bluuebunny/crossref_metadata_2025 huggingface dataset:
satmut_utils: a simulation and variant calling package for multiplexed assays of variant effect
Authors: Hoskins Ian , Sun Song , Cote Atina , Roth Frederick P. , Cenik Can
Published: May 2022

Background

The impact of thousands of individual genetic variants on molecular phenotypes for disease-relevant genes remains unknown. Multiplexed assays for variant effect (MAVEs) are highly scalable methods to annotate the relevant variants. However, current software methods for analyzing MAVEs lack standardized annotation, can require cumbersome configuration, and do not easily scale to large target regions.

Results

Here, we present satmut_utils as a flexible solution for 1) simulation of saturation mutagenesis data; and 2) quantification of variants across four orders of magnitude from multiplexed assay data. Improvements of satmut_utils over existing solutions include support for multiple experimental strategies, unique molecular identifier-based consensus deduplication, and machine learning-based error correction. We developed a rigorous simulation workflow to validate the performance of satmut_utils and carried out the first benchmarking of existing software for variant calling. Finally, we used satmut_utils to determine the mRNA abundance of thousands of coding variants in cystathionine beta-synthase (CBS) by two library preparation methods. We identified an association between variants near chemical cofactor binding sites and decreased mRNA abundance. We also found a correlation between codon optimality and the magnitude of variant effects, emphasizing the potential of single-nucleotide variants to alter mRNA abundance.

Conclusions

satmut_utils enables high-performance analysis of saturation mutagenesis data, achieves unprecedented specificity through novel error correction approaches, and reveals the capability of single-codon variants to alter mRNA abundance in native coding sequences.
Conditional covalent lethality driven by oncometabolite accumulation
Authors: Perez Minervo , Nance Kellie D. , Bak Daniel W. , Gamage Supuni Thalalla , Najera Susana S. , Conte Amy N. , Linehan W. Marston , Weerapana Eranthie , Meier Jordan L.
Published: May 2022

Hereditary leiomyomatosis and renal cell carcinoma (HLRCC) is a cancer predisposition syndrome driven by mutation of the tumor suppressor fumarate hydratase (FH). Inactivation of FH causes accumulation of the electrophilic oncometabolite fumarate. In the absence of methods for reactivation, tumor suppressors can be targeted via identification of synthetic lethal interactions using genetic screens. Inspired by recent advances in chemoproteomic target identification, here we test the hypothesis that the electrophilicity of the HLRCC metabolome may produce unique susceptibilities to covalent small molecules, a phenomenon we term conditional covalent lethality. Screening a panel of chemically diverse electrophiles we identified a covalent ligand, MP-1, that exhibits FH-dependent cytotoxicity. Synthesis and structure-activity profiling identified key molecular determinants underlying the molecule’s effects. Chemoproteomic profiling of cysteine reactivity together with clickable probes validated the ability of MP-1 to engage an array of functional cysteines, including one lying in the Zn-finger domain of the tRNA methyltransferase enzyme TRMT1. TRMT1 overexpression rescues tRNA methylation from inhibition by MP-1 and partially attenuates the covalent ligand’s cytotoxicity. Our studies highlight the potential for covalent metabolites and small molecules to synergistically produce novel synthetic lethal interactions and raise the possibility of applying phenotypic screening with chemoproteomic target identification to identify new functional oncometabolite targets.
Isolated BAP1 loss in malignant pleural mesothelioma predicts immunogenicity with implications for immunotherapeutic response
Authors: Osmanbeyoglu Hatice Ulku , Palmer Drake , Sagan April , Sementino Eleonora , Testa Joseph R
Published: May 2022

Malignant pleural mesothelioma (MPM), an aggressive cancer of the mesothelial cells lining the pleural cavity, lacks effective treatments. Multiple somatic mutations and copy number losses in tumor suppressor genes (TSGs) BAP1, CDKN2A/B, and NF2 are associated with MPM. The impact of single versus multiple losses of TSG on MPM biology, the immune tumor microenvironment, clinical outcomes, and treatment responses are unknown. Tumors with alterations in BAP1 alone were associated with a longer overall patient survival rate compared to tumors with CDKN2A/B and/or NF2 alterations with or without BAP1 and formed a distinct immunogenic subtype with altered transcription factor and pathway activity patterns. CDKN2A/B loss consistently contributed to an adverse clinical outcome. Since the loss of only BAP1 was associated with the PD-1 therapy response signature and higher LAG3 and VISTA gene expression, it is a candidate for immune checkpoint blockade therapy. Our results on the impact of TSG genotypes on MPM and the correlations between TSG alterations and molecular pathways provide a foundation for developing MPM therapies.
Fixation Can Change the Appearance of Phase Separation in Living Cells
Authors: Irgen-Gioro Shawn , Yoshida Shawn , Walling Victoria , Chong Shasha
Published: May 2022

