bluuebunny/crossref_metadata_2025 huggingface dataset:
Single-cell eQTL mapping in yeast reveals a tradeoff between growth and reproduction
Authors: Boocock James , Alexander Noah , Tapia Leslie Alamo , Walter-McNeill Laura , Patel Shivani Prashant , Munugala Chetan , Bloom Joshua S , Kruglyak Leonid
Published: December 2023

Expression quantitative trait loci (eQTLs) provide a key bridge between noncoding DNA sequence variants and organismal traits. The effects of eQTLs can differ among tissues, cell types, and cellular states, but these differences are obscured by gene expression measurements in bulk populations. We developed a one-pot approach to map eQTLs inSaccharomyces cerevisiaeby single-cell RNA sequencing (scRNA-seq) and applied it to over 100,000 single cells from three crosses. We used scRNA-seq data to genotype each cell, measure gene expression, and classify the cells by cell-cycle stage. We mapped thousands of local and distant eQTLs and identified interactions between eQTL effects and cell-cycle stages. We took advantage of single-cell expression information to identify hundreds of genes with allele-specific effects on expression noise. We used cell-cycle stage classification to map 20 loci that influence cell-cycle progression. One of these loci influenced the expression of genes involved in the mating response. We showed that the effects of this locus arise from a common variant (W82R) in the geneGPA1, which encodes a signaling protein that negatively regulates the mating pathway. The 82R allele increases mating efficiency at the cost of slower cell-cycle progression and is associated with a higher rate of outcrossing in nature. Our results provide a more granular picture of the effects of genetic variants on gene expression and downstream traits.
CytoSimplex: Visualizing Single-cell Fates and Transitions on a Simplex
Authors: Liu Jialin , Wang Yichen , Li Chen , Gu Yichen , Ono Noriaki , Welch Joshua D.
Published: December 2023

Cells differentiate to their final fates along unique trajectories, often involving multi-potent progenitors that can produce multiple terminally differentiated cell types. Recent developments in single-cell transcriptomic and epigenomic measurement provide tremendous opportunities for mapping these trajectories. The visualization of single-cell data often relies on dimension reduction methods such as UMAP to simplify high-dimensional single-cell data down into an understandable two-dimensional (2D) form. However, these visualization methods can be misleading and often do not effectively represent the direction of cell differentiation. To address these limitations, we developed a new approach that places each cell from a single-cell dataset within a simplex whose vertices correspond to terminally differentiated cell types. Our approach can quantify and visualize current cell fate commitment and future cell potential. We developed CytoSimplex, a standalone open-source package implemented in R and Python that provides simple and intuitive visualizations of cell differentiation in 2D ternary and three-dimensional (3D) quaternary plots. We believe that CytoSimplex can help researchers gain a better understanding of cell type transitions in specific tissues and characterize developmental processes.

Availability and implementation

The R version of CytoSimplex is available on Github athttps://github.com/welch-lab/CytoSimplex. The Python version of CytoSimplex is available on Github athttps://github.com/welch-lab/pyCytoSimplex.
Tail Length and E525K Dilated Cardiomyopathy Mutant Alter Human β-Cardiac Myosin Super-Relaxed State
Authors: Duno-Miranda Sebastian , Nelson Shane R. , Rasicci David V. , Bodt Skylar L.M. , Cirilo Joseph A. , Vang Duha , Sivaramakrishnan Sivaraj , Yengo Christopher M. , Warshaw David M.
Published: December 2023

Dilated cardiomyopathy (DCM) is characterized by impaired cardiac function due to myocardial hypo-contractility and is associated with point mutations in β-cardiac myosin, the molecular motor that powers cardiac contraction. Myocardial function can be modulated through sequestration of myosin motors into an auto-inhibited "super relaxed" state (SRX), which is further stabilized by a structural state known as the "Interacting Heads Motif" (IHM). Therefore, hypo-contractility of DCM myocardium may result from: 1) reduced function of individual myosin, and/or; 2) decreased myosin availability due to increased IHM/SRX stabilization. To define the molecular impact of an established DCM myosin mutation, E525K, we characterized the biochemical and mechanical activity of wild-type (WT) and E525K human β-cardiac myosin constructs that differed in the length of their coiled-coil tail, which dictates their ability to form the IHM/SRX state. Single-headed (S1) and a short-tailed, double-headed (2HEP) myosin constructs exhibited low (∼10%) IHM/SRX content, actin-activated ATPase activity of ∼5s-1and fast velocities in unloaded motility assays (∼2000nm/s). Double-headed, longer-tailed (15HEP, 25HEP) constructs exhibited higher IHM/SRX content (∼90%), and reduced actomyosin ATPase (<1s-1) and velocity (∼800nm/s). A simple analytical model suggests that reduced velocities may be attributed to IHM/SRX-dependent sequestration of myosin heads. Interestingly, the E525K 15HEP and 25HEP mutants showed no apparent impact on velocity or actomyosin ATPase at low ionic strength. However, at higher ionic strength, the E525K mutation stabilized the IHM/SRX state. Therefore, the E525K-associated DCM human cardiac hypo-contractility may be attributable to reduced myosin head availability caused by enhanced IHM/SRX stability.

