eLife Sciences

Published by eLife Sciences Publications
Online ISSN: 2050-084X
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.
Structure of the ClpXP complex. (A) Side and (B) top-down views of the ClpXP complex bound to a substrate (shown in orange). The six ClpX subunits are each shown in a different color (purple, blue, cyan, green, sage green, and yellow), with ATP shown in red and ADP in pink. The same colors are used throughout the figure. (C) Close-up side view of the pore region in ClpX with arrows indicating the proposed sequential (sage green arrow; Ripstein et al., 2020) and probabilistic (purple arrow; Fei et al., 2020) mechanisms. The tyrosines lining the ClpX pore are colored according to their corresponding ClpX subunit. Five of these tyrosines bind the substrate while the tyrosine of the yellow subunit, which is bound to ADP, does not contact the substrate. The ATP hydrolyzed in each of the models is indicated by a flash. In the sequential model (Ripstein et al., 2020), ATP hydrolysis in the lower subunit allows it to disengage from substrate, transition through the 'yellow state', exchange ADP for ATP, and bind the next two residues of the substrate by docking against the top-most subunit. Because each subunit binds two residues, the net result is ClpX 'walking' up the substrate and translocation of two substrate residues down toward ClpP. In the probabilistic model (Fei et al., 2020), ATP hydrolysis at the top position (sometimes preceded by hydrolysis at other positions) causes the upper subunit to maintain a tight grip on substrate and move it down toward the 'yellow state', thereby translocating substrate by approximately six residues toward ClpP.
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.
Whole striatal dopamine synthesis capacity by group. Individuals who suffered macroscopic perinatal brain injury related to VPT birth had significantly lower dopamine synthesis capacity in the whole striatum compared to other adults born VPT with no macroscopic perinatal brain injury (corrected p=0.023, Cohen's d = 1.36) and full term-born controls (corrected p=0.01, Cohen's d = 1.07). DOI: https://doi.org/10.7554/eLife.29088.004 The following source data is available for figure 1: Source data 1. fig1_source_data.csv-Can be used with create_fig1.m to recreate DOI: https://doi.org/10.7554/eLife.29088.005 
Neonatal, socio-demographic, cognitive and scanning measures.
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Striatal subregion dopamine synthesis capacity †.
Subcortical volumes (mm 3 ) *.
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.
Multi-electrode array monitoring of iPSC-derived glutamatergic neurons. (A-E) Weighted mean firing rate (wMFR) of pooled cell lines from control and KO neurons for each family from week 4 to 8 PNI. (F) Dot plots showing wMFR of each cell line from week 4 to 8 PNI; each dot represents the wMFR of one well, and the color reflects independent experiments. Values are presented as mean ± SEM of several technical and biological replicates, as presented in Supplementary file 3; 'all controls' represents the pool of 311 different control wells from 17 independent experiments; Figure 3 continued on next page
Electrophysiological and protein level variations in EHMT2-deficient neurons. (A) Network burst frequency was recorded using MEA from the EHMT2/UBE2I family from weeks 4-8 PNI; values are presented as mean ± SEM of several technical and biological replicates, as presented in Supplementary file 3; *p<0.05 from multiple t test comparison with Holm-Sidak correction. (B) Patch-clamp recordings of two selected lines, that is, 37E (control) and 38E (mutant); values are presented as mean ± SEM of 14 different neurons from two independent differentiation experiments; *p<0.05 from from unpaired t test two-tailed. (C) Western blot showing a decrease in EHMT2 protein levels in mutant neurons (38B and 38E) compared to their respective control neurons (37E). (D) MEA recordings of the isogenic pair 19-2 and 19-2-EHMT2 StopTag/+ iPSC-derived neurons from weeks 4-11 PNI; values are presented as mean ± SEM of eight different wells for each three independent differentiation experiments; †note that the same data for control 19-2 was used in Figure 4B since it was generated within the same experiments, that is, plates 26, 33 and 37 (see Supplementary file 3); *p<0.05 from multiple t test comparison with Holm-Sidak correction at week 11 (weighted mean firing rate) and weeks 9-11 (network burst frequency). (E) Western blot showing a decrease in EHMT2 protein levels in mutant neurons 19-2-EHMT2 StopTag/+ compared to their respective control (ctrl) neurons 19-2; actin-beta (ACTB) was used as a loading control and the relative intensity of each band is indicated below the blots; pF, picofarad; MW, megaohm; Hz, hertz; pA, picoampere. Figure 6 continued on next page
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 ).