Fixing cells with paraformaldehyde (PFA) is an essential step in numerous biological techniques as it is thought to preserve a snapshot of biomolecular transactions in living cells. Fixed cell imaging techniques such as immunofluorescence have been widely used to detect liquid-liquid phase separation (LLPS)in vivo. Here, we compared images, before and after fixation, of cells expressing intrinsically disordered proteins that are able to undergo LLPS. Surprisingly, we found that PFA fixation can both enhance and diminish putative LLPS behaviors. For specific proteins, fixation can even cause their droplet-like puncta to artificially appear in cells that do not have any detectable puncta in the live condition. Fixing cells in the presence of glycine, a molecule that modulates fixation rates, can reverse the fixation effect from enhancing to diminishing LLPS appearance. We further established a kinetic model of fixation in the context of dynamic protein-protein interactions. Simulations based on the model suggest that protein localization in fixed cells depends on an intricate balance of protein-protein interaction dynamics, the overall rate of fixation, and notably, the difference between fixation rates of different proteins. Consistent with simulations, live-cell single-molecule imaging experiments showed that a fast overall rate of fixation relative to protein-protein interaction dynamics can minimize fixation artifacts. Our work reveals that PFA fixation changes the appearance of LLPS from living cells, presents a caveat in studying LLPS using fixation-based methods, and suggests a mechanism underlying the fixation artifact.
The first glimpse of Homo sapiens hereditary fusion genes
Authors: Zhuo Degen
Published: May 2022

Family-inherited fusion genes have been known to be associated with human disease for decades. However, only a small number of them have been discovered so far. In this report, monozygotic (MZ) twins are used as a genetic model to investigate hereditary fusion genes (HFG). We have analyzed RNA-Seq from 37 MZ twins and discovered 1,180 HFGs, the maximum of which is 608 per haploid genome. Eight HFGs associated with MZ twin inheritance range from 52.7% to 67.6%, some of which are previously-studied cancer fusion genes and indicate hereditary cancer genes. These data suggest that HFGs are major genetic factors for human diseases and complex traits. This study gives us the first glimpse of human HFGs and lays theoretical and technological foundations for future genetic and medical studies.
SARS-CoV-2 Spike N-Terminal Domain modulates TMPRSS2-dependent viral entry and fusogenicity
Authors: Meng Bo , Datir Rawlings , Choi Jinwook ,   CITIID-NIHR BioResource COVID-19 Collaboration, Bradley John , Smith Kenneth GC , Lee Joo Hyeon , Gupta Ravindra K.
Published: May 2022

Over 20 mutations have been identified in the N-Terminal Domain (NTD) of SARS-CoV-2 spike and yet few of them are fully characterised. Here we first examined the contribution of the NTD to infection and cell-cell fusion by constructing different VOC-based chimeric spikes bearing B.1617 lineage (Delta and Kappa variants) NTDs and generating spike pseudotyped lentivirus (PV). We found the Delta NTD on a Kappa or WT background increased spike S1/S2 cleavage efficiency and virus entry, specifically in Calu-3 lung cells and airway organoids, through use of TMPRSS2. We have previously shown Delta spike confers rapid cell-cell fusion kinetics; here we show that increased fusogenicity can be conferred to WT and Kappa variant spikes by transfer of the Delta NTD. Moving to contemporary variants, we found that BA.2 had higher entry efficiency in a range of cell types as compared to BA.1. BA.2 showed higher fusogenic activity than BA.1, but the BA.2 NTD could not confer higher fusion to BA.1 spike. There was low efficiency of TMPRSS2 usage by both BA.1 and BA.2, and chimeras of Omicron BA.1 and BA.2 spikes with a Delta NTD did not result in more efficient use of TMRPSS2 or cell-cell fusogenicity. We conclude that the NTD allosterically modulates S1/S2 cleavage and spike-mediated functions such as entry and cell-cell fusion in a spike context dependent manner, and allosteric interactions may be lost when combining regions from more distantly related spike proteins. These data may explain the lack of successful SARS-CoV-2 inter-variant recombinants bearing breakpoints within spike.
Mutation rates and fitness consequences of mosaic chromosomal alterations in blood
Authors: Watson Caroline J. , Blundell Jamie R.
Published: May 2022

Mosaic chromosomal alterations (mCAs) are commonly detected in many cancers and have been found to arise decades before diagnosis. A quantitative understanding of the rate at which these events occur and their functional consequences could improve cancer risk prediction and yet they remain poorly characterised. Here we use clone size estimates of mCAs from the blood of 500,000 participants in the UK Biobank to estimate the mutation rates and fitness consequences of acquired gain, loss and copy-neutral loss of heterozygosity (CN-LOH) events at the chromosomal arm level. Most mCAs have moderate to high fitness effects, but occur at a low rate, being over 10-fold less common than equivalently fit SNVs. While the majority of mCAs increase in prevalence with age in a way that is consistent with a constant growth rate, we find specific examples of mCAs whose behaviour deviates from this suggesting fitness effects for these mCAs may depend on inherited variants or be influenced by extrinsic factors. We find an association between mCA fitness effect and future blood cancer risk, highlighting the important role mCAs may play in risk stratification.
A detailed landscape of genomic alterations in malignant peripheral nerve sheath tumor cell lines challenges the current MPNST diagnosis
Authors: Magallon-Lorenz Miriam , Terribas Ernest , Fernández Marco , Requena Gerard , Rosas Inma , Mazuelas Helena , Uriarte Itziar , Negro Alex , Castellanos Elisabeth , Blanco Ignacio , DeVries George , Kawashima Hiroyuki , Legius Eric , Brems Hilde , Mautner Viktor , Kluwe Lan , Ratner Nancy , Wallace Margaret , Rodriguez Juana Fernández , Lázaro Conxi , Fletcher Jonathan A , Reuss David , Carrió Meritxell , Gel Bernat , Serra Eduard
Published: May 2022

Background

Malignant peripheral nerve sheath tumors (MPNSTs) are soft tissue sarcomas that arise from the peripheral nervous system. Half of the tumors develop in the context of the genetic disease Neurofibromatosis type 1 (NF1) and the rest are sporadic sarcomas. MPNSTs have a dismal prognosis due to their aggressiveness and tendency to metastasize, and new treatment options are needed. The diagnosis of MPNSTs can be challenging, especially outside of the NF1 context since specific histological criteria have not been completely established. Genomic analysis may both facilitate differential diagnoses and suggest precision medicine strategies.