This research investigates the E525K mutation in human β-cardiac myosin, crucial for heart contraction, and its role in causing Dilated Cardiomyopathy (DCM). It demonstrates that the length of the myosin tail influences its self-inhibition, and the E525K mutation strengthens this effect, potentially reducing heart contractility in DCM.
Nodule organogenesis inMedicago truncatularequires local stage-specific auxin biosynthesis and transport
Authors: Xiao TT , Shen D , Müller S , Liu J , van Seters A , Franssen H , Bisseling T , Kulikova O , Kohlen W
Published: December 2023

The importance of auxin in plant organ development including root nodule formation is well established. Using auxin reporter constructs the spatiotemporal auxin distribution pattern during nodule development has previously been illustrated. However, our understanding of how this pattern is built-up and maintained still remains elusive.

To this end, we studied how the auxin gradient visualized by DR5 expression patterns at different stages of nodule development in Medicago truncatula (Medicago), is correlated with the spatiotemporal expression patterns of known auxin biosynthesis and auxin transport genes. In addition, we record the MtPIN10-GFP expression pattern and polar positioning on the cell plasma membranes during nodule primordium development to investigate the auxin flux. RNA interference and the application of auxin synthesis blockers were used to demonstrate the relevance of biosynthesis and transport at the initial stages of the nodulation process.

Our results show that upon rhizobium inoculation, preceding the first mitotic activity, a specific set of MtYUCs and MtPINs as well as MtLAX2 are expressed in the pericycle contributing to the creation of an auxin maximum. Overall, we demonstrate that dynamic spatiotemporal expression of both, MtYUCs and MtPINs, result in specific auxin outputs in subsequent stages of nodule primordia and nodule meristem formation.
OsUVR8b, rather than OsUVR8a, plays a predominant role in rice UVR8-mediated UV-B response
Authors: Chen Yu-long , Zhong You-bin , Leung David W. M. , Yan Xiao-Yu , Ouyang Meng-ni , Ye Yu-zhen , Li Shi-mei , Peng Xin-xiang , Liu E-e
Published: December 2023

UV RESISTANCE LOCUS 8 (UVR8) has been identified inArabidopsis thalianaas the receptor for UV-B radiation mediating photomorphogenic responses and acclimation to UV-B radiation. However, UVR8-mediated UV-B signaling pathways in rice, that has two proteins (UVR8a and UVR8b) with homology to AtUVR8, remain largely unknown. In this study,UVR8aandUVR8bwere found to be expressed mainly in rice leaves and leaf sheaths, while the level of UVR8b was higher than that of UVR8a. In agreement with prior studies on AtUVR8,uvr8banduvr8a uvr8brice mutants exposed to UV-B showed reduced UVB-induced growth inhibition and upregulation ofCHSandHY5transcripts along with acclimation to UV-B, overexpressing UVR8a or UVR8b enhanced UV-B-induced growth inhibition and acclimation to UV-B, compared to wild-type plants. UV-B was able to enhance the interaction between CONSTITUTIVE PHOTOMORPHOGENESIS1 (COP1) with UVR8a/UVR8b, whereas the interaction intensity of REPRESSOR OF UV-B PHOTOMORPHOGENESIS2 (RUP2) with UVR8a was significantly higher than that with UVR8b. In addition, UVR8a and UVR8b were also found in the nucleus and cytoplasm, but OsUVR8 proteins were localized in nucleus in the absence of UV-B. The level of OsUVR8 monomer showed an invisible change in the leaves of rice seedlings transferred from white light to white light supplemented with UV-B, even UV-B can weaken the interactions of UVR8a or/and UVR8b. Therefore, both UVR8a and UVR8b, that have different location and response modes with Arabidopsis UVR8, function in the response of rice to UV-B radiation, whereas UVR8b plays a predominant role in this process.
Haplo-pheno association forOsNRT1.1paralog in rice reveals superior haplogroup with high nitrate uptake efficiency
Authors: Elangovan Dharani , Pandey Renu , Sharma Sandeep , Balamurugan Balaji , Anand Neha , Das Adhip , Kumar Tarun , Ellur Ranjith Kumar , Kalia Sanjay , Rane Jagadish
Published: December 2023

Haplotype-based breeding approaches hold promise for enhancing crop improvement strategies, allowing for targeted selection of superior genetic combinations to develop high-yielding and resilient varieties. The current study aimed at identification ofNRT1.1nitrate transporter haplotype that could serve as “donors” in haplotype-based breeding. We phenotyped 272 rice accessions in hydroponics with sufficient and low nitrogen (N) for nitrate uptake efficiency. By employing principal component and hierarchical cluster analysis, the accessions were grouped into N efficient, intermediate, and inefficient clusters. Haplotype analysis unveiled the presence of two haplogroups forOsNRT1.1A, three forOsNRT1.1B, and five forOsNRT1.1C. Through haplo-pheno association, the comparison of mean trait values revealed H2 and H3 as the superior haplotypes (SH) for OsNRT1.1A and OsNRT1.1B, respectively. In the case of OsNRT1.1C, H3 and H1 emerged as SH within the N-efficient cluster. Conversely, the inferior haplotypes (IH) consisted of H1 in OsNRT1.1A, H3 in OsNRT1.1B, and H3 and H2 in OsNRT1.1C within the N-inefficient cluster. However, relative expression ofOsNRT1.1(with specific paralogs) in contrasting rice accessions revealed that a few of the inferior accession exhibited higher expression levels in the root but lower in the shoot, which might have contributed to their N-inefficiency. Furthermore, amino acid change at position 403 (Isoleucine to Valine) in inferior accessions influences the active site OsNRT1.1C protein causing N-inefficiency. Ours is the first report on haplotype analysis ofNRT1.1gene demonstrating its genetic diversity, as well as its association with phenotype will have potential implications for improving nitrate uptake efficiency.
The P2 protein of wheat yellow mosaic virus acts as a viral suppressor of RNA silencing to facilitate virus infection in wheat plants
Authors: Chen Dao , Zhang Hui-Ying , Hu Shu-Ming , He Zheng , Wu Yong Qi , Zhang Zong-Ying , Wang Ying , Han Cheng-Gui
Published: December 2023