Chemical structure of compounds. Structures of DRV, GRL-139, GRL-036, GRL-121, and GRL-142. P2-Crn-THF and P2'-Cp-Abt moieties are shown in red and blue, respectively. DOI: https://doi.org/10.7554/eLife.28020.002 
GRL-142 has significantly more potent HIV-1 protease dimerization inhibition activity, much greater thermal stability, and much higher intracellular concentration than DRV. (A) The FRET-based HIV-1 expression assay system detecting HIV-1 PR dimerization and its disruption by DRV or GRL-142. In an attempt to elucidate the dynamics of HIV-1 PR dimerization and the mechanisms of the emergence of HIV-1 resistance against certain PR inhibitors (PIs), we developed an intermolecular FRET-based HIV-1 expression assay system using CYP-and YFP-tagged PR monomers. Using this FRET-based HIV-1 expression assay system, we have previously identified nonpeptidyl small-molecule inhibitors of HIV-1 PR dimerization, including DRV (Koh et al., 2007; Aoki et al., 2012). Various plasmids encoding full-length molecular infectious HIV-1 (HIV-1 NL4-3 ) clones producing CFP-or YFPtagged PR using the PCR-mediated recombination method were prepared. A linker consisting of five alanines was inserted between PR and fluorescent protein. A phenylalanine-proline site (F/P) that HIV-1 PR cleaves was also introduced between the fluorescent protein and reverse transcriptase. (Lower) Structural representations of PR monomers and dimer in association with the linker atoms and fluorescent proteins. FRET occurs only when the fluorescent proteins are 1-10 nm apart. (B) GRL-142 more potently inhibits PR WT dimerization by a factor of ~1000 than DRV. COS7 cells were exposed to various concentrations (0.01 to 1000 nM) of GRL-142 or DRV and were subsequently co-transfected with two plasmids, pHIV-PR WT-CFP and pHIVPR WT-YFP , respectively. After 72 hr, cultured cells were examined in the FRET-based HIV-1 expression assay and the CFP A/B ratios (Y axis) were determined. The arithmetic mean values of the ratios obtained are shown as horizontal bars. A CFP A/B ratio that is greater than one signifies that protease dimerization occurred, whereas a ratio that is less than one signifies that the disruption of protease dimerization occurred (Koh et al., 2007; Aoki et al., 2012). All the experiments were conducted in a blind fashion. The P values were determined using the Wilcoxon rank-sum test (JMP software, SAS, Cary, NC) and were 0.6721 for the CFP A/B ratio in the absence of drug (CFP A/B No-Drug ) versus the CFP A/B ratio in the presence of 10 nM DRV (CFP A/B 10-DRV ), 0.0262 for CFP A/B No-Drug versus CFP A/B 100-DRV , 0.2483 for CFP A/B No-Drug versus CFP A/B 0.01-GRL-142 , 0.0585 for CFP A/B No-Drug versus CFP A/B 0.1-GRL-142 , 0.0145 for CFP A/B No-Drug versus CFP A/B 1-GRL-142 , 0.0042 for CFP A/B No-Drug versus CFP A/B 10-GRL-142 , 0.0056 for CFP A/B No-Drug versus CFP A/
X-ray crystal structure analysis of GRL-142 with HIV-1 protease. (A) X-ray crystal structure of wild-type HIV-1 protease (PR WT ) in complex with DRV or GRL-142. The crystal structure of GRL-142 in complex PR WT was solved (PDB ID: 5TYS). The polar interactions of GRL-142 with protease residues in the active site are shown, and the interactions of DRV with PR WT (PDB ID: 4HLA) are shown for comparison. Cross-section of protease backbone is shown in green and red ribbons. The carbon atoms of GRL-142 and DRV are shown in off-white and green respectively. Nitrogen, oxygen, sulfur, Figure 3 continued on next page 
Antiviral activity of GRL-142 against viruses obtained after in vitro selection.