Methods

We generated a complete genomic resource of a set of widely used human NF1-related and sporadic MPNST cell lines by applying ploidy analysis, whole genome and whole exome sequencing and SNP-array analysis, complemented by methylome-based classification and immunofluorescence of cell identity markers (SOX9, SOX10, S100B).

Results

NF1 MPNST cell lines faithfully recapitulated the genomic copy number profile of primary MPNSTs. Structural variants were key players in the complete inactivation of most recurrently altered tumor suppressor genes (TSGs) (NF1, CDKN2A, SUZ12/EED), while small variants played a minor role in the NF1 context, both concerning TSG inactivation and due to the absence of gain-of-function mutations. In clear contrast, the sporadic cell lines (STS-26T, HS-Sch-2, HS-PSS) did not recapitulate the copy number profile of primary MPNSTs. They carried different TSG inactivation and exhibited gain-of-function mutations by predicted kinase activation or generation of fusion genes. Mutational frequencies and signatures emerged as promising informative tools for aiding in MPNST differential diagnosis. Due to the multiple genomic differences exhibited, we complemented their characterization using a methylome-based classifier. All NF1-related cell lines were assigned within the MPNST group, while sporadic cell lines clustered either with melanomas or with an uncertain MPNST-like sarcoma group. The staining of cell identity markers reinforced the idea of a potential misdiagnose of the MPNSTs used to derive the sporadic cell lines analyzed.

Conclusions

Deep genomic analysis, together with methylome-based sarcoma classification and cell identity marker analysis, challenged the MPNST identity of sporadic cell lines. Results presented here open an opportunity to revise MPNST differential diagnosis and classification.
Herpes simplex virus 1 entry glycoproteins form stable complexes prior to and during membrane fusion
Authors: Pataki Zemplen , Viveros Andrea Rebolledo , Heldwein Ekaterina E.
Published: May 2022

Herpesviruses – ubiquitous pathogens that cause persistent infections – have some of the most complex cell entry mechanisms. Entry of the prototypical herpes simplex virus 1 (HSV-1) requires coordinated efforts of 4 glycoproteins, gB, gD, gH, and gL. The current model posits that the glycoproteins do not interact prior to receptor engagement and that binding of gD to its receptor causes a “cascade” of sequential pairwise interactions, first activating the gH/gL complex and subsequently activating gB, the viral fusogen. But how these glycoproteins interact remains unresolved. Here, using a quantitative split-luciferase approach, we show that pairwise HSV-1 glycoprotein complexes form prior to fusion, remain stable throughout fusion, and do not depend on the presence of the cellular receptor. Based on our findings, we propose a revised “conformational cascade” model of HSV-1 entry. We hypothesize that all 4 glycoproteins assemble into a complex prior to fusion, with gH/gL positioned between gD and gB. Once gD binds to a cognate receptor, the proximity of the glycoproteins within this complex allows for efficient transmission of the activating signal from the receptor-activated gD to gH/gL to gB through sequential conformational changes, ultimately triggering the fusogenic refolding of gB. Our results also highlight previously unappreciated contributions of the transmembrane and cytoplasmic domains to glycoprotein interactions and fusion. Similar principles could be at play in other multicomponent viral entry systems, and the split luciferase approach used here is a powerful tool for investigating protein-protein interactions in these and a variety of other systems.

IMPORTANCE

Herpes simplex virus 1 (HSV-1) infects the majority of humans for life and can cause diseases ranging from painful sores to deadly brain inflammation. No vaccines or curative treatments currently exist. HSV-1 infection of target cells requires coordinated efforts of four viral glycoproteins. But how these glycoproteins interact remains unclear. Using a quantitative protein interaction assay, we found that HSV-1 glycoproteins form stable, receptor-independent complexes. We propose that the 4 proteins form a complex, which could facilitate transmission of the entry-triggering signal from the receptor-binding component to the membrane fusogen component through sequential conformational changes. Similar principles could be applicable across other multicomponent protein systems. A revised model of HSV-1 entry could facilitate the development of therapeutics targeting this process.
Analysis of CheW-like domains provides insights into organization of prokaryotic chemotaxis systems
Authors: Vass Luke R. , Bourret Robert B. , Foster Clay A.
Published: May 2022

The ability to control locomotion in a dynamic environment provides a competitive advantage for microorganisms, thus driving the evolution of sophisticated regulatory systems. Nineteen known categories of chemotaxis systems control motility mediated by flagella or Type IV pili, plus other cellular functions. A key feature that distinguishes chemotaxis systems from generic two-component regulatory systems is separation of receptor and kinase functions into distinct proteins, linked by CheW scaffold proteins. This arrangement allows for formation of varied arrays with remarkable signaling properties. We recently analyzed sequences of CheW-like domains found in CheA kinases and CheW and CheV scaffold proteins. Sixteen Architectures of CheA, CheW, and CheV proteins contain ∼94% of all CheW-like domains and form six Classes with likely functional specializations.

