bluuebunny/crossref_metadata_2025 huggingface dataset:
Strain level centromere variation influences CENP-A association dynamics and centromere function
Authors: Arora Uma P. , Sullivan Beth A. , Dumont Beth L.
Published: May 2022

Centromeres are rapidly evolving chromatin domains that fulfill essential roles in chromosome segregation. Rapid centromere sequence evolution imposes strong selection for compensatory changes in centromere-associated kinetochore proteins, leading to striking co-evolutionary trends across species. However, it remains unknown whether within species centromere sequence diversity leads to functional differences in kinetochore protein association. House mice (Mus musculus) exhibit significant variation in centromere satellite array size and sequence heterogeneity, but the amino acid sequence of CENP-A, a centromere-specific histone variant that specifies centromere identity, is conserved. We hypothesize that centromere satellite sequence variation leads to differences in the localization of CENP-A among house mice, with potential consequences for meiotic drive and genome stability. Using CENP-A chromatin immunoprecipitation with a customized k-mer based, reference-blind bioinformatic analysis strategy, we compare the CENP-A sequence association landscape in four diverse inbred mouse strains (C57BL/6J, CAST/EiJ, LEWES/EiJ, and PWK/PhJ). We uncover significant strain-level diversity in CENP-A associated sequences, with more closely related strains exhibiting more similar CENP-A association profiles. LEWES/EiJ and CAST/EiJ show mild association of CENP-A with the pericentromeric satellite repeat, countering the prevailing notion that functional centromere size is solely determined by the size of the minor satellite array. Strain-specific CENP-A association profiles are enriched for unique suites of transcription factor motifs, hinting at strain differences in centromere transcription. Given the importance of centromere-CENP-A association and centromere transcription for both kinetochore assembly and chromosome segregation fidelity, our findings suggest a potential mechanism for centromere-mediated variation in genome stability among inbred mouse strains.
A computational proposal for tracking multiple molecules in a multi-focus confocal setup
Authors: Jazani Sina , Xu Lance W.Q. , Sgouralis Ioannis , Shepherd Douglas P. , Pressé Steve
Published: May 2022

Tracking single molecules continues to provide new insights into the fundamental rules governing biological function. Despite continued technical advances in fluorescent and non-fluorescent labeling as well as data analysis, direct observations of trajectories and interactions of multiple molecules in dense environments remain aspirational goals. While confocal methods provide a means to deduce dynamical parameters with high temporal resolution, such as diffusion coefficients, they do so at the expense of spatial resolution. Indeed, on account of a confocal volume’s symmetry, typically only distances from the center of the confocal spot can be deduced. Motivated by the need for true three dimensional high speed tracking in densely labeled environments, we propose a computational tool for tracking many fluorescent molecules traversing multiple, closely spaced, confocal measurement volumes providing independent observations. Various realizations of this multiple confocal volumes strategy have previously been used for long term, large area, tracking of one fluorescent molecule in three dimensions. What is more, we achieve tracking by directly using single photon arrival times to inform our likelihood and exploit Hamiltonian Monte Carlo to efficiently sample trajectories from our posterior within a Bayesian nonparametric paradigm. A nonparametric paradigm here is warranted as the number of molecules present are, themselves,a prioriunknown. Taken together, we provide a computational framework to infer trajectories of multiple molecules at once, below the diffraction limit (the width of a confocal spot), in three dimensions at sub-millisecond or faster time scales.
Liver humanized NSG-PiZ mice support the study of chronic hepatitis B virus infection and antiviral therapies
Authors: Colón-Thillet Rossana , Stone Daniel , Loprieno Michelle A. , Klouser Lindsay , Roychoudhury Pavitra , Santo Tracy K. , Xie Hong , Stensland Larry , Upham Sarah L. , Pepper Greg , Huang Meei-Li , Aubert Martine , Jerome Keith R.
Published: May 2022

Hepatitis B virus (HBV) is a pathogen of major public health importance that is largely incurable once a chronic hepatitis B (CHB) infection is established. Only humans and great apes are fully permissive to HBV replication, and this species restriction has impacted HBV research by limiting the utility of small animal models of HBV. To combat the species restriction of HBV and enable more HBV studies in vivo, liver-humanized mouse models have been developed that harbor primary human hepatocytes (PHH) and are fully permissive to HBV infection and replication. Unfortunately, these models can be difficult to establish and are expensive commercially, which has limited their academic use. As an alternative mouse model to study HBV, we evaluated liver-humanized NSG-PiZ mice and showed that they are fully permissive to HBV and can develop CHB. Mice were infected with a precore mutant clinical isolate that has now been serially passaged through 3 generations of mice without loss of fitness. HBV selectively replicates in hCK18+ human hepatocytes within chimeric livers, and HBV+ mice secrete infectious virions and HBsAg into blood, while also harboring covalently closed circular DNA (cccDNA). HBV+ mice remain viremic for at least 169 days, which should enable the study of new curative therapies targeting CHB and respond to antiviral entecavir therapy. The extended duration of viremia is sufficient to enable the study of established and new therapeutic approaches targeting CHB. Furthermore, HBV+ PHH in NSG-PiZ mice can be transduced by the hepatotropic AAV3b and AAV.LK03 vector capsids, which should enable the study of curative gene therapies that target CHB. In summary, our data demonstrates that liver humanized NSG-PiZ mice can be used as a robust and cost-effective alternative to existing CHB models and may enable more academic research labs to study HBV disease pathogenesis and antiviral therapy in a setting that is fully permissive to ongoing replication.
Attention-aware contrastive learning for predicting T cell receptor-antigen binding specificity
Authors: Fang Yiming , Liu Xuejun , Liu Hui
Published: May 2022

