Publications - Publikationer

Neurodevelopmental defects in a mouse model of O-GlcNAc transferase intellectual disability

Mon, 01 Apr 2024 09:14:46 +0200

The addition of O-linked β-N-acetylglucosamine (O-GlcNAc) to proteins (referred to as O-GlcNAcylation) is a modification that is crucial for vertebrate development. O-GlcNAcylation is catalyzed by O-GlcNAc transferase (OGT) and reversed by O-GlcNAcase (OGA). Missense variants of OGT have recently been shown to segregate with an X-linked syndromic form of intellectual disability, OGT-linked congenital disorder of glycosylation (OGT-CDG). Although the existence of OGT-CDG suggests that O-GlcNAcylation is crucial for neurodevelopment and/or cognitive function, the underlying pathophysiologic mechanisms remain unknown. Here we report a mouse line that carries a catalytically impaired OGT-CDG variant. These mice show altered O-GlcNAc homeostasis with decreased global O-GlcNAcylation and reduced levels of OGT and OGA in the brain. Phenotypic characterization of the mice revealed lower body weight associated with reduced body fat mass, short stature and microcephaly. This mouse model will serve as an important tool to study genotype-phenotype correlations in OGT-CDG in vivo and for the development of possible treatment avenues for this disorder.

Error-Corrected Deep Targeted Sequencing of Circulating Cell-Free DNA from Colorectal Cancer Patients for Sensitive Detection of Circulating Tumor DNA

Frydendahl, A., Rasmussen, M. H., Jensen, S. Ø., et al. — Mon, 01 Apr 2024 09:14:46 +0200

Circulating tumor DNA (ctDNA) is a promising biomarker, reflecting the presence of tumor cells. Sequencing-based detection of ctDNA at low tumor fractions is challenging due to the crude error rate of sequencing. To mitigate this challenge, we developed ultra-deep mutation-integrated sequencing (UMIseq), a fixed-panel deep targeted sequencing approach, which is universally applicable to all colorectal cancer (CRC) patients. UMIseq features UMI-mediated error correction, the exclusion of mutations related to clonal hematopoiesis, a panel of normal samples for error modeling, and signal integration from single-nucleotide variations, insertions, deletions, and phased mutations. UMIseq was trained and independently validated on pre-operative (pre-OP) plasma from CRC patients (n = 364) and healthy individuals (n = 61). UMIseq displayed an area under the curve surpassing 0.95 for allele frequencies (AFs) down to 0.05%. In the training cohort, the pre-OP detection rate reached 80% at 95% specificity, while it was 70% in the validation cohort. UMIseq enabled the detection of AFs down to 0.004%. To assess the potential for detection of residual disease, 26 post-operative plasma samples from stage III CRC patients were analyzed. From this we found that the detection of ctDNA was associated with recurrence. In conclusion, UMIseq demonstrated robust performance with high sensitivity and specificity, enabling the detection of ctDNA at low allele frequencies.

Identificación de genes clave implicados en el síndrome de Down mediante terapia génica

Fillat, C., Bofill-De Ros, X., Santos, M., et al. — Fri, 01 Aug 2014 09:14:46 +0200

Viruses have evolved ways of encapsulating and delivering their genes into human cells. Gene therapy takes advantage of this capability to manipulate the viral genome and convert an infectious agent into an effcient vector that delivers therapeutic genes. In the current work we have applied gene therapy approaches based on adeno-associated virus and lentivirus delivery to identify candidate genes (protein-coding or miRNAs) involved in the cognitive defcits in Down Syndrome. We show that the hippocampal injection of the adeno-associated virus AAV2/1-shDyrk1A normalized Dyrk1A expression in the trisomic Ts65Dn mice. As a consequence the regulation of key molecular players in memory and learning processes was rescued and mice showed an attenuation of synaptic plasticity defects and improved effcacy in learning strategies. All together these results reinforce the role of Dyrk1A in cognition. On the other hand, with the lentiviral strategydeveloped to specifcally inhibit miR-155 and miR-802 (Lv-anti-miR155/802), we were able to show a tight control of the miRNAs target Mecp2 suggesting that the downregulation of MeCP2 in Down syndromecould be a contributing factor to the cognitive defects.

Controlling adenoviral replication to induce oncolytic efficacy

Fri, 01 Jan 2010 09:14:46 +0100

Over the last decade, cancer therapy has found itself challenged by the growing field of oncolytic virotherapy.Many different viruses are currently under study, investigating their potential to induce antitumor effects through repeated cycles of viral infection and cell lysis. It was, however, genetically-engineered replication-selective adenoviruses that were the first to enter clinical trials with cancer patients. The difficulties involved in combining selectivity and elevated potency in a single oncolytic adenovirus have led investigators to design and test many different approaches. Different strategies, based on the control of viral replication, are presented in the current review. We discuss how the growing knowledge of cell and tumour biology, with the advances made in adenoviral virology, has inspired the fine-tuning of genetically-engineered adenoviruses Special emphasis is placed on the fundamentals behind the use of certain specific genetic elements, introduced into the viral genome to control viral gene expression and on describing the most important viral gene mutations.

IsomiRs

Bofill-De Ros, X., Yang, A., Gu, S. — Wed, 01 Apr 2020 09:14:46 +0200

MicroRNAs (miRNAs) are a class of small non-coding RNAs that play increasingly appreciated roles in gene regulation. In animals, miRNAs silence gene expression by binding to partially complementary sequences within target mRNAs. It is well-established that miRNAs recognize canonical target sites by base-pairing in the 5′region. However, the development of biochemical methods has identified many novel, non-canonical target sites, suggesting additional modes of miRNA-target association. Here, we review the current knowledge of miRNA-target recognition and how new evidence supports or challenges existing models. We also review the process by which microRNA isoforms achieve functional diversification via modulation of target recognition.

Genome-wide miR-155 and miR-802 target gene identification in the hippocampus of Ts65Dn Down syndrome mouse model by miRNA sponges

Bofill-De Ros, X., Santos, M., Vila-Casadesús, M., et al. — Fri, 06 Nov 2015 09:14:46 +0100

Background: Down syndrome (DS) or trisomy 21 is the result of a genetic dosage imbalance that translates in a broad clinical spectrum. A major challenge in the study of DS is the identification of functional genetic elements with wide impact on phenotypic alterations. Recently, miRNAs have been recognized as major contributors to several disease conditions by acting as post-transcriptional regulators of a plethora of genes. Five chromosome 21 (HSA21) miRNAs have been found overexpressed in DS individuals and could function as key elements in the pathophysiology. Interestingly, in the trisomic Ts65Dn DS mouse model two of these miRNAs (miR-155 and miR-802) are also triplicated and overexpressed in brain. Results: In the current work, we interrogated the impact of miR-155 and miR-802 upregulation on the transcriptome of Ts65Dn brains. We developed a lentiviral miRNA-sponge strategy (Lv-miR155-802T) to identify in vivo relevant miR-155 and miR-802 target mRNAs. Hippocampal injections of lentiviral sponges in Ts65Dn mice normalized the expression of miR-155 and miR-802 and rescued the levels of their targets methyl-CpG-binding protein 2 gene (Mecp2), SH2 (Src homology 2)-containing inositol phosphatase-1 (Ship1) and Forkhead box protein M1 (FoxM1). Transcriptomic data of Lv-miR155-802T miRNA-sponge treated hippocampi correlated with candidate targets highlighting miRNA dosage-sensitive genes. Significant associations were found in a subset of genes (Rufy2, Nova1, Nav1, Thoc1 and Sumo3) that could be experimentally validated. Conclusions: The lentiviral miRNA-sponge strategy demonstrated the genome-wide regulatory effects of miR-155 and miR-802. Furthermore, the analysis combining predicted candidates and experimental transcriptomic data proved to retrieve genes with potential significance in DS-hippocampal phenotype bridging with DS other neurological-associated diseases such as Alzheimer's disease.