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.
Structures of the mimivirus genomic fiber for Cl1a [A-C], Cl3a [D-F], and Cl2
RNA polymerase could be associated to the genomic fiber. A] Micrograph of
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.
Phylogenetic relationships and gene copies in study subjects with partial AZFc deletions. (A) Y-chromosomal lineages indicated with typed terminal markers (left), deleted (white)/retained (black) DAZ and CDY1 gene copies (middle), and secondary rearrangements in the AZFc region (right) of idiopathic male factor infertility (n = 31) and reference cases (n = 13) carrying the gr/gr deletion. The human Y-chromosomal reference sequence has four DAZ and two CDY1 copies; the retained gene copies on each Y chromosome with a gr/gr deletion are shown as filled boxes. Chromosomes Figure 2 continued on next page
Complex structural variants at the Y-chromosomal lineage R1a1-M458 and their effect on andrological parameters. (A) Schematic presentation of the Y chromosome with the r2/r3 inversion compared to the reference sequence. The r2/r3 inversion structure nearly destroys the large palindrome P1 and, consequently, destabilizes the AZFc region since several long DNA amplicons with highly similar DNA sequence (b2, b3, and b4; g2 and g3; y1 and y3) are positioned in the same sequence orientation. This structure promotes non-allelic homologous recombination mediating Figure 3 continued on next page
Summary of the identified Y-chromosomal AZF deletion subtypes.
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.
Flow chart depiction of the assembly pipeline.
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.
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STAT3 silencing inhibited the recruitment of Pol III transcription machinery components to Pol III target loci. (A and B) STAT3 knockdown reduced the occupancies of Pol III transcription machinery components at the DNA locus encoding 5S rRNA. Chromatin immunoprecipitation (ChIP) assays were performed using HepG2 cell lines stably expressing Ctrl shRNA or STAT3 shRNA and antibodies against the indicated factors. Relative enrichment (A) was obtained by comparing the quantity of target DNA in 1 μL of ChIP DNA sample to that in 0.02% of input DNA sample. Fold change (B) was obtained by comparing the mean of relative enrichment of target DNA in a control shRNA sample to that in a STAT3 shRNA sample obtained in A. (C and D) STAT3 knockdown decreased the recruitment of TBP, BRF2, and POLR3A to the DNA locus encoding U6 snRNA. Relative enrichment (C) and fold change (D) were obtained as described in A and B, respectively. (E and F) STAT3 knockdown inhibited the recruitment of Pol III transcription machinery components to the DNA locus encoding 7SL RNA. Relative enrichment (E) and fold change (F) were obtained as described in A and B, respectively. (G and H) STAT3 knockdown dampened the recruitment of Pol III transcription machinery components to the tRNA-Met promoter. Relative enrichment (G) and fold change (H) were obtained as described in A and B, respectively. (I) STAT3 did not bind to the DNA loci encoding 5S rRNA, U6 RNA, 7SL RNA, and tRNA-met but BCL2 promoter. ChIP assays were performed using HepG2 cells and an anti-STAT3 antibody. Relative enrichment (G) was obtained as described in A. (J and K) STAT3 silencing did not affect the expression of Pol III transcription factor subunits. HepG2 (J) and 293T (K) cell lines with STAT3 depletion were used to analyze the expression of pol III transcription factor subunits by western blot. Each column in A, C, E, Figure 3 continued on next page
Effect of STAT3 silencing on genome-wide mRNA expression in HePG2 cells. (A) A Heatmap showing the result of hierarchical cluster analysis based on the mRNA-seq data derive from HepG2 cell lines stably expressing control shRNA or STAT3 shRNA. (B) A volcano plot showing the number of significantly upregulated or downregulated genes obtained from the analysis of the RNA-seq data. (C) STAT3 silencing did not affect the Figure 4 continued on next page
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.