Wheat yellow mosaic virus (WYMV) causes severe viral wheat disease in Asia. The WYMV P1 protein encoded by RNA2 has viral suppressor of RNA silencing (VSR) activity to facilitate virus infection; however, VSR activity has not been identified for P2 protein encoded by RNA2. In this study, P2 protein exhibited strong VSR activity inNicotiana benthamianaat the four-leaf stage, and point mutants P70A and G230A lost VSR activity. Protein P2 interacted with calmodulin (CaM) protein, a gene-silencing associated protein, while point mutants P70A and G230A did not interact with it. Competitive bimolecular fluorescence complementation and competitive co-immunoprecipitation experiments showed that P2 interfered with the interaction between CaM and calmodulin-binding transcription activator 3 (CAMTA3), but the point mutants P70A and G230A could not. Mechanical inoculation of wheat within vitrotranscripts of WYMV infectious cDNA clone further confirmed that VSR-deficient mutants P70A and G230A decreased WYMV infection in wheat plants compared with the wild type. In addition, RNA silencing, temperature, and autophagy had significant effects on accumulation of P2 protein inN. benthamianaleaves. In conclusion, WYMV P2 plays a VSR role in wheat and promotes virus infection by interfering with calmodulin-related antiviral RNAi defense.

One-sentence summary

WYMV P2 protein exerts VSR activity by interfering with the CaM–CAMTA3 interaction to facilitate virus efficient systemic infection in wheat plants.
Differential effects of MATR3 variants on its cryptic splicing repression function
Authors: Khan Mashiat , Chen Xiao Xiao Lily , Dias Michelle , Santos Jhune Rizsan , Kour Sukhleen , You Justin , van Bruggen Rebekah , Youssef Mohieldin M.M. , Wan Ying-Wooi , Liu Zhandong , Rosenfeld Jill A. , Tan Qiumin , Pandey Udai Bhan , Yalamanchili Hari Krishna , Park Jeehye
Published: December 2023

MATR3 is an RNA-binding protein implicated in neurodegenerative and neurodevelopmental diseases. However, little is known regarding the role of MATR3 in cryptic splicing within the context of functional genes and how disease-associated variants impact this function. We show that loss of MATR3 leads to cryptic exon inclusion in many transcripts. We reveal that ALS-linked S85C pathogenic variant reduces MATR3 solubility but does not impair RNA binding. In parallel, we report a novel neurodevelopmental disease-associated M548T variant, located in the RRM2 domain, which reduces protein solubility and impairs RNA binding and cryptic splicing repression functions of MATR3. Altogether, our research identifies cryptic events within functional genes and demonstrates how disease-associated variants impact MATR3 cryptic splicing repression function.
Co-existence of synaptic plasticity and metastable dynamics in a spiking model of cortical circuits
Authors: Yang Xiaoyu , La Camera Giancarlo
Published: December 2023

Evidence for metastable dynamics and its role in brain function is emerging at a fast pace and is changing our understanding of neural coding by putting an emphasis on hidden states of transient activity. Clustered networks of spiking neurons have enhanced synaptic connections among groups of neurons forming structures called cell assemblies; such networks are capable of producing metastable dynamics that is in agreement with many experimental results. However, it is unclear how a clustered network structure producing metastable dynamics may emerge from a fully local plasticity rule, i.e., a plasticity rule where each synapse has only access to the activity of the neurons it connects (as opposed to the activity of other neurons or other synapses). Here, we propose a local plasticity rule producing ongoing metastable dynamics in a deterministic, recurrent network of spiking neurons. The metastable dynamics co-exists with ongoing plasticity and is the consequence of a self-tuning mechanism that keeps the synaptic weights close to the instability line where memories are spontaneously reactivated. In turn, the synaptic structure is stable to ongoing dynamics and random perturbations, yet it remains sufficiently plastic to remap sensory representations to encode new sets of stimuli. Both the plasticity rule and the metastable dynamics scale well with network size, with synaptic stability increasing with the number of neurons. Overall, our results show that it is possible to generate metastable dynamics over meaningful hidden states using a simple but biologically plausible plasticity rule which co-exists with ongoing neural dynamics.
Approaching Maximum Resolution in Structured Illumination Microscopy via Accurate Noise Modeling
Authors: Saurabh Ayush , Brown Peter T. , Bryan J. Shepard , Fox Zachary R. , Kruithoff Rory , Thompson Cristopher , Kural Comert , Shepherd Douglas P. , Pressé Steve
Published: December 2023