It has been verified that only a small fraction of the neoantigens presented by MHC class I molecules on the cell surface can elicit T cells. The limitation can be attributed to the binding specificity of T cell receptor (TCR) to peptide-MHC complex (pMHC). Computational prediction of T cell binding to neoantigens is an challenging and unresolved task. In this paper, we propose an attentive-mask contrastive learning model, ATMTCR, for inferring TCR-antigen binding specificity. For each input TCR sequence, we used Transformer encoder to transform it to latent representation, and then masked a proportion of residues guided by attention weights to generate its contrastive view. Pretraining on large-scale TCR CDR3 sequences, we verified that contrastive learning significantly improved the prediction performance of TCR binding to peptide-MHC complex (pMHC). Beyond the detection of important amino acids and their locations in the TCR sequence, our model can also extracted high-order semantic information underlying the TCR-antigen binding specificity. Comparison experiments were conducted on two independent datasets, our method achieved better performance than other existing algorithms. Moreover, we effectively identified important amino acids and their positional preferences through attention weights, which indicated the interpretability of our proposed model.
Transcriptome profile and clinical characterization of ICOS expression in brain gliomas
Authors: Wang Jin , Shi Fei , Shan Aijun
Published: May 2022

Aims

Inducible Co-Stimulator (ICOS), an immune costimulatory molecule, has been found to play an essential role across various malignancies. This study investigated the transcriptome profile and clinical characterization of ICOS in gliomas.

Methods

Clinical information and transcriptome data of 301 glioma samples were downloaded from the Chinese Glioma Genome Atlas (CGGA) data set for analysis. Furthermore, the results were validated in 697 samples with RNAseq data from the TCGA glioma data set. In addition, single-cell sequencing data from CGGA and GSE 163108 datasets were used to analyze the ICOS expression across different cell types. Statistical analyses and figure production were performed with R-language.

Results

We found that ICOS was significantly upregulated in higher-grade, IDH wildtype, and mesenchymal subtype of gliomas. Functional enrichment analyses revealed that ICOS was mainly involved in glioma-related immune response. Moreover, ICOS showed a robust correlation with other immune checkpoints, including PD1/PD-L1/PD-L2 pathway, CTLA4, ICOSL (ICOS ligand), and IDO1. Furthermore, based on seven clusters of metagenes, GSVA identified that ICOS was tightly associated with HCK, LCK, MHC-I, MHC-II, STAT1, and interferon, especially with LCK, suggesting a strong correlation between ICOS and T-cell activity in gliomas. In cell lineage analysis, ICOS-higher gliomas tended to recruit dendritic cells, monocytes, and macrophages into the tumor microenvironment. Single-cell sequencing analysis indicated that ICOS was highly expressed by regulatory T cells (Treg). Finally, patients with higher ICOS had shortened survival. ICOS was an independent prognosticator for glioma patients.

Conclusions

Higher ICOS was correlated with more malignancy of gliomas and significantly associated with Treg activity among glioma-related immune responses. Moreover, ICOS could contribute as an independent prognostic factor for gliomas. Our study highlighted the role of ICOS in glioma and may facilitate therapeutic strategies targeting ICOS for glioma.
Eulertigs: minimum plain text representation of k-mer sets without repetitions in linear time
Authors: Schmidt Sebastian , Alanko Jarno N.
Published: May 2022

A fundamental operation in computational genomics is to reduce the input sequences to their constituent k-mers. For maximum performance of downstream applications it is important to store the k-mers in small space, while keeping the representation easy and efficient to use (i.e. without k-mer repetitions and in plain text). Recently, heuristics were presented to compute a near-minimum such representation. We present an algorithm to compute a minimum representation in optimal (linear) time and use it to evaluate the existing heuristics. For that, we present a formalisation of arc-centric bidirected de Bruijn graphs and carefully prove that it accurately models the k-mer spectrum of the input. Our algorithm first constructs the de Bruijn graph in linear time in the length of the input strings (for a fixed-size alphabet). Then it uses a Eulerian-cycle-based algorithm to compute the minimum representation, in time linear in the size of the output.

2012 ACM Subject Classification

Applied computing → Computational biology; Theory of computation → Data compression; Theory of computation → Graph algorithms analysis; Theory of computation → Data structures design and analysis
Cerebellar Bergmann Glia Integrating Noxious Information Modulate Nocifensive Behaviors
Authors: Kim Seung Ha , Lee Jaegeon , Roh Seung-Eon , Jang Mirae , Kim Soobin , Lee Ji Hwan , Seo Jewoo , Hwang Jae Yoon , Lee Yong-Seok , Shigetomi Eiji , Lee C. Justin , Koizumi Schuichi , Kim Sun Kwang , Kim Sang Jeong
Published: May 2022

Clinical studies have revealed that the cerebellum is activated by noxious stimuli or pathological pain, and its removal results in somatosensory dysfunction. However, the neural circuits and molecular mechanisms underlying the processing of noxious information in the cerebellum remain unknown. Using two-photon microscopy and optogenetics in mice, we found that the locus coeruleus (LC) terminals in the cerebellar cortex release noradrenaline (NA) in response to cutaneous noxious electrical stimuli. Most Bergmann glia (BG) accumulated this LC-NA noxious information by increasing intracellular calcium in an integrative manner. This global calcium activation of BG, referred to as “flare,” was also elicited in response to an intraplantar capsaicin injection. Chemogenetic inactivation of LC terminals or BG in the cerebellar cortex suppressed BG flares and reduced licking, a nocifensive behavior associated with capsaicin-induced pain. BG-specific knockdown of α-1 adrenergic receptors also suppressed capsaicin-induced BG flares and licking. Chemogenetic activation of BG or an intraplantar capsaicin injection reduced Purkinje cell firings, which disinhibited the output activity of the deep cerebellar nuclei. These results suggest that BG in the cerebellar cortex play an essential role in computing noxious information ascending from the LC and modulate pain-related behaviors by controlling the activity of the cerebellar neural circuits.