Flexible pri-miRNA structures enable tunable production of 5’ isomiRs

Bofill-De Ros, X., Hong, Z., Birkenfeld, B., et al. — Sat, 01 Jan 2022 09:14:46 +0100

The Drosha cleavage of a pri-miRNA defines mature microRNA sequence. Drosha cleavage at alternative positions generates 5’ isoforms (isomiRs) which have distinctive functions. To understand how pri-miRNA structures influence Drosha cleavage, we performed a systematic analysis of the maturation of endogenous pri-miRNAs and their variants both in vitro and in vivo. We show that in addition to previously known features, the overall structural flexibility of pri-miRNA impact Drosha cleavage fidelity. Internal loops and nearby G · U wobble pairs on the pri-miRNA stem induce the use of non-canonical cleavage sites by Drosha, resulting in 5’ isomiR production. By analysing patient data deposited in the Cancer Genome Atlas, we provide evidence that alternative Drosha cleavage of pri-miRNAs is a tunable process that responds to the level of pri-miRNA-associated RNA-binding proteins. Together, our findings reveal that Drosha cleavage fidelity can be modulated by altering pri-miRNA structure, a potential mechanism underlying 5’ isomiR biogenesis in tumours. (Figure presented.).

Late-phase miRNA-controlled oncolytic adenovirus for selective killing of cancer cells

Bofill-De Ros, X., Villanueva, E., Fillat, C. — Thu, 01 Jan 2015 09:14:46 +0100

Tissue-specific detargeting by miRNAs has been demonstrated to be a potent strategy to restrict adenoviral replication to cancer cells. These studies have generated adenoviruses with miRNA target sites placed in the 3'UTR of early gene products. In this work, we have studied the feasibility of providing tissue-specific selectivity to replication-competent adenoviruses through the regulation of the late structural protein fiber (L5 gene). We have engineered a 3'UTR containing eight miR-148a binding sites downstream the L5 coding sequence (Ad-L5-8miR148aT). We present in vitro and in vivo evidences of Ad-L5-8miR148aT miRNA-dependent regulation. In vitro data show that at 72 hours post-infection miR-148a-regulation impaired fiber expression leading to a 70% reduction of viral release. The application of seven consecutive rounds of infection in miR-148a cells resulted in 10.000-fold reduction of viral genomes released. In vivo, liver production of infective viral particles was highly impaired, similarly to that triggered by an adenovirus with miRNA target sites regulating the early E1A gene. Noticeably, mice treated with Ad-L5-8miR148aT showed an attenuation of adenoviral-induced hepatotoxicity but retained full lytic activity in cancer cells and exhibited robust antitumoral responses in patient-derived xenografts. Thus, miRNA-control of late proteins constitutes a novel strategy to provide selectivity to adenoviruses.

RNA targeting using CasRx can prevent aminoglycoside-induced hearing loss

Bofill-De Ros, X. — Tue, 14 Jun 2022 09:14:46 +0200

AGO-bound mature miRNAs are oligouridylated by TUTs and subsequently degraded by DIS3L2

Yang, A., Shao, T. J., Bofill-De Ros, X., et al. — Tue, 01 Dec 2020 09:14:46 +0100

MicroRNAs (miRNAs) associated with Argonaute proteins (AGOs) regulate gene expression in mammals. miRNA 3’ ends are subject to frequent sequence modifications, which have been proposed to affect miRNA stability. However, the underlying mechanism is not well understood. Here, by genetic and biochemical studies as well as deep sequencing analyses, we find that AGO mutations disrupting miRNA 3’ binding are sufficient to trigger extensive miRNA 3’ modifications in HEK293T cells and in cancer patients. Comparing these modifications in TUT4, TUT7 and DIS3L2 knockout cells, we find that TUT7 is more robust than TUT4 in oligouridylating mature miRNAs, which in turn leads to their degradation by the DIS3L2 exonuclease. Our findings indicate a decay machinery removing AGO-associated miRNAs with an exposed 3’ end. A set of endogenous miRNAs including miR-7, miR-222 and miR-769 are targeted by this machinery presumably due to target-directed miRNA degradation.

Guidelines for the optimal design of miRNA-based shRNAs

Bofill-De Ros, X., Gu, S. — Fri, 01 Jul 2016 09:14:46 +0200

RNA interference (RNAi) is an extremely useful tool for inhibiting gene expression. It can be triggered by transfected synthetic small interfering RNA (siRNA) or by expressed small hairpin RNA (shRNA). The cellular machinery processes the latter into siRNA in vivo. shRNA is preferred or required in genetic screens and specific RNAi approaches in gene therapy settings. Despite its many successes, the field of shRNAs faces many challenges. Insufficient knockdowns and off-target effects become obstacles for shRNA usage in many applications. Numerous failures are triggered by pitfalls in shRNA design that is often associated with impoverished biogenesis. Here, based on current understanding of the miRNA maturation pathway, we discuss the principles of different shRNA design (pre-miRNA-like, pri-miRNA-like and Ago-shRNA) with an emphasis on the RNA structure. We also provide detailed instructions for an optimal design of pre-miRNA-like shRNA.

Tumor IsomiR Encyclopedia (TIE)

Bofill-De Ros, X., Luke, B., Guthridge, R., Mudunuri, U., Loss, M., Gu, S. — Wed, 15 Sep 2021 09:14:46 +0200

MicroRNAs (miRNAs) are master regulators of gene expression in cancers. Their sequence variants or isoforms (isomiRs) are highly abundant and possess unique functions. Given their short sequence length and high heterogeneity, mapping isomiRs can be challenging; without adequate depth and data aggregation, low frequency events are often disregarded. To address these challenges, we present the Tumor IsomiR Encyclopedia (TIE): a dynamic database of isomiRs from over 10 000 adult and pediatric tumor samples in The Cancer Genome Atlas (TCGA) and The Therapeutically Applicable Research to Generate Effective Treatments (TARGET) projects. A key novelty of TIE is its ability to annotate heterogeneous isomiR sequences and aggregate the variants obtained across all datasets. Results can be browsed online or downloaded as spreadsheets. Here, we show analysis of isomiRs of miR-21 and miR-30a to demonstrate the utility of TIE.