We surveyed chemotaxis system categories and proteins containing CheW-like domains in ∼1900 prokaryotic species, the most comprehensive analysis to date, revealing new insights. Co-occurrence analyses suggested that many chemotaxis systems occur in non-random combinations within species, implying synergy or antagonism. Furthermore, many Architectures of proteins containing CheW-like domains occurred predominantly with specific categories of chemotaxis systems, suggesting specialized functional interactions. We propose Class 1 (∼80%) and Class 2 (∼20%) CheW proteins exhibit preferences for distinct chemoreceptor structures. Furthermore, rare (∼1%) Class 6 CheW proteins frequently co-occurred with methyl-accepting coiled coil (MAC) proteins, which contain both receptor and kinase functions and so do not require connection via a CheW scaffold but may benefit from arrays. Lastly, rare multi-domain CheW proteins may interact with different receptors than single domain CheW proteins.
Data-driven microscopy allows for automated targeted acquisition of relevant data with higher fidelity
Authors: André Oscar , Ahnlide Johannes Kumra , Norlin Nils , Swaminathan Vinay , Nordenfelt Pontus
Published: May 2022

Light microscopy is a powerful single-cell technique that allows for quantitative spatial information at subcellular resolution. However, unlike flow cytometry and single-cell sequencing techniques, microscopy has issues achieving high-quality population-wide sample characterization while maintaining high resolution. Here, we present a general framework, data-driven microscopy (DDM), that uses population-wide cell characterization to enable data-driven high-fidelity imaging of relevant phenotypes. DDM combines data-independent and data-dependent steps to synergistically enhance data acquired using different imaging modalities. As proof-of-concept, we apply DDM with plugins for improved high-content screening and live adaptive microscopy. DDM also allows for easy correlative imaging in other systems with a plugin that uses the spatial relationship of the sample population for automated registration. We believe DDM will be a valuable approach for reducing human bias, increasing reproducibility, and placing singlecell characteristics in the context of the sample population when interpreting microscopy data, leading to an overall increase in data fidelity.
Dynamic HIV-1 spike motion creates vulnerability for its membrane-bound tripod to antibody attack
Authors: Yang Shuang , Hiotis Giorgos , Wang Yi , Chen Junjian , Wang Jia-huai , Kim Mikyung , Reinherz Ellis L. , Walz Thomas
Published: May 2022

Vaccines targeting HIV-1’s gp160 spike protein are stymied by high viral mutation rates and structural chicanery. gp160’s membrane-proximal external region (MPER) is the target of naturally arising broadly neutralizing antibodies (bnAbs), yet MPER-based vaccines fail to generate bnAbs. Here, nanodisc-embedded spike protein was investigated by cryo-electron microscopy and molecular-dynamics simulations, revealing spontaneous ectodomain tilting that creates vulnerability for HIV-1. While each MPER protomer radiates centrally towards the three-fold axis contributing to a membrane-associated tripod structure that is occluded in the upright spike, tilting provides access to the opposing MPER. Structures of spike proteins with bound 4E10 bnAb Fabs reveal that the antibody binds exposed MPER, thereby altering MPER dynamics, modifying average ectodomain tilt, and imposing strain on the viral membrane and the spike’s transmembrane segments, resulting in the abrogation of membrane fusion and informing future vaccine development.
Activated interstitial macrophages are a predominant target of viral takeover and focus of inflammation in COVID-19 initiation in human lung
Authors: Wu Timothy Ting-Hsuan , Travaglini Kyle J. , Rustagi Arjun , Xu Duo , Zhang Yue , Andronov Leonid , Jang SoRi , Gillich Astrid , Dehghannasiri Roozbeh , Martínez-Colón Giovanny , Beck Aimee , Liu Daniel Dan , Wilk Aaron J. , Morri Maurizio , Trope Winston L. , Bierman Rob , Weissman Irving L. , Shrager Joseph B. , Quake Stephen R. , Kuo Christin S. , Salzman Julia , Moerner W. E. , Kim Peter S. , Blish Catherine A. , Krasnow Mark A.
Published: May 2022

Early stages of deadly respiratory diseases such as COVID-19 have been challenging to elucidate due to lack of an experimental system that recapitulates the cellular and structural complexity of the human lung while allowing precise control over disease initiation and systematic interrogation of molecular events at cellular resolution. Here we show healthy human lung slices culturedex vivocan be productively infected with SARS-CoV-2, and the cellular tropism of the virus and its distinct and dynamic effects on host cell gene expression can be determined by single cell RNA sequencing and reconstruction of “infection pseudotime” for individual lung cell types. This revealed that the prominent SARS-CoV-2 target is a population of activated interstitial macrophages (IMs), which as infection proceeds accumulate thousands of viral RNA molecules per cell, comprising up to 60% of the cellular transcriptome and including canonical and novel subgenomic RNAs. During viral takeover of IMs, there is cell-autonomous induction of a pro-fibrotic program (TGFB1,SPP1), and an inflammatory program characterized by the early interferon response, chemokines (CCL2, 7,8,13, CXCL10) and cytokines (IL6, IL10), along with destruction of cellular architecture and formation of dense viral genomic RNA bodies revealed by super-resolution microscopy. In contrast, alveolar macrophages (AMs) showed neither viral takeover nor induction of a substantial inflammatory response, although both purified AMs and IMs supported production of infectious virions. Spike-dependent viral entry into AMs was neutralized by blockade of ACE2 or Sialoadhesin/CD169, whereas IM entry was neutralized only by DC-SIGN/CD209 blockade. These results provide a molecular characterization of the initiation of COVID-19 in human lung tissue, identify activated IMs as a prominent site of viral takeover and focus of inflammation and fibrosis, and suggest therapeutic targeting of the DC-SIGN/CD209 entry mechanism to prevent IM infection, destruction and early pathology in COVID-19 pneumonia. Our approach can be generalized to define the initiation program and evaluate therapeutics for any human lung infection at cellular resolution.
Histone modifications and DNA methylation act cooperatively in regulating symbiosis genes in the sea anemone Aiptasia
Authors: Nawaz Kashif , Cziesielski Maha J. , Mariappan Kiruthiga G. , Cui Guoxin , Aranda Manuel
Published: May 2022