One Sentence Summary

Bergmann glia mediate noxious information processing in the cerebellum
Guided construction of single cell reference for human and mouse lung
Authors: Guo Minzhe , Morley Michael P. , Wu Yixin , Du Yina , Zhao Shuyang , Wagner Andrew , Kouril Michal , Jin Kang , Gaddis Nathan , Kitzmiller Joseph A. , Stewart Kathleen , Basil Maria C. , Lin Susan M. , Ying Yun , Babu Apoorva , Wikenheiser-Brokamp Kathryn A. , Mun Kyu Shik , Naren Anjaparavanda P. , Lin Sara , Clair Geremy , Adkins Joshua N. , Pryhuber Gloria S. , Misra Ravi S. , Aronow Bruce J. , Tickle Timothy L. , Salomonis Nathan , Sun Xin , Morrisey Edward E. , Whitsett Jeffrey A. , Xu Yan ,   NHLBI LungMAP Consortium
Published: May 2022

Accurate cell type identification is a key and rate-limiting step in single cell data analysis. Single cell references with comprehensive cell types, reproducible and functional validated cell identities, and common nomenclatures are much needed by the research community to optimize automated cell type annotation and facilitate data integration, sharing, and collaboration. In the present study, we developed a novel computational pipeline to utilize the LungMAP CellCards as a dictionary to consolidate single-cell transcriptomic datasets of 104 human lungs and 17 mouse lung samples and constructed “LungMAP CellRef” and “LungMAP CellRef Seed” for both normal human and mouse lungs. “CellRef Seed” has an equivalent prediction power and produces consistent cell annotation as does “CellRef” but improves computational efficiency and simplifies its utilization for fast automated cell type annotation and online visualization. This atlas set incorporates 48 human and 40 mouse well-defined lung cell types catalogued from diverse anatomic locations and developmental time points. Using independent datasets, we demonstrated the utility of our CellRefs for automated cell type annotation analysis of both normal and disease lungs. User-friendly web interfaces were developed to support easy access and maximal utilization of the LungMAP CellRefs. LungMAP CellRefs are freely available to the pulmonary research community through fast interactive web interfaces to facilitate hypothesis generation, research discovery, and identification of cell type alterations in disease conditions.
Borg tandem repeats undergo rapid evolution and are under strong selection to create new intrinsically disordered regions in proteins
Authors: Schoelmerich Marie C. , Sachdeva Rohan , Waldburger Lucas , West-Roberts Jacob , Banfield Jillian F.
Published: May 2022

Borgs are huge, linear extrachromosomal elements associated with anaerobic methane-oxidizing archaea. Striking features of Borg genomes are pervasive tandem direct repeat (TR) regions. Here, we present six new Borg genomes and investigate the characteristics of tandem repeats in all ten complete Borg genomes. We find that TR regions are rapidly evolving, recently formed, arise independently and are virtually absent in hostMethanoperedensgenomes. Flanking partial repeats and A-enriched character constrain the TR formation mechanism. TRs can be in intergenic regions, where they might serve as regulatory RNAs, or in open reading frames (ORFs). TRs in ORFs are under very strong selective pressure, leading to perfect amino acid TRs (aaTRs) that are commonly intrinsically disordered regions. Proteins with aaTRs are often extracellular or membrane proteins, and functionally similar or homologous proteins often have aaTRs composed of the same amino acids. We propose that Borg aaTR-proteins functionally diversifyMethanoperedensand all TRs are crucial for specific Borg-host associations and possibly co-speciation.
3D flightpaths reveal the development of spatial memory in wild hummingbirds
Authors: Pritchard David J. , Hurly T. Andrew , Photopoulou Theoni , Healy Susan D.
Published: May 2022

Many animals learn to relocate important places and reflect this spatial knowledge in their behaviour. Traditionally evidence for learning is examined experimentally by studying spatial memory. However, tools developed for analysing tracking data from widely ranging animals allow a more holistic analysis of behaviour. Here we use the two together in novel combination of experimental and modelling approaches to analyse how patterns of hummingbird movements change as birds learn to find a reward in a location indicated by a pair of landmarks. Using hidden Markov models (HMMs) we identified two movement states which we interpret as Search and Travel and compared these to experimental behavioural measures of spatial memory. When birds had a single training trial to learn a flower’s location, both the behavioural measures and HMMs showed that hummingbirds relied on landmarks to guide search. Hummingbirds focussed hovering around the rewarded location and were more likely to be in the Search state, and more likely to switch from Travel to Search, when closer to the rewarded location, but only when the landmarks were present. When birds had had 12 additional training trials, however, the HMMs and behavioural measures showed differences in how reliant birds were on landmarks. While behaviours like hovering were still strongly affected by removing landmarks, the likelihood of being in or entered the Search state was the same regardless of whether the landmarks were present or removed. These results suggests that hummingbirds rapidly learn to use nearby landmarks to structure where they search, but as birds gain experience the role of these landmarks changes. While familiar local landmarks were still essential for precise search, experienced birds were able to use alternative cues to guide broad-scale transitions between behaviour. HMMs and traditional behavioural measures each capture a different aspect of this learning, with neither approach alone accurately described the role of landmarks in spatial learning.
Improved deterrence of birds using an artificial predator, the RobotFalcon
Authors: Storms Rolf F. , Carere Claudio , Musters Robert , van Gasteren Hans , Verhulst Simon , Hemelrijk Charlotte K.
Published: May 2022