Restoration of microRNA metabolism trigger robust antitumor responses

Bofill-De Ros, X. — Tue, 13 Sep 2022 09:14:46 +0200

Deconvoluting the Effect of Cell-Penetrating Peptides for Enhanced and Controlled Insertion of Large-Scale DNA Nanopores

Zhang, X., Malle, M. G., Thomsen, R. P., et al. — Wed, 17 Apr 2024 09:14:46 +0200

DNA nanopores have emerged as powerful tools for molecular sensing, but the efficient insertion of large DNA nanopores into lipid membranes remains challenging. In this study, we investigate the potential of cell-penetrating peptides (CPPs), specifically SynB1 and GALA, to enhance the insertion efficiency of large DNA nanopores. We constructed SynB1- or GALA-functionalized DNA nanopores with an 11 nm inner diameter and visualized and quantified their membrane insertion using a TIRF microscopy-based single-liposome assay. The results demonstrated that incorporating an increasing number of SynB1 or GALA peptides into the DNA nanopore significantly enhanced the membrane perforation. Kinetic analysis revealed that the DNA nanopore scaffold played a role in prearranging the CPPs, which facilitated membrane interaction and pore formation. Notably, the use of pH-responsive GALA peptides allowed highly efficient and pH-controlled insertion of large DNA pores. Furthermore, single-channel recording elucidated that the insertion process of single GALA-modified nanopores into planar lipid bilayers was dynamic, likely forming transient large toroidal pores. Overall, our study highlights the potential of CPPs as insertion enhancers for DNA nanopores, which opens avenues for improved molecule sensing and the controlled release of cargo molecules.

3′ Uridylation Confers miRNAs with Non-canonical Target Repertoires

Yang, A., Bofill-De Ros, X., Shao, T. J., et al. — Thu, 08 Aug 2019 09:14:46 +0200

Many microRNAs (miRNAs) exist alongside abundant miRNA isoforms (isomiRs), most of which arise from post-maturation sequence modifications such as 3′ uridylation. However, the ways in which these sequence modifications affect miRNA function remain poorly understood. Here, using human miR-27a in cell lines as a model, we discovered that a nonfunctional target site unable to base-pair extensively with the miRNA seed sequence can regain function when an upstream adenosine is able to base-pair with a post-transcriptionally added uridine in the miR-27a tail. This tail-U-mediated repression (TUMR) is abolished in cells lacking the uridylation enzymes TUT4 and TUT7, indicating that uridylation alters miRNA function by modulating target recognition. We identified a set of non-canonical targets in human cells that are specifically regulated by uridylated miR-27a. We provide evidence that TUMR expands the targets of other endogenous miRNAs. Our study reveals a function of uridylated isomiRs in regulating non-canonical miRNA targets. Yang et al. demonstrate that mRNAs lacking a seed pairing are repressed by miR-27a because of base-pairing between an upstream adenosine in the target and a non-templated U tail in miR-27a. They identify a large class of non-canonical targets regulated by uridylated miRNAs and reveal a novel function of 3′ isomiRs.

Stress-induced microrna-708 impairs b-cell function and growth

Rodríguez-Comas, J., Moreno-Asso, A., Moreno-Vedia, J., et al. — Fri, 01 Dec 2017 09:14:46 +0100

The pancreatic β-cell transcriptome is highly sensitive to external signals such as glucose oscillations and stress cues. MicroRNAs (miRNAs) have emerged as key factors in gene expression regulation. Here, we aimed to identify miRNAs that are modulated by glucose in mouse pancreatic islets.We identified miR-708 as the most upregulatedmiRNA in islets cultured at low glucose concentrations, a setting that triggers a strong stress response.miR-708 was also potently upregulated by triggering endoplasmic reticulum (ER) stress with thapsigargin and in islets of ob/ob mice. Low-glucose induction of miR-708 was blocked by treatment with the chemical chaperone 4-phenylbutyrate, uncovering the involvement of ER stress in this response. An integrative analysis identified neuronatin (Nnat) as a potential glucoseregulated target of miR-708. Indeed, Nnat expression was inversely correlated with miR-708 in islets cultured at different glucose concentrations and in ob/ob mouse islets and was reduced after miR-708 overexpression. Consistent with the role of Nnat in the secretory function of β-cells, miR-708 overexpression impaired glucosestimulated insulin secretion (GSIS), which was recovered by NNAT overexpression. Moreover, miR-708 inhibition recovered GSIS in islets cultured at low glucose. Finally, miR-708 overexpression suppressed β-cell proliferation and induced β-cell apoptosis. Collectively, our results provide a novel mechanism of glucose regulation of β-cell function and growth by repressing stress-induced miR-708.

Structural Differences between Pri-miRNA Paralogs Promote Alternative Drosha Cleavage and Expand Target Repertoires

Bofill-De Ros, X., Kasprzak, W. K., Bhandari, Y., et al. — Tue, 08 Jan 2019 09:14:46 +0100

By studying the processing of pri-miR-9 family, Bofill-De Ros et al. demonstrate that the tertiary structure of pri-miRNA triggers alternative biogenesis. Drosha cleaves pri-miR-9-1 at an additional site because of its distorted and flexible lower stem, generating a 5′ isomiR that regulates a distinct set of genes in low-grade glioma.

TENT2, TUT4, and TUT7 selectively regulate miRNA sequence and abundance

Yang, A., Bofill-De Ros, X., Stanton, R., Shao, T. J., Villanueva, P., Gu, S. — Thu, 01 Dec 2022 09:14:46 +0100

TENTs generate miRNA isoforms by 3’ tailing. However, little is known about how tailing regulates miRNA function. Here, we generate isogenic HEK293T cell lines in which TENT2, TUT4 and TUT7 are knocked out individually or in combination. Together with rescue experiments, we characterize TENT-specific effects by deep sequencing, Northern blot and in vitro assays. We find that 3’ tailing is not random but highly specific. In addition to its known adenylation, TENT2 contributes to guanylation and uridylation on mature miRNAs. TUT4 uridylates most miRNAs whereas TUT7 is dispensable. Removing adenylation has a marginal impact on miRNA levels. By contrast, abolishing uridylation leads to dysregulation of a set of miRNAs. Besides let-7, miR-181b and miR-222 are negatively regulated by TUT4/7 via distinct mechanisms while the miR-888 cluster is upregulated specifically by TUT7. Our results uncover the selective actions of TENTs in generating 3’ isomiRs and pave the way to investigate their functions.

Novel, abundant Drosha isoforms are deficient in miRNA processing in cancer cells

Dai, L., Hallmark, L., Bofill De Ros, X., et al. — Sun, 01 Nov 2020 09:14:46 +0100

MicroRNAs (miRNAs) are a class of small noncoding RNAs about 22-nucleotide (nt) in length that collectively regulate more than 60% of coding genes. Aberrant miRNA expression is associated with numerous diseases, including cancer. miRNA biogenesis is licenced by the ribonuclease (RNase) III enzyme Drosha, the regulation of which is critical in determining miRNA levels. We and others have previously revealed that alternative splicing regulates the subcellular localization of Drosha. To further investigate the alternative splicing landscape of Drosha transcripts, we performed PacBio sequencing in different human cell lines. We identified two novel isoforms resulting from partial intron-retention in the region encoding the Drosha catalytic domain. One isoform (AS27a) generates a truncated protein that is unstable in cells. The other (AS32a) produces a full-length Drosha with a 14 amino acid insertion in the RIIID domain. By taking advantage of Drosha knockout cells in combination with a previously established reporter assay, we demonstrated that Drosha-AS32a lacks cleavage activity. Furthermore, neither Drosha-27a nor Drosha-32a were able to rescue miRNA expression in the Drosha knockout cells. Interestingly, both isoforms were abundantly detected in a wide range of cancer cell lines (up to 15% of all Drosha isoforms). Analysis of the RNA-seq data from over 1000 breast cancer patient samples revealed that the AS32a is relatively more abundant in tumours than in normal tissue, suggesting that AS32a may play a role in cancer development.