Cryo-EM structures of F 1 F O ATP synthase inhibited by ADP in three rotary states, and 166 measurement of changes in rotational position between catalytic dwells. A Rotational State-1, pdb-ID 6OQU 167 (25). B State-2, pdb-ID 6OQV, with rotor 120° CCW from A where subunit- is not shown to reveal subunit-. 168 C State-3, pdb-ID 6WNR, with rotor 240° CCW from A showing microscope slide assembly of F 1 F O embedded 169 in a lipid bilayer nanodisc (LBN) for rotation measurements. His 6 -tags (HT) on -subunit C-termini enabled 170 attachment to slide, while the gold nanorod (AuNR) coated with streptavidin (SA) bound to the biotinylated 171 subunit-c ring. D Rotational position of single F 1 F O molecules versus time was monitored by intensity changes 172 of polarized red light scattered from the AuNR in the presence of 1 mM Mg 2+ ATP, which enabled F 1 -ATPase-173 dependent 120° CCW power strokes between catalytic dwells (green bars). Prior to data collection at 200 kHz, a 174 polarizer in the scattered light path was rotated to minimize intensity during one of the three catalytic dwells. 175 Light intensity increased to a maximum upon rotation by 90° during the subsequent CCW 120° power stroke. For 176 each molecule the angular dependence of these power strokes versus time was analyzed. 177
Proportion of low (blue bar), medium (orange bar), and high (green bar) TD formation 327 efficiencies relative to WT low efficiency TD formation. Each was the average of all pH values examined. 328 Vertical bars represent standard error. 329 330
Aqueous Funnel of charged and polar groups can serve as an antenna to supply protons to the 475 input channel. A Transmembrane view of E. coli F O (pdb-ID 6OQR) showing the path of charged and polar 476 residues across the membrane. Outlines indicate space occupied by hydrophobic residues in subunit-a (black 477 line), subunit-b 1 (blue line), and subunit-b 2 (orange line). The inner surface of the Funnel, which is lined with 478 polar residues and loop regions (green), acidic groups (red) and histidines (blue) from subunit-a and the subunit-479 b 1 N-terminus, is exposed to the periplasm at its wide end that narrows to aE219 (yellow) at the bottom. The 480 input channel (yellow) extends from aE219 to aN214 and aQ252, which are proximal to aR210. Between aR210 481 and the Output Channel (dark blue) the leading (pink) and lagging (orange) cD61 groups rotate through a 482 Vestibule lined above and below the cD61 rotation plane by polar sidechains (light blue) that decrease the 483 dielectric constant of the vestibule from that of the lipid bilayer. A protonated cD61 exposed to the lipid bilayer 484 (yellow) is also visible. B Periplasmic surface of F O showing the interior surface of the Funnel (orange oval) 485 lined with charged and polar groups from subunit-a and subunit-b 1 as in A that narrows to aE219 (yellow) at the 486 bottom where the input channel begins. Hydrophobic residues are shown of subunit-a (gray), subunits-b 1 and b 2 487 (black), and the c-ring (white). The cY3 sidechains (orange) are shown to indicate the orientation of the 488 periplasmic surface of the c-ring. 489
