eLife Sciences

Cationic antimicrobial peptides (CAPs) such as defensins are ubiquitously found innate immune molecules that often exhibit broad activity against microbial pathogens and mammalian tumor cells. Many CAPs act at the plasma membrane of cells leading to membrane destabilization and permeabilization. In this study, we describe a novel cell lysis mechanism for fungal and tumor cells by the plant defensin NaD1 that acts via direct binding to the plasma membrane phospholipid phosphatidylinositol 4,5-bisphosphate (PIP2). We determined the crystal structure of a NaD1:PIP2 complex, revealing a striking oligomeric arrangement comprising seven dimers of NaD1 that cooperatively bind the anionic headgroups of 14 PIP2 molecules through a unique 'cationic grip' configuration. Site-directed mutagenesis of NaD1 confirms that PIP2-mediated oligomerization is important for fungal and tumor cell permeabilization. These observations identify an innate recognition system by NaD1 for direct binding of PIP2 that permeabilizes cells via a novel membrane disrupting mechanism. DOI: http://dx.doi.org/10.7554/eLife.01808.001.

Protein-protein interactions are fundamental to many biological processes. Experimental screens have identified tens of thousands of interactions and structural biology has provided detailed functional insight for select 3D protein complexes. An alternative rich source of information about protein interactions is the evolutionary sequence record. Building on earlier work, we show that analysis of correlated evolutionary sequence changes across proteins identifies residues that are close in space with sufficient accuracy to determine the three-dimensional structure of the protein complexes. We evaluate prediction performance in blinded tests on 76 complexes of known 3D structure, predict protein-protein contacts in 32 complexes of unknown structure, and demonstrate how evolutionary couplings can be used to distinguish between interacting and non-interacting protein pairs in a large complex. With the current growth of sequence databases, we expect that the method can be generalized to genome-wide elucidation of protein-protein interaction networks and used for interaction predictions at residue resolution.

Jurassic fossils of a bizarre fly larva that lived in water as a blood-sucking parasite highlight how much can be learnt from the study of the fossils of immature insects.

After acknowledging that power differentials exist, can scientists find inspiration to persevere anyway?

A new method for applying solid stress to aggregates of cells is shedding light on the impact of mechanical forces on cancer cells.

Two interpretations of similar structures for the same molecular machine illustrate the limits of inferring biochemical mechanism from protein structure.

Chromatin remodeling processes can drive acinar cell fate decisions.

Longer poly(A) tails improve translation in early development, but not in mature cells that have higher levels of the protein PABPC.

ELife digest Thirteen million infants are born too early every year. Improved care allows many to survive, but these “preterm infants” still face an increased risk of death and many other complications. Infants born very early, before 32 weeks, are at risk of brain injury because the brain is normally still developing in the later stages of pregnancy. They also have an increased risk of developing mental health problems later in life. Early-life brain injuries in rats cause changes in the production of a chemical called dopamine. Dopamine is a chemical messenger in the brain that reinforces rewarding behaviour. People with schizophrenia and attention deficit hyperactivity disorder (ADHD) have abnormal levels of dopamine. Changes in brain dopamine levels may explain why early-life brain injury is linked to later mental illness. But first scientists must study whether similar changes occur in humans with an early-life brain injury. Now, Froudist-Walsh et al. use brain imaging to show that people born very early who suffered a brain injury have lower dopamine levels than other adults. Imaging techniques were used to scan the brains of 13 adults who were born before 32 weeks and who had a brain injury around birth, 13 adults born before 32 weeks without a brain injury, and 13 adults born at “full term” (around 39 to 40 weeks). Individuals with low dopamine levels reported difficulty concentrating and a lack of motivation and enjoyment in their lives. Both can be warning signs of mental health problems. People born prematurely without a brain injury had normal dopamine levels and did not report such symptoms. More studies may help scientists understand how early brain injuries may cause brain chemical differences later in life, and how these brain changes affect individual’s mental health. They may also help scientists develop treatments to prevent or treat mental illness in people who experienced a brain injury after a very early birth.

Induced pluripotent stem cell (iPSC)-derived neurons are increasingly used to model Autism Spectrum Disorder (ASD), which is clinically and genetically heterogeneous. To study the complex relationship of penetrant and weaker polygenic risk variants to ASD, ‘isogenic’ iPSC-derived neurons are critical. We developed a set of procedures to control for heterogeneity in reprogramming and differentiation, and generated 53 different iPSC-derived glutamatergic neuronal lines from 25 participants from 12 unrelated families with ASD. Heterozygous de novo and rare-inherited presumed-damaging variants were characterized in ASD risk genes/loci. Combinations of putative etiologic variants (GLI3/KIF21A or EHMT2/UBE2I) in separate families were modeled. We used a multi-electrode array, with patch-clamp recordings, to determine a reproducible synaptic phenotype in 25% of the individuals with ASD (other relevant data on the remaining lines was collected). Our most compelling new results revealed a consistent spontaneous network hyperactivity in neurons deficient for CNTN5 or EHMT2. The biobank of iPSC-derived neurons and accompanying genomic data are available to accelerate ASD research. Editorial note: This article has been through an editorial process in which authors decide how to respond to the issues raised during peer review. The Reviewing Editor's assessment is that all the issues have been addressed ( see decision letter ).