QuagmiR

Bofill-De Ros, X., Chen, K., Chen, S., et al. — Wed, 01 May 2019 09:14:46 +0200

MicroRNAs (miRNAs) function as master regulators of gene expression. Recent studies demonstrate that miRNA isoforms (isomiRs) play a unique role in cancer development. Here, we present QuagmiR, the first cloud-based tool to analyze isomiRs from next generation sequencing data. Using a novel and flexible searching algorithm designed for the detection and annotation of heterogeneous isomiRs, it permits extensive customization of the query process and reference databases to meet the user 's diverse research needs.

Specifications of the ACMG/AMP Variant Classification Guidelines for Germline DICER1 Variant Curation

Hatton, J. N., Frone, M. N., Cox, H. C., et al. — Sun, 01 Jan 2023 09:14:46 +0100

Germline pathogenic variants in DICER1 predispose individuals to develop a variety of benign and malignant tumors. Accurate variant curation and classification are essential for reliable diagnosis of DICER1-related tumor predisposition and the identification of individuals who may benefit from surveillance. Since 2015, most labs have followed the American College of Medical Genetics and Genomics and the Association for Molecular Pathology (ACMG/AMP) sequence variant classification guidelines for DICER1 germline variant curation. However, these general guidelines lack gene-specific nuances and leave room for subjectivity. Consequently, a group of DICER1 experts joined ClinGen to form the DICER1 and miRNA-Processing Genes Variant Curation Expert Panel (VCEP) to create DICER1-specific ACMG/AMP guidelines for germline variant curation. The VCEP followed the FDA-approved ClinGen protocol for adapting and piloting these guidelines. A diverse set of 40 DICER1 variants were selected for piloting, including 14 known pathogenic/likely pathogenic (P/LP) variants, 12 known benign/likely benign (B/LB) variants, and 14 variants classified as variants of uncertain significance (VUS) or with conflicting interpretations in ClinVar. Clinically meaningful classifications (i.e., P, LP, LB, or B) were achieved for 82.5% (33/40) of the pilot variants, with 100% concordance among the known P/LP and known B/LB variants. Half of the VUS or conflicting variants were resolved with four variants classified as LB and three as LP. These results demonstrate that the DICER1-specific guidelines for germline variant curation effectively classify known pathogenic and benign variants while reducing the frequency of uncertain classifications. Individuals and labs curating DICER1 variants should consider adopting this classification framework to encourage consistency and improve objectivity.

Recent progress in miRNA biogenesis and decay

Bofill-De Ros, X., Vang Ørom, U. A. — Mon, 01 Jan 2024 09:14:46 +0100

MicroRNAs are a class of small regulatory RNAs that mediate regulation of protein synthesis by recognizing sequence elements in mRNAs. MicroRNAs are processed through a series of steps starting from transcription and primary processing in the nucleus to precursor processing and mature function in the cytoplasm. It is also in the cytoplasm where levels of mature microRNAs can be modulated through decay mechanisms. Here, we review the recent progress in the lifetime of a microRNA at all steps required for maintaining their homoeostasis. The increasing knowledge about microRNA regulation upholds great promise as therapeutic targets.

Olfactory Epithelium Stimulation Using Rhythmic Nasal Air-Puffs Improves the Cognitive Performance of Individuals with Acute Sleep Deprivation

Riazi, H., Nazari, M., Raoufy, M. R., Mirnajafi-Zadeh, J., Shojaei, A. — Mon, 01 Apr 2024 09:14:46 +0200

This study aimed to investigate the effects of intranasal air-puffing on cognitive impairments and brain cortical activity following one night of partial sleep deprivation (PSD) in adults. A total of 26 healthy adults underwent the numerical Stroop test (NST) and electroencephalography (EEG) before and after one night of PSD. Following PSD, subjects in the treatment group (n = 13) received nasal air-puffs (5 Hz, 3 min) before beginning the NST and EEG recording. Administration of nasal air-puffs in the treatment group restored the PSD-induced increase in error rate and decrease in reaction time and missing rate in the NST. Intranasal air-puffs recovered the PSD-induced augmentation of delta and theta power and the reduction of beta and gamma power in the EEG, particularly in the frontal lobes. Intranasal air-puffing also almost reversed the PSD-induced decrease in EEG signal complexity. Furthermore, it had a restorative effect on PSD-induced alteration in intra-default mode network functional connectivity in the beta and gamma frequency bands. Rhythmic nasal air-puffing can mitigate acute PSD-induced impairments in cognitive functions. It exerts part of its ameliorating effect by restoring neuronal activity in cortical brain areas involved in cognitive processing.

Conformational changes in the Niemann-Pick type C1 protein NCR1 drive sterol translocation

Frain, K. M., Dedic, E., Nel, L., et al. — Mon, 01 Apr 2024 09:14:46 +0200

The membrane protein Niemann-Pick type C1 (NPC1, named NCR1 in yeast) is central to sterol homeostasis in eukaryotes. Saccharomyces cerevisiae NCR1 is localized to the vacuolar membrane, where it is suggested to carry sterols across the protective glycocalyx and deposit them into the vacuolar membrane. However, documentation of a vacuolar glycocalyx in fungi is lacking, and the mechanism for sterol translocation has remained unclear. Here, we provide evidence supporting the presence of a glycocalyx in isolated S. cerevisiae vacuoles and report four cryo-EM structures of NCR1 in two distinct conformations, named tense and relaxed. These two conformations illustrate the movement of sterols through a tunnel formed by the luminal domains, thus bypassing the barrier presented by the glycocalyx. Based on these structures and on comparison with other members of the Resistance-Nodulation-Division (RND) superfamily, we propose a transport model that links changes in the luminal domains with a cycle of protonation and deprotonation within the transmembrane region of the protein. Our model suggests that NPC proteins work by a generalized RND mechanism where the proton motive force drives conformational changes in the transmembrane domains that are allosterically coupled to luminal/extracellular domains to promote sterol transport.

A Targetable N-Terminal Motif Orchestrates α-Synuclein Oligomer-to-Fibril Conversion

Santos, J., Cuellar, J., Pallarès, I., et al. — Wed, 01 May 2024 09:14:46 +0200

Oligomeric species populated during α-synuclein aggregation are considered key drivers of neurodegeneration in Parkinson's disease. However, the development of oligomer-targeting therapeutics is constrained by our limited knowledge of their structure and the molecular determinants driving their conversion to fibrils. Phenol-soluble modulin α3 (PSMα3) is a nanomolar peptide binder of α-synuclein oligomers that inhibits aggregation by blocking oligomer-to-fibril conversion. Here, we investigate the binding of PSMα3 to α-synuclein oligomers to discover the mechanistic basis of this protective activity. We find that PSMα3 selectively targets an α-synuclein N-terminal motif (residues 36-61) that populates a distinct conformation in the mono- and oligomeric states. This α-synuclein region plays a pivotal role in oligomer-to-fibril conversion as its absence renders the central NAC domain insufficient to prompt this structural transition. The hereditary mutation G51D, associated with early onset Parkinson's disease, causes a conformational fluctuation in this region, leading to delayed oligomer-to-fibril conversion and an accumulation of oligomers that are resistant to remodeling by molecular chaperones. Overall, our findings unveil a new targetable region in α-synuclein oligomers, advance our comprehension of oligomer-to-amyloid fibril conversion, and reveal a new facet of α-synuclein pathogenic mutations.