Biological images captured by microscopes are characterized by heterogeneous signal-to-noise ratios (SNRs) due to spatially varying photon emission across the field of view convoluted with camera noise. State-of-the-art unsupervised structured illumination microscopy (SIM) reconstruction algorithms, commonly implemented in the Fourier domain, do not accurately model this noise and suffer from high-frequency artifacts, user-dependent choices of smoothness constraints making assumptions on biological features, and unphysical negative values in the recovered fluorescence intensity map. On the other hand, supervised methods rely on large datasets for training, and often require retraining for new sample structures. Consequently, achieving high contrast near the maximum theoretical resolution in an unsupervised, physically principled, manner remains an open problem. Here, we propose Bayesian-SIM (B-SIM), an unsupervised Bayesian framework to quantitatively reconstruct SIM data, rectifying these shortcomings by accurately incorporating known noise sources in the spatial domain. To accelerate the reconstruction process, we use the finite extent of the point-spread-function to devise a parallelized Monte Carlo strategy involving chunking and restitching of the inferred fluorescence intensity. We benchmark our framework on both simulated and experimental images, and demonstrate improved contrast permitting feature recovery at up to 25% shorter length scales over state-of-the-art methods at both high- and low-SNR. B-SIM enables unsupervised, quantitative, physically accurate reconstruction without the need for labeled training data, democratizing high-quality SIM reconstruction and expands the capabilities of live-cell SIM to lower SNR, potentially revealing biological features in previously inaccessible regimes.
Low salinity destabilizes the bacterial community of sugar kelp,Saccharina latissima(Phaeophyceae)
Authors: Schenk Siobhan , Wardrop Connor Glen , Parfrey Laura Wegener
Published: December 2023

As climate change progresses, the intensity and variability of freshwater outflow into the ocean are predicted to increase. The resulting increase in low-salinity events will be a source of stress forSaccharina latissimaand potentiallySaccharina-associated bacteria. Bacteria influence host health and can facilitate or hinder host survival and acclimation to stressful abiotic conditions. Therefore, understanding how bacterial communities change under abiotic stress is critical to understand how abiotic stress will affect kelp physiology. We investigated the effect of low-salinity stress onSaccharina-associated bacteria and the host by surveying the bacterial community associated withSaccharinaand the surrounding environment across naturally occurring salinity gradients during the spring freshet across two years at four field sites with contrasting salinity profiles around Vancouver, Canada (519 samples), coupled with salinity manipulation experiments repeated eight times (269 samples). Overall,Saccharinaharbours a stable core bacterial community, which decreases in relative abundance under abiotic stress. In the field, both salinity and temperature shape the bacterial community, with temperature having higher explanatory power most of the time. In the lab, we confirm that the patterns observed in the field can be replicated by manipulating salinity alone. Decreased relative abundance of core bacteria and increased community dissimilarity in low salinity, in both the lab and field, suggest that host filtering is significantly impaired in low salinity. In the context of a stable host-associated core bacterial community during non-stressful conditions, the change in the community composition observed during conditions of abiotic stress indicates a host stress response.
Unraveling the Phenotypic States of Human innate-like T Cells: Comparative Insights with Conventional T Cells and Mouse Models
Authors: Loh Liyen , Carcy Salomé , Krovi Harsha S. , Domenico Joanne , Spengler Andrea , Lin Yong , Torres Joshua , Palmer William , Norman Paul J. , Stone Matthew , Brunetti Tonya , Meyer Hannah V. , Gapin Laurent
Published: December 2023

The “innate-like” T cell compartment, known as Tinn, represents a diverse group of T cells that straddle the boundary between innate and adaptive immunity, having the ability to mount rapid responses following activation. In mice, this ability is acquired during thymic development. We explored the transcriptional landscape of Tinncompared to conventional T cells (Tconv) in the human thymus and blood using single cell RNA sequencing and flow cytometry. We reveal that in human blood, the majority of Tinncells, including iNKT, MAIT, and Vδ2+Vγ9+T cells, share an effector program characterized by the expression of unique chemokine and cytokine receptors, and cytotoxic molecules. This program is driven by specific transcription factors, distinct from those governing Tconvcells. Conversely, only a fraction of thymic Tinncells displays an effector phenotype, while others share transcriptional features with developing Tconvcells, indicating potential divergent developmental pathways. Unlike the mouse, human Tinncells do not differentiate into multiple effector subsets but develop a mixed type I/type III effector potential. To conduct a comprehensive cross-species analysis, we constructed a murine Tinndevelopmental atlas and uncovered additional species-specific distinctions, including the absence of type II Tinncells in humans, which implies distinct immune regulatory mechanisms across species. The study provides insights into the development and functionality of Tinncells, emphasizing their role in immune responses and their potential as targets for therapeutic interventions.
Not dasycladalean alga, but an Odyssey of the earliest Phanerozoic animal reef-builders
Authors: Yang Aihua , Luo Cui , Han Jian , Zhuravlev Andrey Yu. , Reitner Joachim , Sun Haijing , Zeng Han , Zhao Fangchen , Hu Shixue
Published: December 2023

The compacted macrofossilProtomelission? sp. from the early Cambrian Xiaoshiba Lagerstätte was recently ascribed to early dasycladalean green algae and used to disprove the bryozoan affinity of coeval phosphatized microfossils, which made the puzzling question whether the bryozoans originated in early Cambrian pending again. Our new analyses of multiple specimens which are conspecific withProtomelission? from the Chengjiang Lagerstätte indicate that they are not dasycladaleans but one of the three groups of archaeocyath-like sponges that atypically inhabited siliciclastic substrates. All the archaeocyath-like fossils share the same preservation mode and exhibit archaeocyath-type external skeletal features. Particularly, theProtomellision?-like fossils preserve structures indicative of archaeocyath aquiferous system and ontogeny. They represent the first recognized one-walled archaeocyath sponges in South China and evidence the niche expansion of archaeocyaths on their way of global radiation from Siberia, 518 million years ago. The origin of the bryozoans remains a mystery.
Reference-guided automatic assembly of genomic tandem repeats with only HiFi and Hi-C data enables population-level analysis
Authors: Wen Huaming , Pan Weihua
Published: December 2023