Antiretroviral therapy for HIV-1 infection/AIDS has significantly extended the life expectancy of HIV-1-infected individuals and reduced HIV-1 transmission at very high rates. However, certain individuals who initially achieve viral suppression to undetectable levels may eventually suffer treatment failure mainly due to adverse effects and the emergence of drug-resistant HIV-1 variants. Here, we report GRL-142, a novel HIV-1 protease inhibitor containing an unprecedented 6-5-5-ring-fused crown-like tetrahydropyranofuran, which has extremely potent activity against all HIV-1 strains examined with IC50 values of attomolar-to-picomolar concentrations, virtually no effects on cellular growth, extremely high genetic barrier against the emergence of drug-resistant variants, and favorable intracellular and central nervous system penetration. GRL-142 forms optimum polar, van der Waals, and halogen bond interactions with HIV-1 protease and strongly blocks protease dimerization, demonstrating that combined multiple optimizing elements significantly enhance molecular and atomic interactions with a target protein and generate unprecedentedly potent and practically favorable agents.

Fucose is a common component of eukaryotic cell-surface glycoconjugates, generally added by Golgi-resident fucosyltransferases. Whereas fucosylated glycoconjugates are rare in kinetoplastids, the biosynthesis of the nucleotide sugar GDP-Fuc has been shown to be essential in Trypanosoma brucei. Here we show that the single identifiable T. brucei fucosyltransferase (TbFUT1) is a GDP-Fuc: β-D-galactose α-1,2-fucosyltransferase with an apparent preference for a Galβ1,3GlcNAcβ1-O-R acceptor motif. Conditional null mutants of TbFUT1 demonstrated that it is essential for both the mammalian-infective bloodstream form and the insect vector-dwelling procyclic form. Unexpectedly, TbFUT1 was localized in the mitochondrion of T. brucei and found to be required for mitochondrial function in bloodstream form trypanosomes. Finally, the TbFUT1 gene was able to complement a Leishmania major mutant lacking the homologous fucosyltransferase gene (Guo et al., 2021). Together these results suggest that kinetoplastids possess an unusual, conserved and essential mitochondrial fucosyltransferase activity that may have therapeutic potential across trypanosomatids.

The alpha/B.1.1.7 SARS-CoV-2 lineage emerged in autumn 2020 in the United Kingdom and transmitted rapidly until winter 2021 when it was responsible for most new COVID-19 cases in many European countries. The incidence domination was likely due to a fitness advantage that could be driven by the RBD residue change (N501Y), which also emerged independently in other Variants of Concern such as the beta/B.1.351 and gamma/P.1 strains. Here we present a functional characterization of the alpha/B.1.1.7 variant and show an eight-fold affinity increase towards human ACE-2. In accordance with this, transgenic hACE-2 mice showed a faster disease progression and severity after infection with a low dose of B.1.1.7, compared to an early 2020 SARS-CoV-2 isolate. When challenged with sera from convalescent individuals or anti-RBD monoclonal antibodies, the N501Y variant showed a minor, but significant elevated evasion potential of ACE-2/RBD antibody neutralization. The data suggest that the single asparagine to tyrosine substitution remarkable rise in affinity may be responsible for the higher transmission rate and severity of the B.1.1.7 variant.

Background: The degree of heterotypic immunity induced by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) strains is a major determinant of the spread of emerging variants and the success of vaccination campaigns, but remains incompletely understood. Methods: We examined the immunogenicity of SARS-CoV-2 variant B.1.1.7 (Alpha) that arose in the United Kingdom and spread globally. We determined titres of spike glycoprotein-binding antibodies and authentic virus neutralising antibodies induced by B.1.1.7 infection to infer homotypic and heterotypic immunity. Results: Antibodies elicited by B.1.1.7 infection exhibited significantly reduced recognition and neutralisation of parental strains or of the South Africa variant B.1.351 (Beta) than of the infecting variant. The drop in cross-reactivity was significantly more pronounced following B.1.1.7 than parental strain infection. Conclusions: The results indicate that heterotypic immunity induced by SARS-CoV-2 variants is asymmetric.

Mimivirus is the prototype of the Mimiviridae family of giant dsDNA viruses. Little is known about the organization of the 1.2 Mb genome inside the membrane-limited nucleoid filling the ~0.5 µm icosahedral capsids. Cryo-electron microscopy, cryo-electron tomography and proteomics revealed that it is encased into a ~30 nm diameter helical protein shell surprisingly composed of two GMC-type oxidoreductases, which also form the glycosylated fibrils decorating the capsid. The genome is arranged in 5- or 6-start left-handed super-helices, with each DNA-strand lining the central channel. This luminal channel of the nucleoprotein fiber is wide enough to accommodate oxidative stress proteins and RNA polymerase subunits identified by proteomics. Such elegant supramolecular organization would represent a remarkable evolutionary strategy for packaging and protecting the genome, in a state ready for immediate transcription upon unwinding in the host cytoplasm. The parsimonious use of the same protein in two unrelated substructures of the virion is unexpected for a giant virus with thousand genes at its disposal.

Piwi-interacting RNAs (piRNAs) play essential roles in silencing repetitive elements to promote fertility in metazoans. Studies in worms, flies, and mammals reveal that piRNAs are expressed in a sex-specific manner. However, the mechanisms underlying this sex-specific regulation are unknown. Here we identify SNPC-1.3, a male germline-enriched variant of a conserved subunit of the small nuclear RNA-activating protein complex, as a male-specific piRNA transcription factor in Caenorhabditis elegans . SNPC-1.3 colocalizes with the core piRNA transcription factor, SNPC-4, in nuclear foci of the male germline. Binding of SNPC-1.3 at male piRNA loci drives spermatogenic piRNA transcription and requires SNPC-4. Loss of snpc-1.3 leads to depletion of male piRNAs and defects in male-dependent fertility. Furthermore, TRA-1, a master regulator of sex determination, binds to the snpc-1.3 promoter and represses its expression during oogenesis. Loss of TRA-1 targeting causes ectopic expression of snpc-1.3 and male piRNAs during oogenesis. Thus, sexually dimorphic regulation of snpc-1.3 expression coordinates male and female piRNA expression during germline development.