Effects of milk extracellular vesicles from bovine, human, and caprine origin on rotavirus infectivity in cultured human intestinal cells

Vangsøe, C., Graikini, D., Bojsen, A., Sánchez, L., Trige Rasmussen, J. — Mon, 01 Jul 2024 09:14:46 +0200

Rotavirus is the leading cause of severe gastroenteritis in neonates representing a global health burden. Milk-derived extracellular vesicles [EVs] have emerged as potential antivirals, offering a promising avenue for intervention. The present study evaluated the antiviral activity of EVs sourced from bovine, human, and caprine milk against the infection of the human Wa and bovine WC3 rotavirus strains. EVs induced an endogenous effect in Caco-2 cells, resulting in a significant reduction of rotaviral infection. The antiviral properties of the milk EVs, were found to be associated with synergistic interactions at pre- and post-attachment stages of the infection. Finally, the effect of milk processing on bovine milk EVs efficacy was assessed. It was found that after high-temperature short-time pasteurization EVs retained their activity, while homogenization resulted in no antiviral effect at all. Overall, our findings demonstrate a significant reduction in rotavirus infection in cultured Caco-2 cells when treated with milk EVs.

Single-Molecule-Resolution Approaches in Synaptic Biology

Sun, C. — Thu, 04 Apr 2024 09:14:46 +0200

Synapses between neurons are the primary loci for information transfer and storage in the brain. An individual neuron, alone, can make over 10000 synaptic contacts. It is, however, not easy to investigate what goes on locally within a synapse because many synaptic compartments are only a few hundred nanometers wide in size─close to the diffraction limit of light. To observe the biomolecular machinery and processes within synapses, in situ single-molecule techniques are emerging as powerful tools. Guided by important biological questions, this Perspective will highlight recent advances in using these techniques to obtain in situ measurements of synaptic molecules in three aspects: the cell-biological machinery within synapses, the synaptic architecture, and the synaptic neurotransmitter receptors. These advances showcase the increasing importance of single-molecule-resolution techniques for accessing subcellular biophysical and biomolecular information related to the brain.

N4-acetylcytidine (ac4C) promotes mRNA localization to stress granules

Kudrin, P., Singh, A., Meierhofer, D., Kuśnierczyk, A., Ørom, U. A. V. — Mon, 01 Apr 2024 09:14:46 +0200

Stress granules are an integral part of the stress response that are formed from non-translating mRNAs aggregated with proteins. While much is known about stress granules, the factors that drive their mRNA localization are incompletely described. Modification of mRNA can alter the properties of the nucleobases and affect processes such as translation, splicing and localization of individual transcripts. Here, we show that the RNA modification N4-acetylcytidine (ac4C) on mRNA associates with transcripts enriched in stress granules and that stress granule localized transcripts with ac4C are specifically translationally regulated. We also show that ac4C on mRNA can mediate localization of the protein NOP58 to stress granules. Our results suggest that acetylation of mRNA regulates localization of both stress-sensitive transcripts and RNA-binding proteins to stress granules and adds to our understanding of the molecular mechanisms responsible for stress granule formation.

IDC 2014 chairs' welcome

Iversen, O. S., Thomsen, B. S., Elbæk, L. — Wed, 01 Jan 2014 09:14:46 +0100

The exploration of milk fat crystallization in milk fat globules by confocal Raman microscopy

Mon, 01 Apr 2024 09:14:46 +0200

Crystallization behavior within oil-in-water emulsion is a key factor for the properties and stability of many food products. Confocal Raman microscopy is a promising method to study such complex structures in situ. This study aimed at evaluating the feasibility of confocal Raman microscopy for visualizing milk fat crystallization and establishing a reliable data acquisition and processing methodology. Milk fat globules from raw milk were fixated in an agarose gel and crystallized at different temperatures. Confocal Raman microscopy was applied to collect two-dimensional area scans and supporting images were obtained by polarized light microscopy. The results revealed differences in lipid characteristics, crystal formation, and spatial distribution as a result of crystallization. Specific C-C stretching vibrations at 1063, 1083, and 1125 cm-1 were found to indicate lipid chain mobility and provide quantitative information on crystallinity. Additionally, the study successfully identified the triple-layered milk fat globule membrane. Different approaches for processing spectroscopic data were compared, emphasizing the importance of proper data handling. This novel spectroscopy imaging approach has significant potential in enhancing our understanding of structural heterogeneities of crystallized structures within complex food matrices.

Humanization of a mouse anti-human complement C6 monoclonal antibody as a potential therapeutic for certain complement-mediated diseases

Zhang, L., Armour, K., Chen, J. Y., et al. — Sat, 01 Jun 2024 09:14:46 +0200

The assembly of tissue-damaging membrane attack complexes (MACs; C5b–9) is a major mechanism by which excessive complement activation causes diseases. We previously developed a mouse anti-human C6 monoclonal antibody (mAb) 1C9 that selectively inhibits the assembly of MACs in human and non-human primates. In this project, we found that 1C9 also cross-reacted with rat and guinea pig C6, and determined its binding domains on C6 using different truncated C6 proteins. We then humanized the anti-C6 mAb by molecular modeling and complementarity-determining region grafting. After screening a library of 276 humanized variants with different combinations of humanized light and heavy chains in biophysical assays, we identified clone 3713 with the best developability profile, and an increased affinity against C6 when compared with the parental 1C9 mAb. This humanized 3713 mAb inhibited human, monkey, and rat complement-mediated hemolysis in vitro, and more importantly, it significantly reduced complement-mediated hemolysis in vivo in rats. These results demonstrated the successful humanization of the anti-C6 mAb and suggested that the humanized 3713 mAb could be further developed as a new therapeutic that selectively targets MAC for certain complement-mediated pathological conditions.

Small-molecule positive allosteric modulation of homomeric kainate receptors GluK1-3

Bay, Y., Venskutonytė, R., Frantsen, S. M., et al. — Mon, 01 Apr 2024 09:14:46 +0200

The kainate receptors GluK1-3 (glutamate receptor ionotropic, kainate receptors 1–3) belong to the family of ionotropic glutamate receptors and are essential for fast excitatory neurotransmission in the brain, and are associated with neurological and psychiatric diseases. How these receptors can be modulated by small-molecule agents is not well understood, especially for GluK3. We show that the positive allosteric modulator BPAM344 can be used to establish robust calcium-sensitive fluorescence-based assays to test agonists, antagonists, and positive allosteric modulators of GluK1-3. The half-maximal effective concentration (EC₅₀) of BPAM344 for potentiating the response of 100 μm kainate was determined to be 26.3 μm for GluK1, 75.4 μm for GluK2, and 639 μm for GluK3. Domoate was found to be a potent agonist for GluK1 and GluK2, with an EC₅₀ of 0.77 and 1.33 μm, respectively, upon co-application of 150 μm BPAM344. At GluK3, domoate acts as a very weak agonist or antagonist with a half-maximal inhibitory concentration (IC₅₀) of 14.5 μm, in presence of 500 μm BPAM344 and 100 μm kainate for competition binding. Using H523A-mutated GluK3, we determined the first dimeric structure of the ligand-binding domain by X-ray crystallography, allowing location of BPAM344, as well as zinc-, sodium-, and chloride-ion binding sites at the dimer interface. Molecular dynamics simulations support the stability of the ion sites as well as the involvement of Asp761, Asp790, and Glu797 in the binding of zinc ions. Using electron microscopy, we show that, in presence of glutamate and BPAM344, full-length GluK3 adopts a dimer-of-dimers arrangement.