The symbiotic relationship between cnidarians and dinoflagellates is one of the most widespread endosymbiosis in our oceans and provides the ecological basis of coral-reef ecosystems. Although many studies have been undertaken to unravel the molecular mechanisms underlying these symbioses, we still know little about the epigenetic mechanisms that control the transcriptional responses to symbiosis. Here, we used the model organism Exaiptasia diaphana to study the genome-wide patterns and putative functions of the histone modifications H3K27ac, H3K4me3, H3K9ac, H3K36me3 and H3K27me3 in symbiosis. While we find that their functions are generally conserved, we observed that colocalization of more than one modification and or DNA methylation correlated with significantly higher gene expression, suggesting a cooperative action of histone modifications and DNA methylation in promoting gene expression. Analysis of symbiosis genes revealed that activating histone modifications predominantly associated with symbiosis induced genes involved in glucose metabolism, nitrogen transport, amino acid biosynthesis and organism growth while symbiosis suppressed genes were involved in catabolic processes. Our results provide new insights into the mechanisms of prominent histone modifications and their interaction with DNA methylation in regulating symbiosis in cnidarians.
Simultaneous isolation of proximal and distal lung progenitor cells from individual mice using a 3D printed guide reduces proximal cell contamination of distal lung epithelial cell isolations
Authors: Alsafadi Hani N. , Stegmayr John , Ptasinski Victoria , Silva Iran , Mittendorfer Margareta , Murray Lynne , Wagner Darcy E.
Published: May 2022

The respiratory epithelium consists of multiple, functionally distinct cell-types and is maintained by regionally-specific progenitor populations which repair the epithelium following injury. Several in vitro methods exist for studying lung epithelial repair using primary murine lung epithelial cells, but isolation methods are hampered by a lack of surface markers distinguishing epithelial progenitors along the respiratory epithelium. Here, we developed a 3D-printed lobe divider (3DLD) to aid in simultaneous isolation of proximal versus distal lung epithelial progenitors from individual mice which give rise to differentiated epithelia in multiple in vitro assays. In contrast to 3DLD-isolated distal progenitor cells, classic manual tracheal ligation methods followed by lobe removal resulted in co-isolation of rare proximal progenitors with distal cells which altered the transcriptional landscape of distal organoid cultures. Thus, cell isolation with the 3DLD generates reproducible distal versus proximal progenitor populations and minimizes the potential for contaminating populations to confound in vitro assays.

Highlights

3DLD reproducibly separates lung lobes and extrapulmonary airways (bronchi/trachea)

3DLD cell isolation yields consistent isolation of distal epithelial cells (DECs)

Contamination of proximal cells in classic DEC isolations may alter in vitro results

3DLD allows for simultaneous isolation of proximal and DECs from single animals

eTOC blurb

Alsafadi et al. describes a new method for simultaneous isolation of lung epithelial proximal and distal progenitors using the aid of a 3D printed device (3DLD). Both isolated cell types differentiate in multiple in vitro assays. The 3DLD guide minimized contamination of proximal cells in distal cell isolations whose presence can alter the transcriptional landscape of distal epithelial organoids.
Coupled organoids reveal that signaling gradients drive traveling segmentation clock waves during human axial morphogenesis
Authors: Yaman Yusuf Ilker , Huang Roya , Ramanathan Sharad
Published: May 2022

Axial development of mammals is a dynamic process involving several coordinated morphogenetic events including axial elongation, somitogenesis, and neural tube formation. How different signals control the dynamics of human axial morphogenesis remains largely unknown. By inducing anteroposterior symmetry breaking of spatially coupled epithelial cysts derived from human pluripotent stem cells, we were able to generate hundreds of axially elongating organoids. Each organoid was composed of a neural tube flanked by presomitic mesoderm that was sequentially segmented into somites. Periodic activation of the somite differentiation gene MESP2 coincided in space and time with anteriorly traveling segmentation clock waves in the presomitic mesoderm of the organoids, recapitulating key aspects of somitogenesis. Through timed perturbations of organoids, we demonstrated that FGF and WNT signaling play distinct roles in axial elongation and somitogenesis, and that the segmentation clock waves are driven by FGF signaling gradients. By generating and perturbing organoids that robustly recapitulate the architecture and dynamics of multiple axial tissues in human embryos, this work offers a means to dissect complex mechanisms underlying human embryogenesis.
Cooperative RNA degradation stabilizes intermediate epithelial-mesenchymal states and supports a phenotypic continuum
Authors: Nordick Benjamin , Park Mary Chae-Yeon , Quaranta Vito , Hong Tian
Published: May 2022