Collisions between birds and airplanes, bird strikes, can damage aircrafts, resulting in delays and cancellation of flights, costing the international civil aviation industry more than 1.4 billion U.S. dollars annually. Bird deterrence is therefore crucial, but the effectiveness of all available deterrence methods is limited. For example, live avian predators can be a highly effective deterrent, because potential prey will not habituate to them, but live predators cannot be controlled with sufficient precision. Thus, there is an urgent need for new deterrence methods. To this end we developed the RobotFalcon, a device that we modelled after the peregrine falcon, a cosmopolitan predator that preys on a large range of bird species. Mimicking natural hunting behaviour, we tested the effectiveness of the RobotFalcon to deter flocks of corvids, gulls, starlings and lapwings. We compared its effectiveness with that of a drone, and of conventional methods routinely applied at a military airbase. We show that the RobotFalcon scared away bird flocks from fields immediately, and these fields subsequently remained free of bird flocks for hours. The RobotFalcon outperformed the drone and the best conventional method at the airbase (distress calls). Importantly, there was no evidence that bird flocks habituated to the RobotFalcon. We propose the RobotFalcon to be a practical and ethical solution to drive away bird flocks with all advantages of live predators but without their limitations.

Highlights

We present and test a new method of deterring of deterring birds, the RobotFalcon.

The RobotFalcon chased away flocks fast and prevented early returns.

The RobotFalcon outperformed both a drone and convential methods.

No evidence of habituation to the RobotFalcon was found during the study period.
Developmental timing of programmed DNA elimination in Paramecium tetraurelia recapitulates germline transposon evolutionary dynamics
Authors: Zangarelli Coralie , Arnaiz Olivier , Bourge Mickaël , Gorrichon Kevin , Jaszczyszyn Yan , Mathy Nathalie , Escoriza Loïc , Bétermier Mireille , Régnier Vinciane
Published: May 2022

With its nuclear dualism, the ciliate Paramecium constitutes an original model to study how host genomes cope with transposable elements (TEs). P. tetraurelia harbors two germline micronuclei (MIC) and a polyploid somatic macronucleus (MAC) that develops from the MIC at each sexual cycle. Throughout evolution, the MIC genome has been continuously colonized by TEs and related sequences that are removed from the somatic genome during MAC development. Whereas TE elimination is generally imprecise, excision of ∼45,000 TE-derived Internal Eliminated Sequences (IESs) is precise, allowing for functional gene assembly. Programmed DNA elimination is concomitant with genome amplification. It is guided by non-coding RNAs and repressive chromatin marks. A subset of IESs is excised independently of this epigenetic control, raising the question of how IESs are targeted for elimination. To gain insight into the determinants of IES excision, we established the developmental timing of DNA elimination genome-wide by combining fluorescence-assisted nuclear sorting with next-generation sequencing. Essentially all IESs are excised within only one endoreplication round (32C to 64C), while TEs are eliminated at a later stage. We show that time, rather than replication, controls the progression of DNA elimination. We defined four IES classes according to excision timing. The earliest excised IESs tend to be independent of epigenetic factors, display strong sequence signals at their ends and originate from the most ancient integration events. We conclude that old IESs have been optimized during evolution for early and accurate excision, by acquiring stronger sequence determinants and escaping epigenetic control.
HiCUP-Plus: a fast open-source pipeline for accurately processing large scale Hi-C sequence data
Authors: Kelly S. Thomas , Yuhara Satoshi
Published: May 2022

Hi-C is an unbiased genome-wide assay to study 3D chromosome conformation and gene-regulation. The HiCUP pipeline is an open-source tool to process Hi-C from massively parallel sequencing while accounting for biases specific to the restriction enzyme digests used. It is an excellent solution tailored to analyse this technique, however the latest aligner supported by the current release is Bowtie2. To improve the computational performance and mapping accuracy when using the HiCUP pipeline, we have modified it to optionally call the HiSAT2 and Dragen aligners. This allows using the HiCUP pipeline with 3rd party aligners, including the commercially-licensed high performance Dragen aligner. The HiCUP+ pipeline is modified extensively to be compatible with Dragen outputs while ensuring that the same results as the original pipeline can be reproduced with the Bowtie or Bowtie2 aligners. Using the highly accurate HiSAT2 or Dragen aligners produces larger outputs with a higher proportion of uniquely mapped read pairs. It is therefore feasible to leverage the reduced compute-time of Dragen to reduce compute costs and turnaround-time without compromising quality of results. The HiCUP pipeline and Dragen both compute rich summary information.
Haplotype-phased and chromosome-level genome assembly ofPuccinia polysora, a giga-scale fungal pathogen causing southern corn rust
Authors: Liang Junmin , Li Yuanjie , Dodds Peter N. , Figueroa Melania , Sperschneider Jana , Han Shiling , Tsui Clement K.M. , Zhang Keyu , Li Leifu , Ma Zhanhong , Cai Lei
Published: May 2022