Observing A Protein In Its Natural Habitat

Sun, C., Nissen, P. — Mon, 01 Apr 2024 09:14:46 +0200

Much of our knowledge about proteins inside biological cells comes from ex situ methods, where cellular proteins are extracted from their native environment. This approach has yielded basic information on each protein's structure and function averaged across distinct subcellular compartments, cell states, and, sometimes, cell types. However, the functional demands from different (sub)cellular environments vary. To probe the structural and functional adaptation of proteins in distinct subcellular environments, in situ single-protein characterization has emerged as an important approach. Here we comment on emerging in situ approaches by light and electron microscopy that begin to raise new questions about the distribution, structure, and function of individual protein copies in specific subcellular environments. The advent of in situ single-protein characterization is a key frontier of spatial biology to elucidate the architecture of a single cell.

Staying hungry

Qiao, L., Lin, J., Suzaki, T., Liang, P. — Fri, 01 Mar 2024 09:14:46 +0100

Legumes have evolved specific inventions to enhance nitrogen (N) acquisition by establishing symbiotic interactions with N-fixing rhizobial bacteria. Because symbiotic N fixation is energetically costly, legumes have developed sophisticated mechanisms to ensure carbon–nitrogen balance, in a variable environment, both locally and at the whole plant level, by monitoring nodule number, nodule development, and nodular nitrogenase activity, as well as controlling nodule senescence. Studies of the autoregulation of nodulation and regulation of nodulation by nodule inception (NIN) and NIN-LIKE PROTEINs (NLPs) have provided great insights into the genetic mechanisms underlying the nitrate-induced regulation of root nodulation for adapting to N availability in the rhizosphere. However, many aspects of N-induced pleiotropic regulation remain to be fully explained, such as N-triggered senescence in mature nodules. Wang et al. determined that this process is governed by a transcriptional network regulated by NAC-type transcription factors. Characterization and dissection of these soybean nitrogen-associated NAPs (SNAPs) transcription factor-mastered networks have yielded a roadmap for exploring how legumes rewire nodule functions across a range of N levels, laying the foundation for enhancing the growth of N-deprived crops in agricultural settings.

Uncovering structural themes across cilia microtubule inner proteins with implications for human cilia function

Fri, 01 Mar 2024 09:14:46 +0100

Centrosomes and cilia are microtubule-based superstructures vital for cell division, signaling, and motility. The once thought hollow lumen of their microtubule core structures was recently found to hold a rich meshwork of microtubule inner proteins (MIPs). To address the outstanding question of how distinct MIPs evolved to recognize microtubule inner surfaces, we applied computational sequence analyses, structure predictions, and experimental validation to uncover evolutionarily conserved microtubule- and MIP-binding modules named NWE, SNYG, and ELLEn, and PYG and GFG-repeat by their signature motifs. These modules intermix with MT-binding DM10-modules and Mn-repeats in 24 Chlamydomonas and 33 human proteins. The modules molecular characteristics provided keys to identify elusive cross-species homologs, hitherto unknown human MIP candidates, and functional properties for seven protein subfamilies, including the microtubule seam-binding NWE and ELLEn families. Our work defines structural innovations that underpin centriole and axoneme assembly and demonstrates that MIPs co-evolved with centrosomes and cilia.

Purine nucleosides replace cAMP in allosteric regulation of PKA in trypanosomatid pathogens

Ober, V. T., Githure, G. B., Santos, Y. V., et al. — Sun, 01 Jan 2023 09:14:46 +0100

Cyclic nucleotide binding domains (CNB) confer allosteric regulation by cAMP or cGMP to many signaling proteins, including PKA and PKG. PKA of phylogenetically distant Trypanosoma is the first exception as it is cyclic nucleotide-independent and responsive to nucleoside analogues (Bachmaier et al., 2019). Here, we show that natural nucleosides inosine, guanosine and adenosine are nanomolar affinity CNB ligands and activators of PKA orthologs of the important tropical pathogens Trypanosoma brucei, Trypanosoma cruzi, and Leishmania. The sequence and structural determinants of binding affinity,-specificity and kinase activation of PKAR were established by structure-activity relationship (SAR) analysis, co-crystal structures and mutagenesis. Substitution of two to three amino acids in the binding sites is sufficient for conversion of CNB domains from nucleoside to cyclic nucleotide specificity. In addition, a trypanosomatid-specific C-terminal helix (αD) is required for high affinity binding to CNB-B. The αD helix functions as a lid of the binding site that shields ligands from solvent. Selectivity of guanosine for CNB-B and of adenosine for CNB-A results in synergistic kinase activation at low nanomolar concentration. PKA pulldown from rapid lysis establishes guanosine as the predominant ligand in vivo in T. brucei bloodstream forms, whereas guanosine and adenosine seem to synergize in the procyclic developmental stage in the insect vector. We discuss the versatile use of CNB domains in evolution and recruitment of PKA for novel nucleoside-mediated signaling.

Protocol for generating customizable and reproducible plots of sequencing coverage data using the seqNdisplayR package

Lykke-Andersen, S., Rouvière, J. O., Schmid, M., Gockert, M., Jensen, T. H. — Sat, 01 Jun 2024 09:14:46 +0200

The widespread usage of next-generation sequencing methods for functional genomics studies requires standardized tools for consistent visualization of the associated data. Here, we present seqNdisplayR, an R package for plotting standard sequencing data coverage within a genomic region of interest in a customizable and reproducible manner. We describe steps for installing software, preparing data files, choosing options, and plotting data. This tool is readily available for users with no prior experience with R through the “Shiny app” interface. For complete details on the use and execution of this protocol, please refer to Lykke-Andersen et al.,¹ Gockert et al.,² and Rouviere et al.³

Nitrogen and Nod factor signaling determine Lotus japonicus root exudate composition and bacterial assembly

Tao, K., Jensen, I. T., Zhang, S., et al. — Mon, 01 Apr 2024 09:14:47 +0200

Symbiosis with soil-dwelling bacteria that fix atmospheric nitrogen allows legume plants to grow in nitrogen-depleted soil. Symbiosis impacts the assembly of root microbiota, but it is unknown how the interaction between the legume host and rhizobia impacts the remaining microbiota and whether it depends on nitrogen nutrition. Here, we use plant and bacterial mutants to address the role of Nod factor signaling on Lotus japonicus root microbiota assembly. We find that Nod factors are produced by symbionts to activate Nod factor signaling in the host and that this modulates the root exudate profile and the assembly of a symbiotic root microbiota. Lotus plants with different symbiotic abilities, grown in unfertilized or nitrate-supplemented soils, display three nitrogen-dependent nutritional states: starved, symbiotic, or inorganic. We find that root and rhizosphere microbiomes associated with these states differ in composition and connectivity, demonstrating that symbiosis and inorganic nitrogen impact the legume root microbiota differently. Finally, we demonstrate that selected bacterial genera characterizing state-dependent microbiomes have a high level of accurate prediction.