The existing de novo methods of complete genome assembly are not able to generate large-scale pangenomes with complete assemblies due to the shortcomings such as requiring multiple types of sequencing data of high price, requiring large amount of manual curation, and not being able to achieve haplotype-resolved complete assembly of long tandem repeats in most situations. To solve this problem, in this study, we propose a new genome assembly mode called reference-guided assembly which relies on the reference information to recall the reads for complex genomic regions of interest and assembles them inde novo-like way. As a proof-of-concept, we developed an algorithm TRFill which can reassemble or fill the gaps of tandem repeats in chromosome-level assembly in either haploid or diploid way using only HiFi and Hi-C data. The experimental results on human centromeres and tomato subtelomeres show that TRFill successfully improved the completeness and correctness of about two thirds of the tested tandem repeat sequences. Furthermore, TRFill improved the completeness of subtelomeric tandem repeats by 50% in a recently published tomato pangenome, enabling a population-level analysis of the subtelomeric tandem repeats, which found the ‘local law of sequence similarity of tandem repeats’ providing theoretical basis for reference-guided assembly in turn.
The myeloid cell-driven transdifferentiation of endothelial cells into pericytes promotes the restoration of BBB function and brain self-repair after stroke
Authors: Li Tingbo , Yang Ling , Tu Jiaqi , Hao Yufan , Zhu Zhu , Xiong Yingjie , Gao Qingzhu , Zhou Lili , Xie Guanglei , Zhang Dongdong , Li Xuzhao , Jin Yuxiao , Zhang Yiyi , Zhao Bingrui , Li Nan , Wang Xi , Jia Jie-Min
Published: December 2023

Ischemic stroke, one of the leading causes of death in the world, is accompanied by the dysfunction of the blood-brain barrier (BBB), which aggravates neuron damage. However, the mechanisms underlying the restoration of BBB in the chronic stage after stroke remain unclear. Here, we investigated the changes in the pericyte pool and their effects on BBB function and brain self-repair after stroke. Using lineage tracing, RNA-seq and immunofluorescence staining, we detected endothelial cells (ECs) transdifferentiated into pericytes (E-pericytes) after stroke in the MCAO model. E-pericytes depletion by diphtheria toxin A (DTA) aggravated BBB leakage and exacerbated neurological deficits in the MCAO model. The myeloid cell-driven transdifferentiation of ECs into pericytes accelerated BBB restoration and brain self-repair after stroke via endothelial-mesenchymal transformation (EndoMT). Decreasing the number of E-pericytes by specific knockout of theTgfbr2gene in ECs also aggravated BBB leakage and exacerbated neurological deficits. Specific-ECs overexpression of theTgfbr2gene promoting E-pericytes transdifferentiation reduced BBB leakage and exerted neuroprotective effects. Our findings solve a key question about how changes in the pericyte pool after stroke affect the restoration of BBB function and brain self-repair and may offer a new approach to therapy.
Low-Cost 3D-Printed Tools Towards Robust Longitudinal Multi-Modal Pre-Clinical Imaging
Authors: Allam Nader , Taylor Edward , Vitkin I. Alex
Published: December 2023

1

Window chamber models enable a range of preclinicalin-vivooptical studies with high spatial resolution and contrast, most notably probing the tumour microenvironment (TME). However, there are multiple sources of experimental variability that can affect the quality of the resultant data, especially in the context of longitudinal data acquisition, where accurate registration between images acquired at different times is crucial to understanding changes to the spatially heterogeneous TME. Further, it is challenging to correlate the findings of these models to clinical imaging modalities such as magnetic resonance imaging (MRI), which typically have much lower resolution and derive contrast from different physical mechanisms; yet such correlations may assist in the translation of window-chamber-derived basic preclinical insights into the clinic. Here, we describe the development and construction of a low-cost 3D-printable window chambers and compatible toolset to improve the accuracy, precision, and repeatability of longitudinal pre-clinical imaging and inter-modality co-registration at different spatial resolution scales. Such improvements in our novel multi-modal experimental pipeline may assist researchers in the acquisition and translation of TME biomarkers and other pre-clinical measurements from the window chamber model into the clinic.
Large-Scale Information Retrieval and Correction of Noisy Pharmacogenomic Datasets through Residual Thresholded Deep Matrix Factorization
Authors: Hu Zhiyue Tom , Yu Yaodong , Chen Ruoqiao , Yeh Shan-Ju , Chen Bin , Huang Haiyan
Published: December 2023