Alternating 11° and 25° sub-steps that power F O c-ring ATP synthase direction rotation. A The 549 pH-dependent 11° sub-step occurs when H + transfer from aN214/aQ25-bound water to the unprotonated leading 550 cD61-carboxyl (pink), and from the protonated lagging cD61-carboxyl (orange) to aS199/aE196-bound water. 551 Upon displacement from aR210 by protonation, leading cD61 adopts the closed conformation to enable rotation 552 into the lipid bilayer (yellow). Due to coherent H + movement in the Grotthuss column, each H + entering the input 553 channel (green) from the funnel causes a H + to exit the output channel (blue) to the cytoplasm. Rotation occurs 554 when lagging cD61 is deprotonated because the negatively charged carboxyl moves in response to the decrease 555 in hydrophobicity from the lipid bilayer to the water-containing vestibule (blue), and from the electrostatic 556 attraction to aR210. This decreases the distance between the lagging cD61 carboxyl and the aR210-guanidinium 557 from ~11.5 Å to ~7.5 Å. B The 25° sub-step occurs primarily from the electrostatic interaction between the lagging 558 cD61 carboxy (orange) and the aR210 guanidinium. C Electrostatic attraction decreases the distance between 559 orange cD61 and aR210 from ~7.5 Å to ~3.5 Å to complete a 36° stepwise c-ring rotation, which positions the 560 orange cD61 to become the leading carboxyl for the next pH-dependent 11° sub-step. E. coli F 1 F O cryo-EM 561 structures of rotary sub-states pdb-IDs 5OQS (A and C), and 5OQR (B) are shown as cross-sections of F O with 562 hydrophobic resides of subunit-a (brown) and the c-ring (gray) along the plane defined by cD61 groups as viewed 563 from the periplasm. Protons are alternately colored black and white to show the progression of proton transfer 564 events. 565
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.
H3pT11 responds to nutritional stress. (A) H3pT11 levels in the media containing different concentration of glucose measured by western blots. Wild type (BY4741) yeast cultures at early mid-log phase (OD 0.4) were shifted from YPD to YP media containing 0.02, 0.2, or 2% glucose for 1 hr at 30˚C30˚30˚C. (B) H3pT11 levels in the media containing non-fermentable glycerol measured by western blots. WT cultures were shifted from YPD to YP with 3% glycerol for indicated times. (C) The averaged profiles of H3pT11 at 366 genes, whose H3pT11 levels are increased in YPglycerol (Glycerol) Figure 1 continued on next page 
Sch9 regulates H3pT11 upon nutritional stress. (A) H3pT11 levels in WT, sch9D, ras2D, and tor1D mutants upon media shift to YPglycerol measured by western blots. (B) In vitro kinase assay of TAP purified (Sch9-TAP) Sch9 and CK2 using recombinant H3 as a substrate. (C) (Left) H3pT11 levels in WT, sch9D, cka1D, and sch9Dcka1D at 1 hr after media shift from YPD to YPglycerol analyzed by western blots (Right) The relative ratios of H3pT11 to H3 signals are presented with error bars indicating STD from three biological replicates. (D) The levels of chromatin bound Cka1 (Left) or Sch9 (Right) upon media shifting to YPglycerol measured by western blots. Asterisks (*) indicate nonspecific bands. DOI: https://doi.org/10.7554/eLife.36157.012 
H3pT11 affects CLS by regulation of acetic acid resistance. (A) Relative viability of H3T11A and cka1D mutants compared to their WT strains after exposure to indicated durations and concentrations of acetic acid. (B) Acetic acid resistance of WT, sch9D, cka1D, and sch9Dcka1D. (C) CLS assays of WT and H3T11A strains in buffered (pH 6.0) or unbuffered conditions. (D) H3pT11 levels in WT and cka1D upon 50 mM acetic acid addition analyzed by western blots (upper). The relative band intensities of H3pT11 to H3 signals (lower). (E) H3pT11 levels in WT, sch9D, cka1D, and sch9Dcka1D at 2 hr Figure 6 continued on next page 
Summary models of H3pT11 functions upon stress conditions. Additional files. DOI: https://doi.org/10.7554/eLife.36157.019 
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.