Male infertility is a prevalent condition, affecting 5–10% of men. So far, few genetic factors have been described as contributors to spermatogenic failure. Here, we report the first re-sequencing study of the Y-chromosomal Azoospermia Factor c (AZFc) region, combined with gene dosage analysis of the multicopy DAZ, BPY2, and CDYgenes and Y-haplogroup determination. In analysing 2324 Estonian men, we uncovered a novel structural variant as a high-penetrance risk factor for male infertility. The Y lineage R1a1-M458, reported at 20% frequency in several European populations, carries a fixed ~1.6 Mb r2/r3 inversion, destabilizing the AZFc region and predisposing to large recurrent microdeletions. Such complex rearrangements were significantly enriched among severe oligozoospermia cases. The carrier vs non-carrier risk for spermatogenic failure was increased 8.6-fold (p=6.0×10−4). This finding contributes to improved molecular diagnostics and clinical management of infertility. Carrier identification at young age will facilitate timely counselling and reproductive decision-making.

Laboratory experimental evolution provides a window into the details of the evolutionary process. To investigate the consequences of long-term adaptation, we evolved 205 Saccharomyces cerevisiae populations (124 haploid and 81 diploid) for ~10,000,000 generations in three environments. We measured the dynamics of fitness changes over time, finding repeatable patterns of declining adaptability. Sequencing revealed that this phenotypic adaptation is coupled with a steady accumulation of mutations, widespread genetic parallelism, and historical contingency. In contrast to long-term evolution in E. coli , we do not observe long-term coexistence or populations with highly elevated mutation rates. We find that evolution in diploid populations involves both fixation of heterozygous mutations and frequent loss-of-heterozygosity events. Together, these results help distinguish aspects of evolutionary dynamics that are likely to be general features of adaptation across many systems from those that are specific to individual organisms and environmental conditions.

The biodiversity of tundra areas in northern high latitudes is threatened by invasion of forests under global warming. However, poorly understood nonlinear responses of the treeline ecotone mean the timing and extent of tundra losses are unclear, but policymakers need such information to optimize conservation efforts. Our individual-based model LAVESI, developed for the Siberian tundra-taiga ecotone, can help improve our understanding. Consequently, we simulated treeline migration trajectories until the end of the millennium, causing a loss of tundra area when advancing north. Our simulations reveal that the treeline follows climate warming with a severe, century-long time lag, which is overcompensated by infilling of stands in the long run even when temperatures cool again. Our simulations reveal that only under ambitious mitigation strategies (relative concentration pathway 2.6) will ∼30% of original tundra areas remain in the north but separated into two disjunct refugia.

Over 100 years of studies in Drosophila melanogaster and related species in the genus Drosophila have facilitated key discoveries in genetics, genomics, and evolution. While high-quality genome assemblies exist for several species in this group, they only encompass a small fraction of the genus. Recent advances in long-read sequencing allow high-quality genome assemblies for tens or even hundreds of species to be efficiently generated. Here, we utilize Oxford Nanopore sequencing to build an open community resource of genome assemblies for 101 lines of 93 drosophilid species encompassing 14 species groups and 35 sub-groups. The genomes are highly contiguous and complete, with an average contig N50 of 10.5 Mb and greater than 97% BUSCO completeness in 97/101 assemblies. We show that Nanopore-based assemblies are highly accurate in coding regions, particularly with respect to coding insertions and deletions. These assemblies, along with a detailed laboratory protocol and assembly pipelines, are released as a public resource and will serve as a starting point for addressing broad questions of genetics, ecology, and evolution at the scale of hundreds of species.

The kinesin-3 family member Unc-104/KIF1A is required for axonal transport of many presynaptic components to synapses, and mutation of this gene results in synaptic dysfunction in mice, flies and worms. Our studies at the Drosophila neuromuscular junction indicate that many synaptic defects in unc-104-null mutants are mediated independently of Unc-104’s transport function, via the Wallenda (Wnd)/DLK MAP kinase axonal damage signaling pathway. Wnd signaling becomes activated when Unc-104’s function is disrupted, and leads to impairment of synaptic structure and function by restraining the expression level of active zone (AZ) and synaptic vesicle (SV) components. This action concomitantly suppresses the buildup of synaptic proteins in neuronal cell bodies, hence may play an adaptive role to stresses that impair axonal transport. Wnd signaling also becomes activated when pre-synaptic proteins are over-expressed, suggesting the existence of a feedback circuit to match synaptic protein levels to the transport capacity of the axon.

Deregulation of Pol III products causes a range of diseases, including neural diseases and cancers. However, the factors and mechanisms that modulate Pol III-directed transcription remain to be found, although massive advances have been achieved. Here, we show that STAT3 positively regulates the activities of Pol III-dependent transcription and cancer cell growth. RNA-seq analysis revealed that STAT3 inhibits the expression of TP73, a member of the p53 family. We found that TP73 is not only required for the regulation of Pol III-directed transcription mediated by STAT3 but also independently suppresses the synthesis of Pol III products. Mechanistically, TP73 can disrupt the assembly of TFIIIB subunits and inhibit their occupancies at Pol III target loci by interacting with TFIIIB subunit TBP. MiR-106a-5p can activate Pol III-directed transcription by targeting the TP73 mRNA 3’ UTR to reduce TP 73 expression. We show that STAT3 activates the expression of miR-106a-5p by binding to the miRNA promoter, indicating that the miR-106a-5p links STAT3 with TP73 to regulate Pol III-directed transcription. Collectively, these findings indicate that STAT3 functions as a positive regulator in Pol III-directed transcription by controlling the miR-106a-5p/TP73 axis.