Rust fungi are characterized by large genomes with high repeat content and have two haploid nuclei in most life stages, which makes achieving high-quality genome assemblies challenging. Here, we described a pipeline using HiFi reads and Hi-C data to assemble a gigabase-sized fungal pathogen,Puccinia polysoraf.sp.zeae, to haplotype-phased and chromosome-scale. The final assembled genome is 1.71 Gbp, with ~850 Mbp and 18 chromosomes in each haplotype, being currently one of the two giga-scale fungi assembled to chromosome level. Transcript-based annotation identified 47,512 genes for dikaryotic genome with a similar number for each haplotype. A high level of interhaplotype variation was found with 10% haplotype-specific BUSCO genes, 5.8 SNPs/kbp and structural variation accounting for 3% of the genome size. TheP. polysoragenome displayed over 85% repeat contents, with genome-size expansion and copy number increasing of species-specific orthogroups. Interestingly, these features did not affect overall synteny with otherPucciniaspecies having smaller genomes. Fine-time-point transcriptomics revealed seven clusters of co-expressed secreted proteins that are conserved between two haplotypes. The fact that candidate effectors interspersed with all genes indicated the absence of a “two-speed genome” evolution inP. polysora. Genome resequencing of 79 additional isolates revealed a clonal population structure ofP. polysorain China with low geographic differentiation. Nevertheless, a minor population differentiated from the major population by having mutations on secreted proteins includingAvrRppC, indicating the ongoing virulence to evade recognition byRppC, a major resistance gene in Chinese corn cultivars. The high-quality assembly provides valuable genomic resources for future studies on disease management and the evolution ofP. polysora.
Gene Novelties in Amphioxus Illuminate the Early Evolution of Deuterostomes
Authors: Xiong Qing , Yang Kevin Yi , Zeng Xi , Wang Mingqiang , Ng Patrick Kwok-Shing , Zhou Jun-Wei , Ng Judy Kin-Wing , Law Cherie Tsz-Yiu , Du Qiao , Xu Kejin , Falkenberg Laura Jane , Mao Bingyu , Chen Jun-Yuan , Tsui Stephen Kwok-Wing
Published: May 2022

Amphioxus, as the best living proxy to the chordate ancestor, is considered an irreplaceable model organism for evolutionary studies of chordates and deuterostomes. In this study, a high-quality genome of the Beihai amphioxus,Branchiostoma belcheribeihai, wasde novoassembled and annotated. Within four amphioxus genomes, a wide range of gene novelties were identified, revealing new genes that share unexpectedly high similarities with those from non-metazoan species. These gene innovation events have played roles in a range of amphioxus adaptations, including innate immunity responses, adaptation to anaerobic environments, and regulation of calcium balance. The gene novelties related to innate immunity, such as a group of lipoxygenases and a DEAD-box helicase, boosted amphioxus immune responses. The novel genes for alcohol dehydrogenase and ferredoxin could aid in the anaerobic tolerance of amphioxus. A proximally arrayed cluster of EF-hand calcium-binding protein genes were identified to resemble those of bacteria. The copy number of this gene cluster was linearly correlated to the sea salinity of the collection region, suggesting that it may enhance their survival at different calcium concentrations. Collectively, this comprehensive study on gene novelties of amphioxus reveals insights into the early genome evolution of chordates and deuterostomes and provides valuable resources for future research.
PhiDsc: Protein functional mutation Identification by 3D Structure Comparison
Authors: Hoballa Mohamad Hussein , Eslahchi Changiz
Published: May 2022

Selective pressures that trigger cancer formation and progression shape the mutational landscape of somatic mutations in cancer. Given the limits within which cells are regulated, a growing tumor has access to only a finite number of pathways that it can alter. As a result, tumors arising from different cells of origin often harbor identical genetic alterations. Recent expansive sequencing efforts have identified recurrent hotspot mutated residues in individual genes. Here, we introduce PhiDsc, a novel statistical method developed based on the hypothesis that, functional mutations in a recurrently aberrant gene family can guide the identification of mutated residues in the family’s individual genes, with potential functional relevance. PhiDsc combines 3D structural alignment of related proteins with recurrence data for their mutated residues, to calculate the probability of randomness of the proposed mutation. The application of this approach to the RAS and RHO protein families returned known mutational hotspots as well as previously unrecognized mutated residues with potentially altering effect on protein stability and function. These mutations were located in, or in proximity to, active domains and were indicated as protein-altering according to six in silico predictors. PhiDsc is freely available at https://github.com/hobzy987/PhiDSC-DALI.
Structure-Guided Identification of Critical Residues in the Vacuolar Na+,K+/H+ Antiporter NHX1 from Arabidopsis thaliana
Authors: Rombola-Caldentey Belen , Mendoza Imelda , Quintero Francisco J. , Pardo José M.
Published: May 2022