Pharmacogenomics studies are attracting an increasing amount of interest from researchers in precision medicine. The advances in high-throughput experiments and multiplexed approaches allow the large-scale quantification of drug sensitivities in molecularly characterized cancer cell lines (CCLs), resulting in a number of open drug sensitivity datasets for drug biomarker discovery. However, a significant inconsistency in drug sensitivity values among these datasets has been noted. Such inconsistency indicates the presence of substantial noise, subsequently hindering downstream analyses. To address the noise in drug sensitivity data, we introduce a robust and scalable deep learning framework, Residual Thresholded Deep Matrix Factorization (RT-DMF). This method takes a single drug sensitivity data matrix as its sole input and outputs a corrected and imputed matrix. Deep Matrix Factorization (DMF) excels at uncovering subtle patterns, due to its minimal reliance on data structure assumptions. This attribute significantly boosts DMF’s ability to identify complex hidden patterns among nuisance effects in the data, thereby facilitating the detection of signals that are therapeutically relevant. Furthermore, RT-DMF incorporates an iterative residual thresholding (RT) procedure, which plays a crucial role in retaining signals more likely to hold therapeutic importance. Validation using simulated datasets and real pharmacogenomics datasets demonstrates the effectiveness of our approach in correcting noise and imputing missing data in drug sensitivity datasets (open source package available athttps://github.com/tomwhoooo/rtdmf).
Optimized Mass Spectrometry Detection of Thyroid Hormones and Polar Metabolites in Rodent Cerebrospinal Fluid
Authors: Fame Ryann M. , Ali Ilhan , Lehtinen Maria K. , Kanarek Naama , Petrova Boryana
Published: December 2023

Background

Thyroid hormones (TH) are required for brain development and function. Cerebrospinal fluid (CSF), which bathes the brain and spinal cord, contains TH as free or transthyretin (TTR)-bound. Tight thyroid hormone level regulation in the central nervous system is essential for developmental gene expression that governs neurogenesis, myelination, and synaptogenesis. This integrated function of TH highlights the importance of developing precise and reliable methods for assessing TH levels in CSF. Methods: we report an optimized LC-MS based method to measure thyroid hormones in rodent CSF and serum, applicable to both fresh and frozen samples. Results: We find distinct differences in CSF thyroid hormone in pregnant dams vs. non-pregnant adults and in embryonic vs. adult CSF. Further, targeted LC-MS metabolic profiling uncovers distinct central carbon metabolism in the CSF of these populations. Conclusions: TH detection and metabolite profiling of related metabolic pathways open new avenues of rigorous research into CSF thyroid hormone and will inform future studies on metabolic alterations in CSF during normal development.
Evaluation of the fitness benefit conferred by RNA cis-regulators toStreptococcus pneumoniaeduring infection
Authors: Warrier Indu , Perry Ariana , Hubbell Sara M. , Eichelman Matthew , van Opijnen Tim , Meyer Michelle M.
Published: December 2023

Bacteria have evolved complex transcriptional regulatory networks, as well as many diverse regulatory strategies at the RNA level, to enable more efficient use of metabolic resources and rapid response to changing conditions. However, most RNA-based regulatory mechanisms are not well conserved across different bacterial species despite controlling genes important for virulence or essential biosynthetic processes. Here, we characterize the activity of, and assess the fitness benefit conferred by, twelve cis-acting regulatory RNAs (including several riboswitches and a T-box), in the opportunistic pathogenStreptococcus pneumoniaeTIGR4. By evaluating native locus mutants of each regulator that result in constitutively active and repressed expression, we establish that growth defects in planktonic culture are associated with constitutive repression of gene expression, while constitutive activation of gene expression is rarely deleterious. In contrast, in mouse nasal carriage and pneumonia models, strains with both constitutively active and repressed gene expression are significantly less fit than matched control strains. Furthermore, two RNA-regulated pathways, FMN synthesis/transport and pyrimidine synthesis/transport display the greatest sensitivity to mis-regulation or constitutive gene repression in both planktonic culture andin vivoenvironments. Our findings suggest that despite lack of an obvious phenotypes associated with gene over-expressionin vitro, the fitness benefit conferred on bacteria via fine-tuned metabolic regulation through cis-acting regulatory RNAs is often substantialin vivo, and therefore easily sufficient to drive the evolution and maintenance of diverse RNA regulatory mechanisms.
The mutational landscape ofStaphylococcus aureusduring colonisation
Authors: Coll Francesc , Blane Beth , Bellis Katherine , Matuszewska Marta , Jamrozy Dorota , Toleman Michelle , Geoghegan Joan A , Parkhill Julian , Massey Ruth C , Peacock Sharon J , Harrison Ewan M
Published: December 2023

Staphylococcus aureusis an important human pathogen but is primarily a commensal of the human nose and skin. Survival during colonisation is likely one of the major drivers ofS. aureusevolution. Here we use a genome-wide mutation enrichment approach to analyse a genomic dataset of 3,060S. aureusisolates from 791 individuals to show that despite limited within-host genetic diversity, an excess of protein-altering mutations can be found in genes encoding key metabolic pathways, in regulators of quorum-sensing and in known antibiotic targets. Nitrogen metabolism and riboflavin synthesis are the metabolic processes with strongest evidence of adaptation. Further evidence of adaptation to nitrogen availability was revealed by enrichment of mutations in the assimilatory nitrite reductase and urease, including mutations that enhance growth with urea as the sole nitrogen source. Inclusion of an additional 4,090 genomes from 802 individuals revealed eight additional genes includingsasA/sraP,pstA, andrsbUwith signals adaptive variation that warrant further characterisation. Our study provides the most comprehensive picture to date of the heterogeneity of adaptive changes that occur in the genomes ofS. aureusduring colonisation, revealing the likely importance of nitrogen metabolism, loss of quorum sensing and antibiotic resistance for successful human colonisation.
Understanding Molecular Links of Vascular Cognitive Impairment: Selective Interaction between Mutant APP, TP53, and MAPKs
Authors: Zeylan Melisa Ece , Senyuz Simge , Keskin Ozlem , Gursoy Attila
Published: December 2023