Most cellular ATP is made by rotary F 1 F O ATP synthases using proton translocation-generated clockwise torque on the F O c-ring rotor, while F 1 -ATP hydrolysis can force counterclockwise rotation and proton pumping. The F O torque-generating mechanism remains elusive even though the F O interface of stator subunit-a, which contains the transmembrane proton half-channels, and the c-ring is known from recent F 1 F O structures. Here, single-molecule F 1 F O rotation studies determined that the pKa values of the half-channels differ, show that mutations of residues in these channels change the pKa values of both half-channels, and reveal the ability of F O to undergo single c-subunit rotational stepping. These experiments provide evidence to support the hypothesis that proton translocation through F O operates via a Grotthuss mechanism involving a column of single water molecules in each half-channel linked by proton translocation-dependent c-ring rotation. We also observed pH-dependent 11° ATP synthase-direction sub-steps of the E. coli c 10 -ring of F 1 F O against the torque of F 1 -ATPase-dependent rotation that result from H ⁺ transfer events from F O subunit-a groups with a low pKa to one c-subunit in the c-ring, and from an adjacent c-subunit to stator groups with a high pKa. These results support a mechanism in which alternating proton translocation-dependent 11° and 25° synthase-direction rotational sub-steps of the c 10 -ring occur to sustain F 1 F O ATP synthesis.

Changes in available nutrients are inevitable events for most living organisms. Upon nutritional stress, several signaling pathways cooperate to change the transcription program through chromatin regulation to rewire cellular metabolism. In budding yeast, histone H3 threonine 11 phosphorylation (H3pT11) acts as a marker of low glucose stress and regulates the transcription of nutritional stress responsive genes. Understanding how this histone modification 'senses' external glucose changes remains elusive. Here, we show that Tda1, the yeast orthologue of human Nuak1, is a direct kinase for H3pT11 upon low glucose stress. Yeast AMPK directly phosphorylates Tda1 to govern Tda1 activity, while CK2 regulates Tda1 nuclear localization. Collectively, AMPK and CK2 signaling converge on histone kinase Tda1 to link external low glucose stress to chromatin regulation.

Upon nutritional stress, the metabolic status of cells is changed by nutrient signaling pathways to ensure survival. Altered metabolism by nutrient signaling pathways has been suggested to influence cellular lifespan. However, it remains unclear how chromatin regulation is involved in this process. Here, we found that histone H3 threonine 11 phosphorylation (H3pT11) functions as a marker for nutritional stress and aging. Sch9 and CK2 kinases cooperatively regulate H3pT11 under stress conditions. Importantly, H3pT11 defective mutants prolonged chronological lifespan (CLS) by altering nutritional stress responses. Thus, the phosphorylation of H3T11 by Sch9 and CK2 links a nutritional stress response to chromatin in the regulation of CLS.

Cas7-11 is a Type III-E CRISPR Cas effector that confers programmable RNA cleavage and has potential applications in RNA interference. Cas7-11 encodes a single polypeptide containing four Cas7- and one Cas11-like segments that obscures the distinction between the multi-subunit Class 1 and the single-subunit Class-2 CRISPR-Cas systems. We report a cryo-EM structure of the active Cas7-11 from Desulfonema ishimotonii (DiCas7-11) that reveals the molecular basis for RNA processing and interference activities. DiCas7-11 arranges its Cas7- and Cas11-like domains in an extended form that resembles the backbone made up by four Cas7 and one Cas11 subunits in the multi-subunit enzymes. Unlike the multi-subunit enzymes, however, the backbone of DiCas7-11 contains evolutionarily different Cas7 and Cas11 domains, giving rise to their unique functionality. The first Cas7-like domain nearly engulfs the last 15 direct repeat nucleotides in processing and recognition of the CRISPR RNA, and its free-standing fragment retains most of the activity. Both the second and the third Cas7-like domains mediate target RNA cleavage in a metal-dependent manner. The structure and mutational data indicate that the long variable insertion to the fourth Cas7 domain has little impact to RNA processing or targeting, suggesting the possibility for engineering a compact and programmable RNA interference tool.

Background: Vaccination is one of the most effective public health interventions. We investigate the impact of vaccination activities for Haemophilus influenzae type b, hepatitis B, human papillomavirus, Japanese encephalitis, measles, Neisseria meningitidis serogroup A, rotavirus, rubella, Streptococcus pneumoniae, and yellow fever over the years 2000–2030 across 112 countries. Methods: Twenty-one mathematical models estimated disease burden using standardised demographic and immunisation data. Impact was attributed to the year of vaccination through vaccine-activity-stratified impact ratios. Results: We estimate 97 (95%CrI[80, 120]) million deaths would be averted due to vaccination activities over 2000–2030, with 50 (95%CrI[41, 62]) million deaths averted by activities between 2000 and 2019. For children under-5 born between 2000 and 2030, we estimate 52 (95%CrI[41, 69]) million more deaths would occur over their lifetimes without vaccination against these diseases. Conclusions: This study represents the largest assessment of vaccine impact before COVID-19-related disruptions and provides motivation for sustaining and improving global vaccination coverage in the future. Funding: VIMC is jointly funded by Gavi, the Vaccine Alliance, and the Bill and Melinda Gates Foundation (BMGF) (BMGF grant number: OPP1157270 / INV-009125). Funding from Gavi is channelled via VIMC to the Consortium’s modelling groups (VIMC-funded institutions represented in this paper: Imperial College London, London School of Hygiene and Tropical Medicine, Oxford University Clinical Research Unit, Public Health England, Johns Hopkins University, The Pennsylvania State University, Center for Disease Analysis Foundation, Kaiser Permanente Washington, University of Cambridge, University of Notre Dame, Harvard University, Conservatoire National des Arts et Métiers, Emory University, National University of Singapore). Funding from BMGF was used for salaries of the Consortium secretariat (authors represented here: TBH, MJ, XL, SE-L, JT, KW, NMF, KAMG); and channelled via VIMC for travel and subsistence costs of all Consortium members (all authors). We also acknowledge funding from the UK Medical Research Council and Department for International Development, which supported aspects of VIMC's work (MRC grant number: MR/R015600/1).