Cation/Proton Antiporters (CPA) acting in all biological membranes help regulate the volume and pH of cells and of intracellular organelles. A key issue with these proteins is their structure-function relationships since they present intrinsic regulatory features that rely on structural determinants, including pH-sensitivity and the stoichiometry of ion exchange. Crystal structures are only available for prokaryotic CPA, whereas the eukaryotic ones have been modeled using the former as templates. Here we show an updated and improved structural model of the tonoplast-localized K+,Na+/H+ antiporter NHX1 of Arabidopsis as a representative of the vacuolar NHX family that is key to the accumulation of K+ into plant vacuoles. Conserved residues judged as functionally important were mutated and the resulting protein variants were tested for activity in the yeast Saccharomyces cerevisiae. Results indicate that residue N184 in the ND-motif characteristic of CPA1 could be replaced by the DD-motif of CPA2 family members with minimal consequences on activity, yet this residue may help to regulate the optimal pH range of the exchanger. Attempts to alter the electroneutrality of AtNHX1 by different combinations of amino acid replacements at N184, R353 and R390 residues resulted in inactive or partly active proteins with differential ability to control the vacuolar pH of the yeast.
The Wnt/TCF7L1 transcriptional repressor axis drives primitive endoderm formation by antagonizing naive and formative pluripotency
Authors: Athanasouli Paraskevi , Balli Martina , De Jaime-Soguero Anchel , Boel Annekatrien , Papanikolaou Sofia , van der Veer Bernard K. , Janiszewski Adrian , Francis Annick , El Laithy Youssef , Lo Nigro Antonio , Aulicino Francesco , Koh Kian Peng , Pasque Vincent , Cosma Maria Pia , Verfaille Catherine , Zwijsen An , Heindryckx Bjorn , Nikolaou Christoforos , Lluis Frederic
Published: May 2022

Early during preimplantation development and in heterogeneous mouse embryonic stem cells (mESC) culture, pluripotent cells are specified towards either the primed epiblast or the primitive endoderm (PE) lineage. Canonical Wnt signaling is crucial for safeguarding naive pluripotency and embryo implantation, yet the role and relevance of canonical Wnt inhibition during early mammalian development remains unknown. Here, we demonstrate that transcriptional repression exerted by Wnt/TCF7L1 promotes PE differentiation of mESCs and in preimplantation inner cell mass. Time-series RNA sequencing and promoter occupancy data reveal that TCF7L1 binds and represses genes encoding essential naive pluripotency factors and indispensable regulators of the formative pluripotency program, including Otx2 and Lef1. Consequently, TCF7L1 promotes pluripotency exit and suppresses epiblast lineage formation, thereby driving cells into PE specification. Conversely, deletion of Tcf7l1 abrogates PE differentiation without restraining epiblast priming. Taken together, our study underscores the importance of transcriptional Wnt inhibition in regulating lineage segregation in ESCs and preimplantation embryo development as well as identifies TCF7L1 as key regulator of this process.
Two different mental disc forms in the Tashan caveGarra:different species or ecotypes?
Authors: Segherloo Iraj Hashemzadeh , Chaloshtory Sajad Najafi , Naser Murtada D. , Yasser Amaal G. , Tabatabaei Seyedeh Narjes , Piette-Lauziere Gabriel , Mashtizadeh Amirhossein , Elmi Amirmohammad , Sedighi Omid , Changizi Alieh , Hallerman Eric , Bernatchez Louis
Published: May 2022

In the Tashan Cave barbGarra tashanensis, inhabiting a small cave in south west Iran, two mental disc forms were observed. To assess their phylogenetic relationships, disc-less and disc-bearing individuals were analyzed using mitochondrial cytochrome oxidase subunit I (COI) partial DNA sequences. Both mental disc forms nested within one clade with absolute bootstrap support (BS= 100), and the genetic distances between the disc-bearing and disc-less individuals (0.3-0.7%) was considerably lower than inter-species mtDNA sequence distances reported among members of the genusGarra. Hence, the observed mental disc variation was not inferred to be a taxonomic feature or a consequence of character displacement. Instead, it was inferred as a case of character release to diversify among ecological niches in the limited subterranean habitat, which should be clarified in follow-up ecological and population genetic studies in more detail.
Extensive mosaicism by somatic L1 retrotransposition in normal human cells
Authors: Nam Chang Hyun , Youk Jeonghwan , Kim Jeong Yeon , Lim Joonoh , Park Jung Woo , Oh Soo A , Lee Hyun Jung , Park Ji Won , Jeong Seung-Yong , Lee Dong-Sung , Oh Ji Won , Han Jinju , Lee Junehawk , Kwon Hyun Woo , Kim Min Jung , Ju Young Seok
Published: May 2022

Over the course of an individual’s lifetime, genomic alterations accumulate in somatic cells. However, the mutational landscape by retrotranspositions of long interspersed nuclear element-1 (L1), a widespread mobile element in the human genome, is poorly understood in normal cells. Here, we explored the whole-genome sequences of 892 single-cell clones established from various tissues collected from 28 individuals. Remarkably, 88% of colorectal epithelial cells acquired somatic L1 retrotranspositions (soL1Rs), carrying ∼3 events per cell on average with substantial intra- and inter-individual variances, which was accelerated at least 10-fold during tumourigenesis. Breakpoints of soL1Rs suggested that a few variant mechanisms can be involved in the L1 retrotransposition processes. Fingerprinting of donor L1s using source-specific unique sequences revealed 34 hot L1s, 44% of which were newly discovered in this study, and many ultra-rare hot L1s in the human population showed higher retrotransposition potential in somatic lineages than common sources. Multi-dimensional analysis of soL1Rs with early embryonic developmental relationships, genome-wide methylation, and gene expression profiles of the clones demonstrated that (1) soL1Rs occur from early embryogenesis at a substantial rate, (2) epigenetic activation of hot L1s is stochastically acquired during the wave of early global epigenomic reprogramming, rather than by the sporadic loss-of-methylation at the late stage, and (3) most L1 transcripts in the cytoplasm do not generate soL1Rs in somatic lineages. In summary, this study provides insights into the retrotransposition dynamics of L1s in the human genome and the resultant somatic mosaicism in normal human cells.
Lack of evidence for increased transcriptional noise in aged tissues
Authors: Ibáñez-Solé Olga , Ascensión Alex M. , Araúzo-Bravo Marcos J. , Izeta Ander
Published: May 2022