Phase-type distributions in mathematical population genetics

Hobolth, A., Rivas-González, I., Bladt, M., Futschik, A. — Sat, 01 Jun 2024 09:14:47 +0200

A phase-type distribution is the time to absorption in a continuous- or discrete-time Markov chain. Phase-type distributions can be used as a general framework to calculate key properties of the standard coalescent model and many of its extensions. Here, the ‘phases’ in the phase-type distribution correspond to states in the ancestral process. For example, the time to the most recent common ancestor and the total branch length are phase-type distributed. Furthermore, the site frequency spectrum follows a multivariate discrete phase-type distribution and the joint distribution of total branch lengths in the two-locus coalescent-with-recombination model is multivariate phase-type distributed. In general, phase-type distributions provide a powerful mathematical framework for coalescent theory because they are analytically tractable using matrix manipulations. The purpose of this review is to explain the phase-type theory and demonstrate how the theory can be applied to derive basic properties of coalescent models. These properties can then be used to obtain insight into the ancestral process, or they can be applied for statistical inference. In particular, we show the relation between classical first-step analysis of coalescent models and phase-type calculations. We also show how reward transformations in phase-type theory lead to easy calculation of covariances and correlation coefficients between e.g. tree height, tree length, external branch length, and internal branch length. Furthermore, we discuss how these quantities can be used for statistical inference based on estimating equations. Providing an alternative to previous work based on the Laplace transform, we derive likelihoods for small-size coalescent trees based on phase-type theory. Overall, our main aim is to demonstrate that phase-type distributions provide a convenient general set of tools to understand aspects of coalescent models that are otherwise difficult to derive. Throughout the review, we emphasize the versatility of the phase-type framework, which is also illustrated by our accompanying R-code. All our analyses and figures can be reproduced from code available on GitHub.

Structural basis for competitive binding of productive and degradative co-transcriptional effectors to the nuclear cap-binding complex

Dubiez, E., Pellegrini, E., Finderup Brask, M., et al. — Mon, 01 Jan 2024 09:14:47 +0100

The nuclear cap-binding complex (CBC) coordinates co-transcriptional maturation, transport, or degradation of nascent RNA polymerase II (Pol II) transcripts. CBC with its partner ARS2 forms mutually exclusive complexes with diverse “effectors” that promote either productive or destructive outcomes. Combining AlphaFold predictions with structural and biochemical validation, we show how effectors NCBP3, NELF-E, ARS2, PHAX, and ZC3H18 form competing binary complexes with CBC and how PHAX, NCBP3, ZC3H18, and other effectors compete for binding to ARS2. In ternary CBC-ARS2 complexes with PHAX, NCBP3, or ZC3H18, ARS2 is responsible for the initial effector recruitment but inhibits their direct binding to the CBC. We show that in vivo ZC3H18 binding to both CBC and ARS2 is required for nuclear RNA degradation. We propose that recruitment of PHAX to CBC-ARS2 can lead, with appropriate cues, to competitive displacement of ARS2 and ZC3H18 from the CBC, thus promoting a productive rather than a degradative RNA fate.

HSV-1 ICP27 targets the TBK1-activated STING signalsome to inhibit virus-induced type I IFN expression

Christensen, M. H., Jensen, S. B., Miettinen, J. J., et al. — Fri, 01 Jul 2016 09:14:47 +0200

Herpes simplex virus (HSV) 1 stimulates type I IFN expression through the cGAS–STING–TBK1 signaling axis. Macrophages have recently been proposed to be an essential source of IFN during viral infection. However, it is not known how HSV-1 inhibits IFN expression in this cell type. Here, we show that HSV-1 inhibits type I IFN induction through the cGAS–STING–TBK1 pathway in human macrophages, in a manner dependent on the conserved herpesvirus protein ICP27. This viral protein was expressed de novo in macrophages with early nuclear localization followed by later translocation to the cytoplasm where ICP27 prevented activation of IRF3. ICP27 interacted with TBK1 and STING in a manner that was dependent on TBK1 activity and the RGG motif in ICP27. Thus, HSV-1 inhibits expression of type I IFN in human macrophages through ICP27-dependent targeting of the TBK1-activated STING signalsome.

Predicting nuclear G-quadruplex RNA-binding proteins with roles in transcription and phase separation

Luige, J., Armaos, A., Tartaglia, G. G., Ørom, U. A. V. — Fri, 01 Mar 2024 09:14:47 +0100

RNA-binding proteins are central for many biological processes and their characterization has demonstrated a broad range of functions as well as a wide spectrum of target structures. RNA G-quadruplexes are important regulatory elements occurring in both coding and non-coding transcripts, yet our knowledge of their structure-based interactions is at present limited. Here, using theoretical predictions and experimental approaches, we show that many chromatin-binding proteins bind to RNA G-quadruplexes, and we classify them based on their RNA G-quadruplex-binding potential. Combining experimental identification of nuclear RNA G-quadruplex-binding proteins with computational approaches, we build a prediction tool that assigns probability score for a nuclear protein to bind RNA G-quadruplexes. We show that predicted G-quadruplex RNA-binding proteins exhibit a high degree of protein disorder and hydrophilicity and suggest involvement in both transcription and phase-separation into membrane-less organelles. Finally, we present the G4-Folded/UNfolded Nuclear Interaction Explorer System (G4-FUNNIES) for estimating RNA G4-binding propensities at http://service.tartaglialab.com/new_submission/G4FUNNIES.

An RNA origami robot that traps and releases a fluorescent aptamer

Vallina, N. S., McRae, E. K.S., Geary, C., Andersen, E. S. — Fri, 01 Mar 2024 09:14:47 +0100

RNA nanotechnology aims to use RNA as a programmable material to create self-assembling nanodevices for application in medicine and synthetic biology. The main challenge is to develop advanced RNA robotic devices that both sense, compute, and actuate to obtain enhanced control over molecular processes. Here, we use the RNA origami method to prototype an RNA robotic device, named the “Traptamer,” that mechanically traps the fluorescent aptamer, iSpinach. The Traptamer is shown to sense two RNA key strands, acts as a Boolean AND gate, and reversibly controls the fluorescence of the iSpinach aptamer. Cryo–electron microscopy of the closed Traptamer structure at 5.45-angstrom resolution reveals the mechanical mode of distortion of the iSpinach motif. Our study suggests a general approach to distorting RNA motifs and a path forward to build sophisticated RNA machines that through sensing, computing, and actuation modules can be used to precisely control RNA functionalities in cellular systems.