MicroRNA-122 (miR-122) is the most abundant microRNA in hepatocytes and a central player in liver biology and disease. Herein, we report a previously unknown role for miR-122 in hepatocyte intrinsic innate immunity. Restoring miR-122 levels in hepatoma cells markedly enhanced the activation of interferons (IFNs) in response to a variety of viral nucleic acids or simulations, especially of hepatitis C virus RNA and poly (I:C). Mechanistically, miR-122 down-regulated the phosphorylation (Tyr705) of STAT3 and thereby removed the negative regulation of STAT3 on IFN-signaling. While STAT3 represses IFN expression by inhibiting interferon regulatory factor 1 (IRF1), miR-122 targets MERTK, FGFR1 and IGF1R, three receptor tyrosine kinases (RTKs) that directly promote STAT3 phosphorylation. This work identifies a miR-122-RTKs/STAT3-IRF1-IFNs regulatory circuitry, which may play a pivotal role in regulating hepatocyte innate immunity. These findings renewed our knowledge about miR-122's function and have important implications for treating hepatitis viruses.

Dietary restriction (DR) extends healthy lifespan in diverse species. Age and nutrient-related changes in the abundance of microRNAs (miRNAs) and their processing factors have been linked to organismal longevity. However, the mechanisms by which they modulate lifespan and the tissue-specific role of miRNA-mediated networks in DR-dependent enhancement of lifespan remains largely unexplored. We show that two neuronally enriched and highly conserved microRNAs, miR-125 and let-7 mediate the DR response in Drosophila melanogaster . Functional characterization of miR-125 demonstrates its role in neurons while its target chinmo acts both in neurons and the fat body to modulate fat metabolism and longevity. Proteomic analysis revealed that Chinmo exerts its DR effects by regulating the expression of FATP, CG2017, CG9577, CG17554, CG5009, CG8778, CG9527 , and FASN1 . Our findings identify miR-125 as a conserved effector of the DR pathway and open the avenue for this small RNA molecule and its downstream effectors to be considered as potential drug candidates for the treatment of late-onset diseases and biomarkers for healthy aging in humans.

In an effort to identify human endothelial cell (EC)-enriched lncRNAs,~500 lncRNAs were shown to be highly restricted in primary human ECs. Among them, lncEGFL7OS, located in the opposite strand of the EGFL7/miR-126 gene, is regulated by ETS factors through a bidirectional promoter in ECs. It is enriched in highly vascularized human tissues, and upregulated in the hearts of dilated cardiomyopathy patients. LncEGFL7OS silencing impairs angiogenesis as shown by EC/fibroblast co-culture, in vitro/in vivo and ex vivo human choroid sprouting angiogenesis assays, while lncEGFL7OS overexpression has the opposite function. Mechanistically, lncEGFL7OS is required for MAPK and AKT pathway activation by regulating EGFL7/miR-126 expression. MAX protein was identified as a lncEGFL7OS-interacting protein that functions to regulate histone acetylation in the EGFL7/miR-126 promoter/enhancer. CRISPR-mediated targeting of EGLF7/miR-126/lncEGFL7OS locus inhibits angiogenesis, inciting therapeutic potential of targeting this locus. Our study establishes lncEGFL7OS as a human/primate-specific EC-restricted lncRNA critical for human angiogenesis.

CAG/CTG trinuncleotide repeats are fragile sequences that when expanded form DNA secondary structures and cause human disease. We evaluated CAG/CTG repeat stability and repair outcomes in histone H2 mutants in S. cerevisiae. Although the two copies of H2A are nearly identical in amino acid sequence, CAG repeat stability depends on H2A copy 1 (H2A.1) but not copy 2 (H2A.2). H2A.1 promotes high-fidelity homologous recombination, sister chromatid recombination (SCR), and break-induced replication whereas H2A.2 does not share these functions. Both decreased SCR and the increase in CAG expansions were due to the unique Thr126 residue in H2A.1 and hta1Δ or hta1-T126A mutants were epistatic to deletion of the Polδ subunit Pol32, suggesting a role for H2A.1 in D-loop extension. We conclude that H2A.1 plays a greater repair-specific role compared to H2A.2 and may be a first step towards evolution of a repair-specific function for H2AX compared to H2A in mammalian cells.

Background: Patients affected by different types of autoimmune diseases, including common conditions such as Multiple Sclerosis (MS) and Rheumatoid Arthritis (RA), are often treated with immunosuppressants to suppress disease activity. It is not fully understood how the SARS-CoV-2 specific humoral and cellular immunity induced by infection and/or upon vaccination is affected by immunosuppressants. Methods: The dynamics of cellular immune reactivation upon vaccination of SARS-CoV-2 experienced MS patients treated with the humanized anti-CD20 monoclonal antibody ocrelizumab (OCR) and RA patients treated with methotrexate (MTX) monotherapy were analyzed at great depth via high-dimensional flow cytometry of whole blood samples upon vaccination with the SARS-CoV-2 mRNA-1273 (Moderna) vaccine. Longitudinal B and T cell immune responses were compared to SARS-CoV-2 experienced healthy controls (HC) before and 7-days after the first and second vaccination. Results: OCR-treated MS patients exhibit a preserved recall response of CD8 ⁺ T central memory cells following first vaccination compared to healthy controls and a similar CD4 ⁺ circulating T follicular helper 1 and T helper 1 dynamics, whereas humoral and B cell responses were strongly impaired resulting in absence of SARS-CoV-2 specific humoral immunity. MTX treatment significantly delayed antibody levels and B reactivation following the first vaccination, including sustained inhibition of overall reactivation marker dynamics of the responding CD4 ⁺ and CD8 ⁺ T cells. Conclusion: Together, these findings indicate that SARS-CoV-2 experienced MS-OCR patients still benefit from vaccination by inducing a broad CD8 ⁺ T cell response which can contribute to milder disease outcome. A delayed dynamics of vaccine-induced immunological recall in RA-MTX patients support repeated vaccine strategies to protect against future variants of concern, especially for these patients. Funding: This research project was supported by ZonMw (The Netherlands Organization for Health Research and Development, #10430072010007), the European Union’s Horizon 2020 research and innovation program under the Marie Skłodowska-Curie grant agreement (#792532 and #860003), the European Commission (SUPPORT-E, #101015756) and by PPOC (#20_21 L2506), the NHMRC Leadership Investigator Grant (#1173871).

