one of the largest multibrain region bulk transcriptional datasets for this species and characterized sex-biased gene expression patterns. We demonstrate that these patterns are similar to those in humans and are associated with overlapping regulatory mechanisms, biological processes, and genes implicated in sex-biased human disorders, including autism. We also show that sex-biased genes exhibit greater genetic variance for expression and more tissue-specific expression patterns, which may facilitate the rapid evolution of sex-biased genes. Our findings provide insights into the biological mechanisms underlying sex-biased disease and validate the rhesus macaque model for the study of these conditions.

neuroscience↗

bioRxiv2022

Sex differences in early and term placenta are conserved in adult tissues

BackgroundPregnancy complications vary based on the fetuss genetic sex, which may, in part, be modulated by the placenta. Further, developmental differences early in life can have lifelong health outcomes. Yet, sex differences in gene expression within the placenta at different time points throughout pregnancy and comparisons to adult tissues remains poorly characterized. MethodsHere, we collect and characterize sex differences in gene expression in term placentas ( [≥] 36.6 weeks; 23 male XY and 27 female XX). These are compared with sex differences in previously collected first trimester placenta samples and 42 non-reproductive adult tissues from GTEx. ResultsWe identify 268 and 53 sex differentially expressed genes in the uncomplicated late first trimester and term placentas, respectively. Of the 53 sex differentially expressed genes observed in the term placentas, 31 are also sex differentially expressed genes in the late first trimester placentas. Furthermore, sex differences in gene expression in term placentas are highly correlated with sex differences in the late first trimester placentas. We found that sex differential gene expression in the term placenta is significantly correlated with sex differences in gene expression in 42 non-reproductive adult tissues (correlation coefficient ranged from 0.892 to 0.957), with the highest correlation in brain tissues. Sex differences in gene expression were largely driven by gene expression on the sex chromosomes. We further show that some gametologous genes (genes with functional copies on X and Y) will have different inferred sex differences if the X-linked gene expression in females is compared to the sum of the X-linked and Y-linked gene expression in males. ConclusionsWe find that sex differences in gene expression are conserved in late first trimester and term placentas and that these sex differences are conserved in adult tissues. We demonstrate that there are sex differences associated with innate immune response in late first trimester placentas but there is no significant difference in gene expression of innate immune genes between sexes in healthy full term placentas. Finally, sex differences are predominantly driven by expression from sex-linked genes. HighlightsO_LISex differences in gene expression in late first trimester placentas are positively correlated with sex differences in gene expression in full term placentas; sex differences develop early and are maintained. C_LIO_LISex differences in gene expression on the sex chromosomes in the placenta are correlated to sex differences in adult tissues. C_LIO_LISex-linked gametolog genes require additional methodological approaches for accurate quantification. C_LI

genomics↗

bioRxiv2022

A molecular plugin rescues GroEL/ES substrates from pre-folding oxidation

Hsp60 chaperonins and their Hsp10 cofactors assist protein folding in all living cells, constituting the paradigmatic example of molecular chaperones. Despite extensive investigations of their structure and mechanism, crucial questions regarding how these chaperonins promote folding remain unsolved. Here, we report that the bacterial Hsp60 chaperonin GroEL forms a stable, functionally relevant complex with the chaperedoxin CnoX, a protein combining a chaperone and a redox function. Binding of GroES (Hsp10) to GroEL induces CnoX release. Cryo-electron microscopy provided crucial structural information on the GroEL-CnoX complex, showing that CnoX binds GroEL outside the substrate-binding site via a highly conserved C-terminal -helix. Furthermore, the identification of complexes in which CnoX, bound to GroEL, forms mixed-disulfides with GroEL substrates indicates that CnoX likely functions as a redox quality-control plugin for GroEL. Proteins sharing structural features with CnoX exist in eukaryotes, which suggests that Hsp60 molecular plugins have been conserved through evolution.

biochemistry↗

bioRxiv2022

GENESPACE: syntenic pan-genome annotations for eukaryotes

The development of multiple high-quality reference genome sequences in many taxonomic groups has yielded a high-resolution view of the patterns and processes of molecular evolution. Nonetheless, leveraging information across multiple reference haplotypes remains a significant challenge in nearly all eukaryotic systems. These challenges range from studying the evolution of chromosome structure, to finding candidate genes for quantitative trait loci, to testing hypotheses about speciation and adaptation in nature. Here, we address these challenges through the concept of a pan-genome annotation, where conserved gene order is used to restrict gene families and define the expected physical position of all genes that share a common ancestor among multiple genome annotations. By leveraging pan-genome annotations and exploring the underlying syntenic relationships among genomes, we dissect presence-absence and structural variation at four levels of biological organization: among three tetraploid cotton species, across 300 million years of vertebrate sex chromosome evolution, across the diversity of the Poaceae (grass) plant family, and among 26 maize cultivars. The methods to build and visualize syntenic pan-genome annotations in the GENESPACE R package offer a significant addition to existing gene family and synteny programs, especially in polyploid, outbred and other complex genomes.

genomics↗