Sounding an Alarm over Spatial Biology

Orr, M. E., Kulasinghe, A., Kolar, G. R., et al. — Fri, 01 Mar 2024 09:14:47 +0100

Trypanosoma brucei Invariant Surface Glycoprotein 75 Is an Immunoglobulin Fc Receptor Inhibiting Complement Activation and Antibody-Mediated Cellular Phagocytosis

Mikkelsen, J. H., Stødkilde, K., Jensen, M. P., et al. — Mon, 15 Apr 2024 09:14:47 +0200

Various subspecies of the unicellular parasite Trypanosoma brucei cause sleeping sickness, a neglected tropical disease affecting millions of individuals and domestic animals. Immune evasion mechanisms play a pivotal role in parasite survival within the host and enable the parasite to establish a chronic infection. In particular, the rapid switching of variant surface glycoproteins covering a large proportion of the parasite's surface enables the parasite to avoid clearance by the adaptive immune system of the host. In this article, we present the crystal structure and discover an immune-evasive function of the extracellular region of the T. brucei invariant surface gp75 (ISG75). Structural analysis determined that the ISG75 ectodomain is organized as a globular head domain and a long slender coiled-coil domain. Subsequent ligand screening and binding analysis determined that the head domain of ISG75 confers interaction with the Fc region of all subclasses of human IgG. Importantly, the ISG75-IgG interaction strongly inhibits both activation of the classical complement pathway and Ab-dependent cellular phagocytosis by competing with C1q and host cell FcγR CD32. Our data reveal a novel immune evasion mechanism of T. brucei, with ISG75 able to inactivate the activities of Abs recognizing the parasite surface proteins.

Evolution of the germline mutation rate across vertebrates

Bergeron, L. A., Besenbacher, S., Zheng, J., et al. — Thu, 09 Mar 2023 09:14:47 +0100

The germline mutation rate determines the pace of genome evolution and is an evolving parameter itself¹. However, little is known about what determines its evolution, as most studies of mutation rates have focused on single species with different methodologies². Here we quantify germline mutation rates across vertebrates by sequencing and comparing the high-coverage genomes of 151 parent–offspring trios from 68 species of mammals, fishes, birds and reptiles. We show that the per-generation mutation rate varies among species by a factor of 40, with mutation rates being higher for males than for females in mammals and birds, but not in reptiles and fishes. The generation time, age at maturity and species-level fecundity are the key life-history traits affecting this variation among species. Furthermore, species with higher long-term effective population sizes tend to have lower mutation rates per generation, providing support for the drift barrier hypothesis³. The exceptionally high yearly mutation rates of domesticated animals, which have been continually selected on fecundity traits including shorter generation times, further support the importance of generation time in the evolution of mutation rates. Overall, our comparative analysis of pedigree-based mutation rates provides ecological insights on the mutation rate evolution in vertebrates.

Identification of constrained sequence elements across 239 primate genomes

Kuderna, L. F.K., Ulirsch, J. C., Rashid, S., et al. — Thu, 25 Jan 2024 09:14:47 +0100

Noncoding DNA is central to our understanding of human gene regulation and complex diseases^1,2, and measuring the evolutionary sequence constraint can establish the functional relevance of putative regulatory elements in the human genome^3–9. Identifying the genomic elements that have become constrained specifically in primates has been hampered by the faster evolution of noncoding DNA compared to protein-coding DNA¹⁰, the relatively short timescales separating primate species¹¹, and the previously limited availability of whole-genome sequences¹². Here we construct a whole-genome alignment of 239 species, representing nearly half of all extant species in the primate order. Using this resource, we identified human regulatory elements that are under selective constraint across primates and other mammals at a 5% false discovery rate. We detected 111,318 DNase I hypersensitivity sites and 267,410 transcription factor binding sites that are constrained specifically in primates but not across other placental mammals and validate their cis-regulatory effects on gene expression. These regulatory elements are enriched for human genetic variants that affect gene expression and complex traits and diseases. Our results highlight the important role of recent evolution in regulatory sequence elements differentiating primates, including humans, from other placental mammals.

Trails

Rivas-González, I., Schierup, M. H., Wakeley, J., Hobolth, A. — Thu, 01 Feb 2024 09:14:47 +0100

Genome-wide genealogies of multiple species carry detailed information about demographic and selection processes on individual branches of the phylogeny. Here, we introduce TRAILS, a hidden Markov model that accurately infers time-resolved population genetics parameters, such as ancestral effective population sizes and speciation times, for ancestral branches using a multi-species alignment of three species and an outgroup. TRAILS leverages the information contained in incomplete lineage sorting fragments by modelling genealogies along the genome as rooted three-leaved trees, each with a topology and two coalescent events happening in discretized time intervals within the phylogeny. Posterior decoding of the hidden Markov model can be used to infer the ancestral recombination graph for the alignment and details on demographic changes within a branch. Since TRAILS performs posterior decoding at the base-pair level, genome-wide scans based on the posterior probabilities can be devised to detect deviations from neutrality. Using TRAILS on a human-chimp-gorilla-orangutan alignment, we recover speciation parameters and extract information about the topology and coalescent times at high resolution.

The impact of hUC MSC-derived exosome-nanoliposome hybrids on α-synuclein fibrillation and neurotoxicity

Aliakbari, F., Marzookian, K., Parsafar, S., et al. — Fri, 05 Apr 2024 09:14:47 +0200

Amyloid aggregation of α-synuclein (αSN) protein amplifies the pathogenesis of neurodegenerative diseases (NDs) such as Parkinson's disease (PD). Consequently, blocking aggregation or redirecting self-assembly to less toxic aggregates could be therapeutic. Here, we improve brain-specific nanocarriers using a hybrid of exosomes (Ex) from human umbilical cord mesenchymal stem cells (hUC MSCs) and nanoliposomes containing baicalein (Ex-NLP-Ba) and oleuropein (Ex-NLP-Ole). The hybrids contained both lipid membranes, Ex proteins, and baicalein or oleuropein. Fluorescence resonance energy transfer analysis confirmed their proper integration. The hybrids reduced the extent of αSN fibrillation and interfered with secondary nucleation and disaggregation. They not only reduced αSN pathogenicity but also enhanced drug internalization into cells, surpassing the efficacy of NLP alone, and also crossed the blood-brain barrier in a cellular model. We conclude that Ex can be successfully extracted and efficiently merged with NLPs while retaining its original properties, demonstrating great potential as a theranostic drug delivery vehicle against NDs like PD.

A region of suppressed recombination misleads neoavian phylogenomics

Mirarab, S., Rivas-González, I., Feng, S., et al. — Tue, 09 Apr 2024 09:14:47 +0200

Genomes are typically mosaics of regions with different evolutionary histories. When speciation events are closely spaced in time, recombination makes the regions sharing the same history small, and the evolutionary history changes rapidly as we move along the genome. When examining rapid radiations such as the early diversification of Neoaves 66 Mya, typically no consistent history is observed across segments exceeding kilobases of the genome. Here, we report an exception. We found that a 21-Mb region in avian genomes, mapped to chicken chromosome 4, shows an extremely strong and discordance-free signal for a history different from that of the inferred species tree. Such a strong discordance-free signal, indicative of suppressed recombination across many millions of base pairs, is not observed elsewhere in the genome for any deep avian relationships. Although long regions with suppressed recombination have been documented in recently diverged species, our results pertain to relationships dating circa 65 Mya. We provide evidence that this strong signal may be due to an ancient rearrangement that blocked recombination and remained polymorphic for several million years prior to fixation. We show that the presence of this region has misled previous phylogenomic efforts with lower taxon sampling, showing the interplay between taxon and locus sampling. We predict that similar ancient rearrangements may confound phylogenetic analyses in other clades, pointing to a need for new analytical models that incorporate the possibility of such events.