During the development, tight regulation of the expansion of neural progenitor cells (NPCs) and their differentiation into neurons is crucial for normal cortical formation and function. In this study, we demonstrate that microRNA (miR)-128 regulates the proliferation and differentiation of NPCs by repressing pericentriolar material 1 (PCM1). Specifically, overexpression of miR-128 reduced NPC proliferation but promoted NPC differentiation into neurons both in vivo and in vitro. In contrast, the reduction of endogenous miR-128 elicited the opposite effects. Overexpression of miR-128 suppressed the translation of PCM1, and knockdown of endogenous PCM1 phenocopied the observed effects of miR-128 overexpression. Furthermore, concomitant overexpression of PCM1 and miR-128 in NPCs rescued the phenotype associated with miR-128 overexpression, enhancing neurogenesis but inhibiting proliferation, both in vitro and in utero. Taken together, these results demonstrate a novel mechanism by which miR-128 regulates the proliferation and differentiation of NPCs in the developing neocortex.

High-grade serous ovarian cancer is characterized by extensive copy number alterations, among which the amplification of MYC oncogene occurs in nearly half of tumors. We demonstrate that ovarian cancer cells highly depend on MYC for maintaining their oncogenic growth, indicating MYC as a therapeutic target for this difficult-to-treat malignancy. However, targeting MYC directly has proven difficult. We screen small molecules targeting transcriptional and epigenetic regulation, and find that THZ1 - a chemical inhibiting CDK7, CDK12, and CDK13 - markedly downregulates MYC. Notably, abolishing MYC expression cannot be achieved by targeting CDK7 alone, but require the combined inhibition of CDK7, CDK12, and CDK13. In 11 patient derived xenografts models derived from heavily pre-treated ovarian cancer patients, administration of THZ1 induces significant tumor growth inhibition with concurrent abrogation of MYC expression. Our study indicates that targeting these transcriptional CDKs with agents such as THZ1 may be an effective approach for MYC-dependent ovarian malignancies.

Tuberculosis is a respiratory disease that is treatable with antibiotics. An increasing prevalence of resistance means that to ensure a good treatment outcome it is desirable to test the susceptibility of each infection to different antibiotics. Conventionally this is done by culturing a clinical sample and then exposing aliquots to a panel of antibiotics, Using 96-well broth micro dilution plates with each well containing a lyophilised predetermined amount of an antibiotic is a convenient and cost-effective way to measure the MICs of several drugs at once for a clinical sample. Although accurate, this is still an expensive and slow process that requires highly skilled and experienced laboratory scientists. Here we show that, through the BashTheBug project hosted on the Zooniverse citizen science platform, a crowd of volunteers can reproducibly and accurately determine the MICs for 13 drugs and that simply taking the median or mode of 11-17 independent classifications is sufficient. There is therefore a potential role for crowds to support (but not supplant) the role of experts in antibiotic susceptibility testing.

Cell identity is characterized by a distinct combination of gene expression, cell morphology, and cellular function established as progenitor cells divide and differentiate. Following establishment, cell identities can be unstable and require active and continuous maintenance throughout the remaining life of a cell. Mechanisms underlying the maintenance of cell identities are incompletely understood. Here, we show that the gene ctbp-1, which encodes the transcriptional corepressor C-t erminal b inding p rotein-1 (CTBP-1), is essential for the maintenance of the identities of the two AIA interneurons in the nematode Caenorhabditis elegans. ctbp-1 is not required for the establishment of the AIA cell fate but rather functions cell-autonomously and can act in later larval stage and adult worms to maintain proper AIA gene expression, morphology and function. From a screen for suppressors of the ctbp-1 mutant phenotype, we identified the gene egl-13, which encodes a SOX family transcription factor. We found that egl-13 regulates AIA function and aspects of AIA gene expression, but not AIA morphology. We conclude that the CTBP-1 protein maintains AIA cell identity in part by utilizing EGL-13 to repress transcriptional activity in the AIAs. More generally, we propose that transcriptional corepressors like CTBP-1 might be critical factors in the maintenance of cell identities, harnessing the DNA-binding specificity of transcription factors like EGL-13 to selectively regulate gene expression in a cell-specific manner.