Vascular cognitive impairment (VCI) is an understudied cerebrovascular disease. As it can result in a significant amount of functional and cognitive disabilities, it is vital to reveal proteins related to it. Our study focuses on revealing proteins related to this complex disease by deciphering the crosstalk between cardiovascular and cognitive diseases. We build protein-protein interaction networks related to cardiovascular and cognitive diseases. After merging these networks, we analyze the network to extract the hub proteins and their interactors. We found the clusters on this network and built the structural protein-protein interaction network of the most connected cluster on the network. We analyzed the interactions of this network with molecular modeling via PRISM. PRISM predicted several interactions that can be novel in the context of VCI-related interactions. Two mutant forms of APP (V715M and L723P), previously not connected to VCI, were discovered to interact with other proteins. Our findings demonstrate that two mutant forms of APP interact differently with TP53 and MAPK’s. Furthermore, TP53, AKT1, PARP1, and FGFR1 interact with MAPKs through their mutant conformations. We hypothesize that these interactions might be crucial for VCI. We suggest that these interactions and proteins can act as early VCI markers or as possible therapeutic targets.
pyRBDome: A comprehensive computational platform for enhancing and interpreting RNA-binding proteome data
Authors: Chu Liang-Cui , Christopoulou Niki , McCaughan Hugh , Winterbourne Sophie , Cazzola Davide , Wang Shichao , Litvin Ulad , Brunon Salomé , Harker Patrick J.B. , McNae Iain , Granneman Sander
Published: December 2023

High-throughput proteomics approaches have revolutionised the identification of RNA-binding proteins (RBPome) and RNA-binding sequences (RBDome) across organisms. Yet the extent of noise, including false-positives, associated with these methodologies, is difficult to quantify as experimental approaches for validating the results are generally low throughput. To address this, we introduce pyRBDome, a pipeline for enhancing RNA-binding proteome datain silico. It aligns the experimental results with RNA-binding site (RBS) predictions from distinct machine learning tools and integrates high-resolution structural data when available. Its statistical evaluation of RBDome data enables quick identification of likely genuine RNA-binders in experimental datasets. Furthermore, by leveraging the pyRBDome results, we have enhanced the sensitivity and specificity of RBS detection through training new ensemble machine learning models. pyRBDome analysis of a human RBDome dataset, compared with known structural data, revealed that while UV cross-linked amino acids were more likely to contain predicted RBSs, they infrequently bind RNA in high-resolution structures. This discrepancy underscores the limitations of structural data as benchmarks, positioning pyRBDome as a valuable alternative for increasing confidence in RBDome datasets.
Human rhinovirus 16 infection modifies the microRNA landscape during epithelial cell infection
Authors: Bosner Pax , Cappleman Victoria , Tomicic Alka , Kyyaly Aref , Jubrail Jamil
Published: December 2023

Human rhinovirus is a major driver of disease exacerbations for patients with asthma and chronic obstructive pulmonary disease. Importantly, the virus can disrupt immune cell functioning leading to secondary bacterial infections in these patients. MicroRNAs are important regulators of gene expression and control viral pathogenesis and immune cell functions. However, the role of different miRNAs during rhinovirus infections remains poorly defined. The aim of this study was to analyze the impact of RV16 on epithelial cell responses and identify miRNAs with altered expression levels in response to the viral infection and their target genes. HeLa-Ohio cells exposed to RV16 showed deficiencies in bacterial clearance and induced changes in the expression of several novel miRNAs identified using a custom panel of 88 different miRNAs and further analyzed using our newly developed computer program. Our experiments identified a panel of 9 differentially regulated miRNAs and our computer program narrowed this down to two that could represent biomarkers for RV16 infection. Our results also showed that upon upregulation of these miRNAs (miR-101-3p and miR-30b-5p) by RV16 there was a downregulation in EZH2, RARG and PTPN13 that could correlate with a productive viral infection and a worsened immune response. Taken together, our findings suggest that RV16 modifies the miRNA landscape in epithelial cells and that miR-101-3p and miR-30b-5p could represent key biomarkers for either early or late RV16 infection.
Proton Relaxometry of Tree Leaves at Hypogeomagnetic Fields
Authors: Fabricant Anne M. , Put Piotr , Barskiy Danila A.
Published: December 2023

We report on a cross-species proton-relaxometry study inex vivotree leaves using nuclear magnetic resonance (NMR) at 7 μT. Apart from the intrinsic interest of probing nuclear-spin relaxation in biological tissues at magnetic fields below Earth field, our setup enables comparative analysis of plant water dynamics without the use of expensive commercial spectrometers. In this work, we focus on leaves from common Eurasian evergreen and deciduous tree families: Pinaceae (pine, spruce), Taxaceae (yew), Betulaceae (hazel), Prunus (cherry), and Fagaceae (beech, oak). Using a nondestructive protocol, we measure their effective protonT2relaxation times as well as track the evolution of water content associated with leaf dehydration. Newly developed “gradiometric quadrature” detection and data-processing techniques are applied in order to increase the signal-to-noise ratio (SNR) of the relatively weak measured signals. We find that while measured relaxation times do not vary significantly among tree genera, they tend to increase as leaves dehydrate. Such experimental modalities may have particular relevance for future drought-stress research in ecology, agriculture, and space exploration.
Byproducts of inflammatory radical metabolism provide transient nutrient niches for microbes in the inflamed gut
Authors: Spiga Luisella , Winter Maria G. , Muramatsu Matthew K. , Rojas Vivian K. , Chanin Rachael B. , Zhu Wenhan , Hughes Elizabeth R. , Taylor Savannah J. , Faber Franziska , Porwollik Steffen , Carvalho Tatiane F. , Qin Tian , Santos Renato L. , Andrews-Polymenis Helene , McClelland Michael , Winter Sebastian E.
Published: December 2023