Aging is often associated with a loss of cell type identity that results in an increase in transcriptional noise in aged tissues. If this phenomenon reflects a fundamental property of aging remains an open question. Transcriptional changes at the cellular level are best detected by single-cell RNA sequencing (scRNAseq). However, the diverse computational methods used for the quantification of age-related loss of cellular identity have prevented reaching meaningful conclusions by direct comparison of existing scRNAseq datasets. To address these issues we created Decibel, a Python toolkit that implements side-to-side four commonly used methods for the quantification of age-related transcriptional noise in scRNAseq data. Additionally, we developed Scallop, a novel computational method for the quantification of membership of single cells to their assigned cell type cluster. Cells with a greater Scallop membership score are transcriptionally more stable. Application of these computational tools to seven aging datasets showed large variability between tissues and datasets, suggesting that increased transcriptional noise is not a universal hallmark of aging. To understand the source of apparent loss of cell type identity associated with aging, we analyzed cell type-specific changes in transcriptional noise and the changes in cell type composition of the mammalian lung. No robust pattern of cell type-specific transcriptional noise alteration was found across aging lung datasets. In contrast, age-associated changes in cell type composition of the lung were consistently found, particularly of immune cells. These results suggest that claims of increased transcriptional noise of aged tissues should be reformulated.
Fibroblasts-derived from Pluripotent Cells Harboring a Single Allele Knockout in Two Pluripotency Genes Exhibit DNA Methylation Abnormalities and pluripotency induction Defects
Authors: Lasry Rachel , Maoz Noam , Cheng Albert W. , Tov Nataly Yom , Kulenkampff Elisabeth , Azagury Meir , Yang Hui , Ople Cora , Markoulaki Styliani , Faddah Dina A. , Makedonski Kirill , Sabbag Ofra , Jaenisch Rudolf , Buganim Yosef
Published: May 2022

A complete knockout (KO) of a single key pluripotency gene has been shown to drastically affect embryonic stem cell (ESC) function and epigenetic reprogramming. However, knockin (KI)/KO of a reporter gene only in one of two alleles in a single pluripotency gene is considered harmless and is largely used in the stem cell field. Here, we sought to understand the impact of simultaneous elimination of a single allele in two ESC key genes on pluripotency potential and acquisition. We established multiple pluripotency systems harboring KI/KO in a single allele of two different pluripotency genes (i.e. Nanog+/-; Sall4+/-, Nanog+/-; Utf1+/-, Nanog+/-; Esrrb+/- and Sox2+/-; Sall4+/-). Interestingly, although these double heterozygous mutant lines maintain their stemness and contribute to chimeras equally to their parental control cells, fibroblasts derived from these systems show a significant reduction in their capability to induce pluripotency either by Oct4, Sox2, Klf4 and Myc (OSKM) or by nuclear transfer (NT). Tracing the expression of Sall4 and Nanog, as representative key pluripotency targeted genes, at early phases of reprogramming could not explain the seen delay/blockage. Further exploration identifies abnormal methylation landscape around pluripotent and developmental genes in the double heterozygous mutant fibroblasts. Accordingly, treatment with 5-azacytidine two days prior to transgene induction rescues the reprogramming defects. This study emphasizes the importance of maintaining two intact alleles for pluripotency induction and suggests that insufficient levels of key pluripotency genes leads to DNA methylation abnormalities in the derived-somatic cells later on in development.
wrmXpress: A modular package for high-throughput image analysis of parasitic and free-living worms
Authors: Wheeler Nicolas J. , Gallo Kendra J. , Garncarz Elena J. , Ryan Kaetlyn T. , Chan John D. , Zamanian Mostafa
Published: May 2022

Advances in high-throughput and high-content imaging technologies require concomitant development of analytical software capable of handling large datasets and generating relevant phenotypic measurements. Several tools have been developed to analyze drug response phenotypes in parasitic and free-living worms, but these are siloed and often limited to specific instrumentation, worm species, and single phenotypes. No effort has been made to unify tools for analyzing high-content phenotypic imaging data of worms and provide a platform for future extensibility. We have developed wrmXpress, a unified framework for analyzing a variety of phenotypes matched to high-content experimental assays of free-living and parasitic nematodes and flatworms. We demonstrate its utility for analyzing a suite of phenotypes, including motility, development/size, and feeding, and establish the package as a platform upon which to build future custom phenotypic modules, including those that incorporate deep learning techniques. We show that wrmXpress can serve as an analytical workhorse for anthelmintic screening efforts across schistosomes, filarial nematodes, and free-living model nematodes, and holds promise for enabling collaboration among investigators with diverse interests.
A neural theory for counting memories
Authors: Dasgupta Sanjoy , Hattori Daisuke , Navlakha Saket
Published: May 2022