Macrophages are a highly adaptive population of innate immune cells. Polarization with IFNγ and LPS into the 'classically activated' M1 macrophage enhances pro-inflammatory and microbicidal responses, important for eradicating bacteria such as Mycobacterium tuberculosis . By contrast, 'alternatively activated' M2 macrophages, polarized with IL-4, oppose bactericidal mechanisms and allow mycobacterial growth. These activation states are accompanied by distinct metabolic profiles, where M1 macrophages favor near exclusive use of glycolysis, whereas M2 macrophages up-regulate oxidative phosphorylation (OXPHOS). Here we demonstrate that activation with IL-4 and IL-13 counterintuitively induces protective innate memory against mycobacterial challenge. In human and murine models, prior activation with IL-4/13 enhances pro-inflammatory cytokine secretion in response to a secondary stimulation with mycobacterial ligands. In our murine model, enhanced killing capacity is also demonstrated. Despite this switch in phenotype, IL-4/13 trained murine macrophages do not demonstrate M1-typical metabolism, instead retaining heightened use of OXPHOS. Moreover, inhibition of OXPHOS with oligomycin, 2-deoxy glucose or BPTES all impeded heightened pro-inflammatory cytokine responses from IL-4/13 trained macrophages. Lastly, this work identifies that IL-10 attenuates protective IL-4/13 training, impeding pro-inflammatory and bactericidal mechanisms. In summary, this work provides new and unexpected insight into alternative macrophage activation states in the context of mycobacterial infection.

UNC-13 proteins play an essential role in synaptic transmission by recruiting synaptic vesicles (SVs) to become available for release, which is termed SV priming. Here we show that the C2A domain of UNC-13L, like the corresponding domain in mammalian Munc13-1, displays two conserved binding modes: forming C2A/C2A homodimers, or forming a heterodimer with the zinc finger domain of UNC-10/RIM (C2A/RIM). Functional analysis revealed that UNC-13L’s C2A promotes synaptic transmission by regulating a post-priming process. Stimulus-evoked release but not SV priming, was impaired in unc-10 mutants deficient for C2A/RIM heterodimerization, leading to decreased release probability. Disrupting C2A/C2A homodimerization in UNC-13L-rescued animals had no effect on synaptic transmission, but fully restored the evoked release and the release probability of unc-10/RIM mutants deficient for C2A/RIM heterodimerization. Thus, our results support the model that RIM binding C2A releases UNC-13L from an autoinhibitory homodimeric complex to become fusion-competent by functioning as a switch only.

1300-nm three-photon calcium imaging has emerged as a useful technique to allow calcium imaging in deep brain regions. Application to large-scale neural activity imaging entails a careful balance between recording fidelity and tissue heating. We calculated and experimentally verified the excitation pulse energy to achieve the minimum photon count required for the detection of calcium transients in GCaMP6s-expressing neurons for 920-nm two-photon and 1320-nm three-photon excitation, respectively. Brain tissue heating by continuous three-photon imaging was simulated with Monte Carlo method and experimentally validated with immunohistochemistry. We observed increased immunoreactivity with 150 mW excitation power at 1.0- and 1.2-mm imaging depths. Based on the data, we explained how three-photon excitation achieves better calcium imaging fidelity than two-photon excitation in the deep brain and quantified the imaging depth where three-photon microscopy should be applied. Our analysis presents a translatable model for the optimization of three-photon calcium imaging based on experimentally tractable parameters.

The proper development and function of neuronal circuits relies on a tightly regulated balance between excitatory and inhibitory (E/I) synaptic transmission, and disrupting this balance can cause neurodevelopmental disorders, e.g. schizophrenia. microRNA-dependent gene regulation in pyramidal neurons is important for excitatory synaptic function and cognition, but its role in inhibitory interneurons is poorly understood. Here, we identify miR138-5p as a regulator of short-term memory and inhibitory synaptic transmission in the mouse hippocampus. Sponge-mediated miR138-5p inactivation specifically in mouse parvalbumin (PV)-expressing interneurons impairs spatial recognition memory and enhances GABAergic synaptic input onto pyramidal neurons. Cellular and behavioural phenotypes associated with miR138-5p inactivation are paralleled by an upregulation of the schizophrenia-associated Erbb4 , which we validated as a direct miR138-5p target gene. Our findings suggest that miR138-5p is a critical regulator of PV interneuron function in mice, with implications for cognition and schizophrenia. More generally, they provide evidence that microRNAs orchestrate neural circuit development by fine-tuning both excitatory and inhibitory synaptic transmission.

Dendritic spines are the central postsynaptic machinery that determines synaptic function. The F-actin within dendritic spines regulates their dynamic formation and elimination. Rai14 is an F‑actin-regulating protein with a membrane‑shaping function. Here, we identified the roles of Rai14 for the regulation of dendritic spine dynamics associated with stress-induced depressive-like behaviors. Rai14-deficient neurons exhibit reduced dendritic spine density in the Rai14 +/- mouse brain, resulting in impaired functional synaptic activity. Rai14 was protected from degradation by complex formation with Tara, and accumulated in the dendritic spine neck, thereby enhancing spine maintenance. Concurrently, Rai14 deficiency in mice altered gene expression profile relevant to depressive conditions and increased depressive-like behaviors. Moreover, Rai14 expression was reduced in the prefrontal cortex of the mouse stress model, which was blocked by antidepressant treatment. Thus, we propose that Rai14-dependent regulation of dendritic spines may underlie the plastic changes of neuronal connections relevant to depressive-like behaviors.

The core CoREST complex (LHC) contains histone deacetylase HDAC1 and histone demethylase LSD1 held together by the scaffold protein CoREST. Here we analyze the purified LHC with modified peptide and reconstituted semisynthetic mononucleosome substrates. LHC demethylase activity toward methyl-Lys4 in histone H3 is strongly inhibited by H3 Lys14 acetylation, and this appears to be an intrinsic property of the LSD1 subunit. Moreover, the deacetylase selectivity of LHC unexpectedly shows a marked preference for H3 acetyl-Lys9 versus acetyl-Lys14 in nucleosome substrates but this selectivity is lost with isolated acetyl-Lys H3 protein. This diminished activity of LHC to Lys-14 deacetylation in nucleosomes is not merely due to steric accessibility based on the pattern of sensitivity of the LHC enzymatic complex to hydroxamic acid-mediated inhibition. Overall, these studies have revealed how a single Lys modification can confer a composite of resistance in chromatin to a key epigenetic enzyme complex involved in gene silencing.