Epithelial-mesenchymal transition (EMT) is a change in cell shape and mobility that occurs during normal development or cancer metastasis. Multiple intermediate EMT states reflecting hybrid epithelial and mesenchymal phenotypes were observed in various physiological and pathological conditions. Previous theoretical models explaining the intermediate EMT states rely on multiple regulatory loops involving transcriptional feedback. These models produce three or four attractors with a given set of rate constants, which is incompatible with experimentally observed non-genetic heterogeneity reflecting a continuum-like EMT spectrum. EMT is regulated by many microRNAs that typically bind transcripts of EMT-related genes via multiple binding sites. It was unclear whether post-transcriptional regulations associated with the microRNA binding sites alone can stabilize intermediate EMT states. Here, we used models describing the post-transcriptional regulations with elementary reaction networks, finding that cooperative RNA degradation via multiple microRNA binding sites can generate four-attractor systems without transcriptional feedback. We identified many specific, experimentally supported instances of network structures predicted to permit intermediate EMT states. Furthermore, transcriptional feedback and the newly identified intermediates-enabling circuits can be combined to produce even more intermediate EMT states in both modular and emergent manners. Finally, multisite-mediated cooperative RNA degradation can increase the distribution of gene expression in the EMT spectrum and support the phenotypic continuum without the need of higher noise. Our work reveals a previously unknown role of cooperative RNA degradation and microRNA in EMT, providing a theoretical framework that can help to bridge the gap between mechanistic models and single-cell experiments.
Identification of cancer drivers from tumor-only RNA-seq with RNA-VACAY
Authors: Akutagawa Jon , Mak Allysia J , Aspden Julie L , Brooks Angela N
Published: May 2022

Detecting somatic mutations is a cornerstone of cancer genomics and clinical genotyping; however, there has been little systematic evaluation of the utility of RNA sequencing (RNA-seq) for somatic variant detection and driver mutation analysis. Variants found in RNA-Seq are also expressed, reducing the identification of passenger mutations and would not suffer from annotation bias observed in whole-exome sequencing (WES). We developed RNA-VACAY, a containerized pipeline that automates somatic variant calling from tumor RNA-seq data, alone, and evaluated its performance on simulated data and 1,349 RNA-seq samples with matched whole-genome sequencing (WGS). RNA-VACAY was able to detect at least 1 putative driver gene in 15 out of 16 cancer types and identified known driver mutations in 5’ and 3’ UTRs. The computational cost and time to generate and analyze RNA-seq data is lower than WGS or WES, which decreases the resources necessary for somatic variant detection. This study demonstrates the utility of RNA-seq to detect cancer drivers.
Analysis and Identification of Necroptosis Landscape on Therapy and Prognosis in Bladder Cancer
Authors: Zhao Zihan , Jiang Ning , Zhang Yulin , Bai Yuhao , Liu Tianyao , Li Tianhang , Guo Hongqian , Yang Rong
Published: May 2022

Bladder cancer (BLCA) is one of the most common malignant tumors of the urinary system, but current therapeutic strategy based on chemotherapy and immune checkpoint inhibitors (ICIs) therapy cannot meet the treatment needs, which mainly owing to the endogenous or acquired apoptotic resistance of cancer cells. Targeting necroptosis provides a novel strategy for chemotherapy, targeted drugs, and improves the efficacy of ICIs because of strong immunogenicity of necroptosis. Therefore, we systemically analyzed necroptosis landscape on therapy and prognosis in BLCA. We firstly divided BLCA patients from The Cancer Genome Atlas (TCGA) database into two necroptosis-related clusters (C1 and C2). Necroptosis C2 showed a significantly better prognosis than C1, and the differential genes of C2 and C1 were mainly related to the immune response according to GO and KEGG analysis. Next, we constructed a novel necroptosis related genes (NRGs) signature consisting of SIRT6, FASN, GNLY, FNDC4, SRC, ANXA1, AIM2, and IKBKB to predict the survival of TCGA-BLCA cohort, the accuracy of NRGsocre was also verified by external datasets. In addition, a nomogram combining NRGscore and several clinicopathological features was established to predict the BLCA patient’s OS more accurately and conveniently. We also found that NRGscore was significantly related to the infiltration levels of CD8 T cells, NK cells, and iDC cells, the gene expression of CTLA4, PD1, TIGIT, and LAG3 of TME, the sensitivity to chemotherapy and targeted agents in BLCA patients. In conclusion, the NRGscore has an excellent performance in evaluating the prognosis, clinicopathologic features, tumor microenvironment (TME) and therapeutic sensitivity of BLCA patients, which could be utilized as a guide for chemotherapy, ICIs therapy, and combination therapy.
Glutamine 666 renders murine ADAM10 an inefficient S. aureus α-toxin receptor
Authors: von Hoven Gisela , Meyenburg Martina , Neukirch Claudia , Siedenschnur Daniel , Husmann Matthias
Published: May 2022