Engineering a compact Desulfonema ishimotonii Cas7-11 (DiCas7-11). (a) Schematic of domain organization of wild-type and an insertion deletion variant DiCas7-11-Δint1. The region removed is colored in purple and numbered. (b) Cartoon representation of DiCas7-11 overlaying with density map resulted from focused classification using a mask around the insertion domain. The insertion structure model is from AlphaFold prediction. (c) Target RNA cleavage by DiCas7-11 (WT) and DiCas7-11-Δint1(Δint1) are analyzed on a polyacrylamide urea gel. Cleavage products (P) are stained by SYBR Gold and imaged by ChemiDoc MP and are indicated by green triangles. The online version of this article includes the following source data for figure 4: Source data 1. Polyacrylamide gel image showing target RNA cleavage activity by DiCas7-11-Δint1.
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.
Mean predicted deaths due to the 10 Vaccine Impact Modelling Consortium (VIMC) pathogens per 100,000 population per country for years 2000-2019 under the no vaccination and with vaccination (routine immunisations; RI only) scenarios. Countries are arranged by World Health Organisation (WHO) African (AFRO), Eastern Mediterranean (EMRO), European (EURO), Pan American (PAHO), South-East Asian (SEARO), and Western Pacific (WPRO) regions. The difference (i.e. deaths averted) between these two scenarios are shown in Table 2 and Figure 2.
Deaths averted per year of vaccination for hepatitis B (HepB), Haemophilus influenzae type b (Hib), human papillomavirus (HPV), Japanese encephalitis (JE), measles, Neisseria meningitidis serogroup A (MenA), Streptococcus pneumoniae (PCV), rotavirus (Rota), rubella, and yellow fever (YF). The bars show the number of deaths averted (in millions) in each vaccination year. Error bars indicate 95% CI. The line shows the number of fully vaccinated persons (FVPs; in millions) achieved in each year's vaccination activities.
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).
figure supplement 2
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.
Chinmo is nutrient-dependent downstream effector of the DR-pathway. (A) Chinmo protein levels increase in adult fat tissue of w 1118 flies that were fed a DR diet for 10 days. Confocal images of dissected fat body from adult w 1118 flies that were fed an AL or DR diet for 10 days. Immunostaining was performed with anti-Chinmo, anti-La (nuclear protein), and Dapi. Scale bar, 10 mm. (B, C) Quantitation of Chinmo protein levels in adult fat tissue nuclear and peripheral (non-nuclear) regions as determined by measuring pixel intensity in 10 cells in five samples per condition. Error bars represent Figure 3 continued on next page
Export of Chinmo from the nucleus is dependent on deacetylation of Chinmo by dSir2. (A) Chinmo redistributes to the cytoplasm upon cell starvation. Kc167 cells were transfected with Tubulin Gal4 and UAS-GFP::Chinmo constructs and redistribution of Chinmo in fed (upper panel) and starved (lower panel) cells was examined by immunostaining with GFP antibody (Chinmo), Fmr1 (cytoplasmic marker) and Dapi (blue). Chinmo is predominantly localized to the nucleus in the fed state and redistributes to the cytoplasm in starved Kc167 cells. (B, C) Localization of GFP::Chinmo in Figure 4 continued on next page