“I’ve never smelled anything like this.” “I’ve seen you once before.” “I’ve heard this song many times.” Keeping track of the number of times different stimuli have been experienced is a critical computation for behavior. This computation occurs ubiquitously across sensory modalities, and naturally without reward or punishment. However, the neural circuitry that mediates this computation remains unknown. Here, we propose a theoretical two-layer neural circuit that can store counts of stimulus occurrence frequencies. This circuit implements a data structure, called a count sketch, that is commonly used in computer science to maintain item frequencies in streaming data. Our first model implements the count sketch data structure using Hebbian synapses and outputs stimulus-specific frequencies. Our second model uses anti-Hebbian plasticity and only tracks frequencies within four count categories (“1-2-3-many”), which we suggest makes a better trade-off between the number of categories that need to be distinguished and the potential ethological value of those categories. Using real-world datasets, we show how both models can closely track the frequencies of different stimuli experienced, while being robust to noise, thus expanding the traditional novelty-familiarity memory axis from binary to continuous. Finally, we show that an implementation of the “1-2-3-many” count sketch — including network architecture, synaptic plasticity rule, and output neuron that encodes count categories — exists in a novelty detection circuit in the insect mushroom body, and we argue that similar circuit motifs also appear in mammals, suggesting that basic memory counting machinery may be broadly conserved.
Analysis of Cyp51 protein sequences shows 4 major Cyp51 gene family groups across Fungi
Authors: Celia-Sanchez Brandi N , Mangum Brandon , Brewer Marin , Momany Michelle
Published: May 2022

Azole drugs target fungal sterol biosynthesis and are used to treat millions of human fungal infections each year. Resistance to azole drugs has emerged in multiple fungal pathogens including Candida albicans, Cryptococcus neoformans, Histoplasma capsulatum, and Aspergillus fumigatus. The most well-studied resistance mechanism in A. fumigatus arises from missense mutations in the coding sequence combined with a tandem repeat in the promoter of cyp51A, which encodes a cytochrome P450 enzyme in the fungal sterol biosynthesis pathway. Filamentous members of Ascomycota such as A. fumigatus have either one or two of three Cyp51 paralogs (Cyp51A, Cyp51B, and Cyp51C). Most previous research in A. fumigatus has focused on Cyp51A due to its role in azole resistance. We used the A. fumigatus Cyp51A protein sequence as the query in database searches to identify Cyp51 proteins across Fungi. We found 435 Cyp51 proteins in 301 species spanning from early-diverging fungi (Blastocladiomycota, Chytridiomycota, Zoopagomycota and Mucormycota) to late-diverging fungi (Ascomycota and Basidiomycota). We found these sequences formed 4 major Cyp51 groups: Cyp51, Cyp51A, Cyp51B, and Cyp51C. Surprisingly, we found all filamentous Ascomycota had a Cyp51B paralog, while only 50% had a Cyp51A paralog. We created maximum likelihood trees to investigate the evolution of Cyp51 in Fungi. Our results suggest Cyp51 is present in all fungi with three paralogs emerging in Pezizomycotina, including Cyp51C which appears to have diverged from the progenitor of the Cyp51A and Cyp51B groups.

Author Summary

Each year millions of people are infected by a fungal pathogen and receive antifungal treatment with azole drugs. Resistance to azole drugs is becoming increasingly prevalent and is mostly caused by mutations in the azole drug target, Cyp51. Aspergillus fumigatus is an airborne fungal pathogen that causes more than 600,000 deaths every year. Azole resistance in A. fumigatus is primarily driven by a promoter repeat coupled with mutations in cyp51A. In our study, we found 435 Cyp51 proteins in 4 major groups across Fungi, with some species having multiple Cyp51 proteins (Cyp51, Cyp51A, Cyp51B, and Cyp51C). Although most research in A. fumigatus has focused on Cyp51A, we found Cyp51B in all filamentous Ascomycota fungi showing it is more conserved than Cyp51A and likely plays a vital role in these fungi.

Author Summary (Shortened)

Resistance to azole drugs is becoming increasingly prevalent and is mostly caused by mutations in the azole drug target, Cyp51. Azole resistance in Aspergillus fumigatus is primarily driven by a promoter repeat coupled with mutations in cyp51A. We found 435 Cyp51 proteins in 4 major groups across Fungi, with some species having multiple Cyp51 proteins (Cyp51, Cyp51A, Cyp51B, and Cyp51C). Although most research focuses on Cyp51A, we found Cyp51B in all filamentous Ascomycota fungi showing it’s more conserved than Cyp51A.
Structural analysis of cholesterol binding and sterol selectivity by ABCG5/G8
Authors: Farhat Danny , Rezaei Fatemeh , Ristovski Milica , Yang Yidai , Stancescu Albert , Dzimkova Lucia , Samnani Sabrina , Couture Jean-François , Lee Jyh-Yeuan
Published: May 2022

The ATP-binding cassette (ABC) sterol transporters are responsible for maintaining cholesterol homeostasis in mammals by participating in reverse cholesterol transport (RCT) or transintestinal cholesterol efflux (TICE). The heterodimeric ABCG5/G8 carries out selective sterol excretion, preventing the abnormal accumulation of plant sterols in human bodies, while homodimeric ABCG1 contributes to the biogenesis and metabolism of high-density lipoproteins. A sterol-binding site on ABCG5/G8 was proposed at the interface of the transmembrane domain and the core of lipid bilayers. In this study, we have determined the crystal structure of ABCG5/G8 in a cholesterol-bound state. The structure combined with amino acid sequence analysis shows that in the proximity of the sterol-binding site, a highly conserved phenylalanine array supports functional implications for ABCG cholesterol/sterol transporters. Lastly, in silico docking analysis of cholesterol and stigmasterol (a plant sterol) suggests sterol-binding selectivity on ABCG5/G8, but not ABCG1. Together, our results provide a structural basis for cholesterol binding on ABCG5/G8 and the sterol selectivity by ABCG transporters.