Louis Pasteur’s experiments on tartaric acid laid the foundation for our understanding of molecular chirality, but major questions remain. By comparing the optical activity of naturally-occurring tartaric acid with chemically-synthesized paratartaric acid, Pasteur realized that naturally-occurring tartaric acid contained only L-tartaric acid while paratartaric acid consisted of a racemic mixture of D- and L-tartaric acid. Curiously, D-tartaric acid has no known natural source, yet several gut bacteria specifically degrade D-tartaric acid. Here, we investigated the oxidation of monosaccharides by inflammatory reactive oxygen and nitrogen species. We found that this reaction yields an array of alpha hydroxy carboxylic acids, including tartaric acid isomers. Utilization of inflammation- derived D- and L-tartaric acid enhanced colonization bySalmonellaTyphimurium andE. coliin murine models of gut inflammation. Our findings suggest that byproducts of inflammatory radical metabolism, such as tartrate and other alpha hydroxy carboxylic acids, create transient nutrient niches for enteric pathogens and other potentially harmful bacteria. Furthermore, this work illustrates that inflammatory radicals generate a zoo of molecules, some of which may erroneously presumed to be xenobiotics.
Assessment of ferroptosis inducers and Nrf2 inhibitors as radiosensitisers in 2D and 3D breast cancer cell cultures
Authors: Alzufairi AA , Souilhol C , Jordan-Mahy N , Cross NA
Published: December 2023

Ferroptosis is a form of programmed cell death that is modulated in some cancer cells as a pro-survival mechanism. Induction of ferroptosis is a potential anti-cancer strategy, and enhancement of ferroptosis using ferroptosis inducers has the potential to enhance current anti-tumour mechanisms. In this study, we assessed the effect of the ferroptosis inducers Erastin, RSL-3 and FIN-56 on radiosensitivity in 2D cell culture, and in 3D alginate tumour spheroids from breast cancer cell lines. Since some tumours modulate ferroptosis via increased Nrf2 production, and MCF-7 and MDA-MB-231 both produce Nrf2 protein, we also assessed the effects of the Nrf2 inhibitor ML385 on radiosensitivity. MDA-MB-231 was highly sensitive to all ferroptosis inducers, and ferroptosis was reversed by the ferroptosis inhibitors Ferrostatin-1, Liproxstatin-1 and Deferoxamine. MCF-7 was resistant to all ferroptosis inducers. MDA-MB-231 and MCF-7 cells were sensitive to irradiation in 2D cell culture but resistant to irradiation in 3D alginate spheroids. Ferroptosis inducers did not synergistically enhance irradiation-induced cell death in 2D cell cultures. There was also no robust enhancement to irradiation effects with ferroptosis inducers in 2D or 3D cell culture. Ferroptosis inducers did, however, show a heterogeneous response in 3D cell culture, in that isogenic spheroids responded differently within the same spheroid. The Nrf2 inhibitor ML385 showed no synergistic enhancement of ferroptotic cell death when combined with irradiation. These studies suggest targeting ferroptosis does not induce short-term enhancement of ferroptotic cell death.
Targeted protein degradation systems to enhance Wnt signaling
Authors: Sampathkumar Parthasarathy , Jung Heekyung , Chen Hui , Zhang Zhengjian , Suen Nicholas , Yang Yiran , Huang Zhong , Lopez Tom , Benisch Robert , Lee Sung-Jin , Ye Jay , Yeh Wen-Chen , Li Yang
Published: December 2023

Molecules that facilitate targeted protein degradation (TPD) offer great promise as novel therapeutics. The human hepatic lectin asialoglycoprotein receptor (ASGR) is selectively expressed on hepatocytes. We have previously engineered an anti-ASGR1 antibody-mutant RSPO2 (RSPO2RA) fusion protein (called SWEETSTM) to drive tissue-specific degradation of ZNRF3/RNF43 E3-ubiquitin ligases, which achieved hepatocyte-specific enhanced Wnt signaling, proliferation, and restored liver function in mouse models, and an antibody-RSPO2RA fusion molecule is currently in human clinical trials. In the current study, we identified two new ASGR1 and ASGR1/2 specific antibodies, 8M24 and 8G8. High-resolution crystal structures of ASGR1:8M24 and ASGR2:8G8 complexes revealed that these antibodies bind to distinct epitopes on opposing sides of ASGR, away from the substrate binding site. Both antibodies enhanced Wnt-activity when assembled as SWEETS molecules with RSPO2RA through specific effects sequestering E3 ligases. In addition, 8M24-RSPO2RA and 8G8-RSPO2RA efficiently downregulate ASGR1 through TPD mechanisms. These results demonstrate the possibility of combining different therapeutic effects and degradation mechanisms in a single molecule.