Ectopic expression of chinmo in adult neurons and fat body mimics miR-125 mutant DR phenotype. UAS chinmo was expressed in adult neurons and adult fat tissue using the steroid (RU-486) inducible gene switch Gal4 drivers. (A) Female flies that were fed an RU-486 supplemented diet for 5 days displayed increased levels of Chinmo in neuronal cells, as detected by Chinmo. Woc (nuclear marker) and Dapi staining of dissected adult fly brains. (B, D) 3X ElavGS >UAS chinmo (B) or 3X ElavGS >UAS Flag chinmo (D) flies that were not fed RU-486 show a significant increase in lifespan upon DR (DR, blue line) as compared to 3X ELavGS >UAS chinmo (B) or 3X ElavGS >UAS Flag chinmo (D) flies that were fed an ad libitum (AL) diet (red line)(3X ElavGS >UAS chinmo: AL-, n = 98; median lifespan = 32 d; DR-, n = 99; median lifespan = 42; 3X ElavGS >UAS Flag-chinmo: AL-, n = 107; median lifespan = 44 d; DR-, n = 99; median lifespan = 70 d). RU-486 fed 3X ElavGS >UAS chinmo flies or 3X ElavGS >UAS Flag-chinmo display a significantly dampened DR-dependent increase in lifespan (blue and red dotted lines) (3X ElavGS >UAS chinmo: AL+, n = 100; median lifespan = 10 d; DR+, n = 95; median lifespan = 16 d; 3X ElavGS >UAS Flag-chinmo: AL+, n = 137; median lifespan = 18 d; DR+, n = 100; median lifespan = 26 d). (C, E) FB GS >UAS chinmo or FB GS >UAS Flag-chinmo flies that were fed RU-486 displayed a stronger DR phenotype relative to 3X ElavGS >UAS Chinmo or 3X ElavGS >UAS Flag chinmo (blue and red dotted lines)(FB GS >UAS chinmo: AL-, n = 186; median lifespan = 26 d; DR-, n = 187; median lifespan = 52 d; AL+, n = 213; median lifespan = 16 d; DR+, n = 209; median lifespan = 16 d; FB GS >UAS Flag-chinmo: AL-, n = 96; median lifespan = 42; DR-, n = 91; median lifespan = 54; AL+, n = 83; median lifespan = 14 d; DR+, n = 73; median lifespan = 14 d) (Compare p values and Χ 2 in panels B/D with C/E) compared to the flies that were fed the Ethanol (blue and red solid lines). For statistical comparison of survival curves, p values and Χ 2 were calculated with log rank test. (F, G) Quantitation of triglyceride (TAG) stored levels in AL-RU-486 (Solid red bars), AL + RU-486 (Red dotted Figure 6 continued on next page
Ectopic expression of chinmo in adult neurons represses transcription of genes involved in fat metabolic processes. (A) Quantitation of chinmo mRNA levels in RNA extracted from 3X ElavGS >UAS chinmo flies that were fed a solvent (light blue bars) or RU-486 (pink bars) diet for 5 and 10 days. (B, C) Quantitation of chinmo mRNA levels in RNA extracted from head tissue (B) and decapitated body tissue (C) of 3X ElavGS >UAS chinmo flies that were fed ethanol (blue bar) or RU-486 (pink bar) diet for 10 days. Expression levels were normalized to Actin5c. Values are mean ± SD, n = 3. (D) Venn diagram of 1175 proteins identified across the two groups (-RU-486 and +RU-486) and 91% were common between the two groups. (E) Figure 7 continued on next page
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).