S. aureus is one of the most important causes of infectious diseases in hospitalized individuals and outpatients. The majority of clinical isolates secretes large amounts of the small membrane pore-forming α-toxin, alias α-hemolysin, which serves as an important virulence factor of this organism. The identification of A Disintegrin And Metalloprotease (ADAM10) as its high affinity receptor held great promise for a better understanding of the processes underlying membrane damage by α-toxin. Twelve years on however, the molecular details of initial toxin binding to target cells remain elusive. Because we noted that several murine cell lines were resilient to α-toxin, we considered the possibility that murine ADAM10 could be less efficient a receptor, as compared to human or bovine orthologues. Accordingly, we sought to identify amino acid residues in ADAM10, which could explain species-dependent functionality as receptor for α-toxin. Our work led to the finding that replacement of a single glutamine residue (Q666) in murine ADAM10 with corresponding glutamic acid (E665) of human or bovine ADAM10 enhances significantly the binding and consequent cytotoxicity of α-toxin. Consistently, a synthetic peptide comprising E665 mitigated α-toxin-dependent hemolysis. In multicellular organisms, E665 is highly conserved, but mice and several other members of the taxon glires evolved glutamine at the corresponding position. The residue is located in a short membrane proximal, extracellular region of ADAM10. Taken together, available structural information, in silico docking, and functional data suggests that α-toxin monomers could bind to cellular membranes via this so-called stalk region of ADAM10 and phosphocholine.
Sleep does not influence schema-facilitated motor memory consolidation
Authors: Reverberi S. , Dolfen N. , van Roy A. , Albouy G. , King B.R.
Published: May 2022

Study Objectives

Novel information is rapidly learned when it is compatible with previous knowledge. This “schema” effect, initially described for declarative memories, was recently extended to the motor memory domain. Importantly, this beneficial effect was only observed 24 hours – but not immediately – following motor schema acquisition. Given the established role of sleep in memory consolidation, we hypothesized that sleep following the initial learning of a schema is necessary for the subsequent rapid integration of novel motor information.

Methods

Two experiments were conducted to investigate the effect of diurnal and nocturnal sleep on schema-mediated motor sequence memory consolidation. In Experiment 1, participants first learned an 8-element motor sequence through repeated practice (Session 1). They were then afforded a 90-minute nap opportunity (N=25) or remained awake (N=25) before learning a second motor sequence (Session 2) which was highly compatible with that learned prior to the sleep/wake interval. Experiment 2 was similar; however, Sessions 1 and 2 were separated by a 12-hour interval that included nocturnal sleep (N=28) or only wakefulness (N=29).

Results

For both experiments, we found no group differences in motor sequence performance (reaction time and accuracy) following the sleep/wake interval. Furthermore, in Experiment 1, we found no correlation between sleep features (non-REM sleep duration, spindle and slow wave activity) and post-sleep behavioral performance.

Conclusions

The results of this research suggest that integration of novel motor information into a cognitive-motor schema does not specifically benefit from post-learning sleep.
FAMetA: a mass isotopologue-based tool for the comprehensive analysis of fatty acid metabolism
Authors: Alcoriza-Balaguer María Isabel , García-Cañaveras Juan Carlos , Benet Marta , Vidal Oscar Juan , Lahoz Agustín
Published: May 2022

The use of stable isotope tracers and mass spectrometry (MS) is the gold standard method for the analysis of fatty acids (FAs) metabolism. Yet current state-of-the-art tools provide limited and difficult to interpret information about FA biosynthetic routes. Here we present FAMetA, an R-package and a web-based application (www.fameta.es) that use 13C mass-isotopologue profiles to estimate FA import, de novo lipogenesis, elongation, and desaturation in a user-friendly platform. The FAMetA workflow covers all the functionalities needed for MS data analyses. To illustrate its utility, different in vitro and in vivo experimental settings are used in which FA metabolism is modified. Thanks to the comprehensive characterisation of FA biosynthesis and the easy-to-interpret graphical representations compared to previous tools, FAMetA discloses unnoticed insights into how cells reprogramme their FA metabolism and, when combined with FASN, SCD1 and FADS2 inhibitors, it enables the straightforward identification of new FAs by the metabolic reconstruction of their synthesis route.
Genome assembly and analysis of the flavonoid and phenylpropanoid biosynthetic pathways in Fingerroot ginger (Boesenbergia rotunda)
Authors: Taheri Sima , How Teo Chee , Heslop-Harrison John S. , Schwarzacher Trude , Seong Tan Yew , Yee Wee Wei , Khalid Norzulaani , Biswas Manosh Kumar , Mutha Naresh V R , Mohd-Yusuf Yusmin , Gan Han Ming , Harikrishna Jennifer Ann
Published: May 2022

Boesenbergia rotunda(Zingiberaceae), is a high-value culinary and ethno-medicinal plant of Southeast Asia. The rhizomes of this herb have high flavanone and chalcone content. Here we report genome analysis ofB. rotundatogether with a complete genome sequence as a hybrid assembly.B. rotundahas an estimated genome size of 2.4 Gb which was assembled as 27,491 contigs with N50 size of 12.386 Mb. The highly heterozygous genome encodes 71,072 protein-coding genes and has 72% repeat content, with class I TEs occupying ∼67% of the assembled genome. FluorescenceIn SituHybridization of the 18 chromosome pairs at metaphase showed six sites of 45S rDNA and two sites of 5S rDNA. SSR analysis identified 238,441 gSSRs and 4,604 EST-SSRs with 49 SSR markers common among related species. Genome-wide methylation percentages ranged from 73% CpG, 36% CHG and 34% CHH in leaf to 53% CpG, 18% CHG and 25% CHH in embryogenic callus. Panduratin A biosynthetic unigenes were most highly expressed in watery callus.B rotundahas a relatively large genome with high heterozygosity and TE content. This assembly and data (PRJNA71294) comprise a source for further research on the functional genomics ofB. rotunda, the evolution of the ginger plant family and the potential genetic selection or improvement of gingers.
DNA double strand breaks lead to de novo transcription and translation of damage-induced long RNAs in planta
Authors: Schreiber Tom , Tripathee Sunita , Iwen Thomas , Prange Anja , Vahabi Khabat , Grützner Ramona , Horn Claudia , Marillonnet Sylvestre , Tissier Alain
Published: May 2022