The intermediate filament protein keratin 14 (K14) provides vital structural support in basal keratinocytes of epidermis. Recent studies evidenced a role for K14-dependent disulfide bonding in the organization and dynamics of keratin IFs in skin keratinocytes. Here we report that knock-in mice harboring a cysteine-to-alanine substitution at Krt14's codon 373 (C373A) exhibit alterations in disulfide-bonded K14 species and a barrier defect secondary to enhanced proliferation, faster transit time and altered differentiation in epidermis. A proteomics screen identified 14-3-3 as K14 interacting proteins. Follow-up studies showed that YAP1, a transcriptional effector of Hippo signaling regulated by 14-3-3sigma in skin keratinocytes, shows aberrant subcellular partitioning and function in differentiating Krt14 C373A keratinocytes. Residue C373 in K14, which is conserved in a subset of keratins, is revealed as a novel regulator of keratin organization and YAP function in early differentiating keratinocytes, with an impact on cell mechanics, homeostasis and barrier function in epidermis.

Adolescence is a common time for initiation of alcohol use and development of alcohol use disorders. The present study investigates neuroanatomical predictors for trajectories of future alcohol use based on a novel voxel-wise whole-brain structural equation modeling framework. In 1814 healthy adolescents of the IMAGEN sample, the Alcohol Use Disorder Identification Test (AUDIT) was acquired at three measurement occasions across five years. Based on a two-part latent growth curve model, we conducted whole-brain analyses on structural MRI data at age 14, predicting change in alcohol use score over time. Higher grey-matter volumes in the caudate nucleus and the left cerebellum at age 14 years were predictive of stronger increase in alcohol use score over 5 years. The study is the first to demonstrate the feasibility of running separate voxel-wise structural equation models thereby opening new avenues for data analysis in brain imaging.

Motors proteins of the conserved kinesin-14 family have important roles in mitotic spindle organization and chromosome segregation. Previous studies have indicated that kinesin-14 motors are non-processive enzymes, working in the context of multi-motor ensembles that collectively organize microtubule networks. Here we show that the yeast kinesin-14 Kar3 generates processive movement as a heterodimer with the non-motor proteins Cik1 or Vik1. By analyzing the single-molecule properties of engineered motors we demonstrate that the non-catalytic domain has a key role in the motility mechanism by acting as a "foothold" that allows Kar3 to bias translocation towards the minus end. This mechanism rivals the speed and run length of conventional motors, can support transport of the Ndc80 complex in vitro and is critical for Kar3 function in vivo. Our findings provide an example for a non-conventional translocation mechanism and help to explain how Kar3 can substitute for key functions of Dynein in the yeast nucleus.

Iron is essential for survival of most organisms. All organisms have thus developed mechanisms to sense, acquire and sequester iron. In C. elegans, iron uptake and sequestration are regulated by HIF-1. We previously showed that hif-1 mutants are developmentally delayed when grown under iron limitation. Here we identify nhr-14, encoding a nuclear receptor, in a screen conducted for mutations that rescue the developmental delay of hif-1 mutants under iron limitation. nhr-14 loss upregulates the intestinal metal transporter SMF-3 to increase iron uptake in hif-1 mutants. nhr-14 mutants display increased expression of innate immune genes and DAF-16/FoxO-Class II genes, and enhanced resistance to Pseudomonas aeruginosa. These responses are dependent on the transcription factor PQM-1, which localizes to intestinal cell nuclei in nhr-14 mutants. Our data reveal how C. elegans utilizes nuclear receptors to regulate innate immunity and iron availability, and show iron sequestration as a component of the innate immune response.

Shade avoidance syndrome enables shaded plants to grow and compete effectively against their neighbors. In Arabidopsis, the shade-induced de-phosphorylation of the transcription factor PIF7 (PHYTOCHROME-INTERACTING FACTOR 7) is the key event linking light perception to stem elongation. However, the mechanism through which phosphorylation regulates the activity of PIF7 is unclear. Here, we show that shade light induces the de-phosphorylation and nuclear accumulation of PIF7. Phosphorylation-resistant site mutations in PIF7 result in increased nuclear localization and shade-induced gene expression, and consequently augment hypocotyl elongation. PIF7 interacts with 14-3-3 proteins. Blocking the interaction between PIF7 and 14-3-3 proteins or reducing the expression of 14-3-3 proteins accelerates shade-induced nuclear localization and de-phosphorylation of PIF7, and enhances the shade phenotype. By contrast, the 14-3-3 overexpressing line displays an attenuated shade phenotype. These studies demonstrate a phosphorylation-dependent translocation of PIF7 when plants are in shade and a novel mechanism involving 14-3-3 proteins, mediated by the retention of PIF7 in the cytoplasm that suppresses the shade response.

The cytoskeleton network of eukaryotic cells is essential for diverse cellular processes, including vesicle trafficking, cell motility and immunity, thus is a common target for bacterial virulence factors. A number of effectors from the bacterial pathogen Legionella pneumophila have been shown to modulate the function of host actin cytoskeleton to construct the Legionella-containing vacuole (LCV) permissive for its intracellular replication. In this study, we found that the Dot/Icm effector Lem8 (Lpg1290) is a protease whose activity is catalyzed by a Cys-His-Asp motif known to be associated with diverse biochemical activities. Intriguingly, we found that Lem8 interacts with the host regulatory protein 14-3-3ζ, which activates its protease activity. Furthermore, Lem8 undergoes self-cleavage in a process that requires 14-3-3ζ. We identified the Pleckstrin homology-like domain-containing protein Phldb2 involved in cytoskeleton organization as a target of Lem8 and demonstrated that Lem8 plays a role in the inhibition of host cell migration by attacking Phldb2.