DNA double strand breaks (DSBs) are lethal threats that need to be repaired. Although many of the proteins involved in the early steps of DSB repair have been characterized, recent reports indicate that damage induced long and small RNAs also play an important role in DSB repair. Here, using a Nicotiana benthamiana transgenic line originally designed as a reporter for targeted knock-ins, we show that DSBs generated by Cas9 induce the transcription of long stable RNAs (damage-induced long RNAs - dilRNAs) that are translated into proteins. Using an array of single guide RNAs we show that the initiation of transcription takes place in the vicinity of the DSB. Single strand DNA nicks are not able to induce transcription, showing that cis DNA damage-induced transcription is specific for DSBs. Our results support a model in which a default and early event in the processing of DSBs is transcription into RNA which, depending on the genomic and genic context, can undergo distinct fates, including translation into protein, degradation or production of small RNAs. Our results have general implications for understanding the role of transcription in the repair of DSBs and, reciprocally, reveal DSBs as yet another way to regulate gene expression.
Comprehensive analysis of gene regulatory dynamics, fitness landscape, and population evolution during sexual reproduction
Authors: Okubo Kenji , Kaneko Kunihiko
Published: May 2022

The fitness landscape is a critical concept in evolutionary biology and genetics that depicts fitness in the genotype space and visualizes the relationship between genotype and fitness. However, the fitness landscape is challenging to characterize because the quantitative relationships between genotype and phenotype and their association to fitness has not been comprehensively well described. To address this challenge, we adopted gene regulatory networks to determine gene expression dynamics. We analyzed how phenotype and fitness are shaped by the genotype in two-gene networks. A two-by-two matrix provided the two-gene regulatory network in which a vector with two angle values (Θ) was introduced to characterize the genotype. Mapping from this angle vector to phenotypes allowed for the classification of steady-state expression patterns of genes into seven types. We then studied all possible fitness functions given by the Boolean output from the on/off expression of the two genes. The possible fitness landscapes were obtained as a function of the genetic parameters Θ. Finally, the evolution of the population distribution under sexual reproduction was investigated in the obtained landscape. We found that the distribution was restricted to a convex region within the landscape, resulting in the branching of population distribution, including the speciation process.
A mechanism for the elimination of IgE plasma cells
Authors: Wade-Vallance Adam K. , Yang Zhiyong , Libang Jeremy B. , Robinson Marcus J. , Tarlinton David M. , Allen Christopher D. C.
Published: May 2022

The proper regulation of IgE production safeguards against allergic disease, highlighting the importance of mechanisms that restrict IgE plasma cell (PC) survival. IgE PCs have unusually high surface B cell receptor (BCR) expression, yet the functional consequences of ligating this receptor are unknown. Here, we found that BCR ligation induced BCR signaling in IgE PCs followed by their elimination. In cell culture, exposure of IgE PCs to cognate antigen or anti-BCR antibodies induced apoptosis. IgE PC depletion correlated with the affinity, avidity, amount, and duration of antigen exposure and required the BCR signalosome components Syk, BLNK, and PLCγ2. In mice with a PC-specific impairment of BCR signaling, the abundance of IgE PCs was selectively increased. Conversely, BCR ligation by injection of cognate antigen or anti-IgE depleted IgE PCs. These findings establish a mechanism for the elimination of IgE PCs through BCR ligation. This has important implications for allergen tolerance and immunotherapy as well as anti-IgE monoclonal antibody treatments.
Multi-Omic Integration by Machine Learning (MIMaL) Reveals Protein-Metabolite Connections and New Gene Functions
Authors: Dickinson Quinn , Aufschnaiter Andreas , Ott Martin , Meyer Jesse G.
Published: May 2022

Cells respond to environments by regulating gene expression to exploit resources optimally. Recent advances in technologies allow the ability to gather information of cellular states of its components, measuring abundances of transcripts, their translation, the accumulation of proteins, lipids and metabolites. These highly complex datasets reflect the state of the different layers in a biological system. Multi-omics is the integration of these disparate methods and data to gain a clearer picture of the biological state. Multi-omic studies of the proteome and metabolome are becoming more common as mass spectrometry technology continues to be democratized. However, knowledge extraction through integration of these data remains challenging. Here we show that connections between omic layers can be discovered through a combination of machine learning and model interpretation. We find that model interpretation values connecting proteins to metabolites are valid experimentally and reveal also largely new connections. Further, clustering the magnitudes of protein control over all metabolites enabled prediction of gene five gene functions, each of which was validated experimentally. We accurately predicted that two uncharacterized genes in yeast modulate mitochondrial translation, YJR120W and YLD157C.We also predict and validate functions for several incompletely characterized genes, including SDH9, ISC1, and FMP52. Our work demonstrates that multi-omic analysis with machine learning (MIMaL) views multi-omic data through a new lens to reveal new insight that was not possible using existing methods.