miR-128 modulates the proliferation and differentiation of NPCs in vitro. (A) Schematic representation of the cell proliferation assay procedures. (B-E) Ectopic expression of miR-128 decreases proliferation, while that of miR-Zip-128 increases NPC proliferation. NPCs were electroporated with the indicated plasmids and pulse-labeled with BrdU for 6 hr. NPCs were immunostained with an antibody against BrdU. The arrowheads indicate BrdU and GFP double-positive cells. Scale bars, 10 mm. Quantification of the number of GFP-BrdU double-positive cells relative to the total number of GFP-positive cells (C, E). (F) Schematic representation of the cell differentiation assay procedures. (G-J) Ectopic expression of miR128 increases neurogenesis, while that of miR-Zip-128 decreases the neurogenesis of NPCs. NPCs were electroporated with the indicated plasmids and immunostained with antibodies against TUJ1. The arrowheads indicate TUJ1 + GFP + cells. Scale bars, 50 mm. Quantification of the number of the GFPTUJ1 double-positive cells relative to the number of GFP-positive cells (H, J). More than 1500 GFP-positive cells were counted for each condition. At least three sets of independent experiments were performed. The values represent the mean ± s.d. (n = 3). Student's t-test, differences were considered significant at ***p<0.001 and **p<0.01. DOI: 10.7554/eLife.11324.005 Figure 2 continued on next page 
PCM1 knockdown decreases NPC neurogenesis. (A,B) PCM1 knockdown in NPCs decreases neural proliferation. NPCs were electroporated with a plasmid expressing PCM1 shRNA or with a control vector and pulse-labeled with BrdU for 6 hr. NPCs were immunostained with an antibody against BrdU. The arrowheads indicate BrdU and GFP double-positive cells. Scale bars, 10 mm. Quantification of the number of GFP-BrdU doublepositive cells relative to the total number of GFP-positive cells (B). (C,D) PCM1 knockdown in NPCs increases neurogenesis. NPCs were electroporated with a plasmid expressing PCM1 shRNA or with a control vector and then immunostained with antibodies against TUJ1. The arrowheads indicate TUJ1 + GFP + cells. Scale bars, 50 mm. Quantification of the number of GFP-TUJ1 double-positive cells relative to the number of GFP-positive cells (D). More than 1500 GFP-positive cells were counted for each condition. At least three sets of independent experiments were performed. The values represent the mean ± s.d. (n = 3). Student's t-test, differences were considered significant at ***p<0.001. DOI: 10.7554/eLife.11324.024 The following figure supplements are available for figure 5: Figure supplement 1. Efficient knockdown of PCM1. DOI: 10.7554/eLife.11324.025 Figure supplement 2. PCM1 knockdown in NPCs did not trigger apoptotic cell death. DOI: 10.7554/eLife.11324.026 Figure supplement 3. PCM1 knockdown in NPCs increases neurogenesis. DOI: 10.7554/eLife.11324.027 Figure supplement 4. Overexpression of PCM1 increases NPC proliferation. DOI: 10.7554/eLife.11324.028 Figure supplement 5. Overexpression of PCM1 decreases NPC neuronal differentiation. DOI: 10.7554/eLife.11324.029 Figure supplement 6. Overexpression of PCM1 in NPCs did not trigger apoptotic cell death. DOI: 10.7554/eLife.11324.030 Figure supplement 7. PCM1 regulates NPC proliferation and differentiation in vivo. DOI: 10.7554/eLife.11324.031 
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.
figure supplement 2A). These data indicate that CDK7 is indeed a target of THZ1 in 243 downregulating MYC. Although overexpression of mutant CDK7 (C312S) fails to rescue cell 244 growth inhibition conferred by THZ1 (Figure 4-figure supplement 2B), it significantly rescues 245 cell growth inhibition by the selective CDK7 inhibitor, YKL-1-116 (Figure 4-figure supplement 246 2B-2C). We would expect that a greater degree of rescue might require the elimination of 247 endogenous CDK7. In addition, considering that THZ1 targets both CDK7 and CDK12/13, we 248 speculate that co-overexpression of mutant CDK7 (C312S), mutant CDK12 (C1039S), and 249 mutant CDK13 may rescue cell growth inhibition by THZ1. Unfortunately, we failed to 250 successfully achieve overexpression of CDK12/13, likely due to their large sizes (1490 aa for 251 CDK12, and 1512 aa for CDK13), which might be needed to see rescue of cell growth inhibition 252 by THZ1.THZ1 abolishes the growth of patient-derived ovarian tumors 253
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.
Heatmap showing how all the individual BashTheBug classifications (n=214,164) compare to the dilution measured by the laboratory scientist using the Thermo Fisher Vizion instrument after 14 days incubation (n=12,488). (A) The probability that a single volunteer exactly agrees with the Expert +AMyGDA dataset varies with the dilution. (B) The distribution of all dilutions in the Expert +AMyGDA dataset after 14 days incubation. The differences are due to different drugs having different numbers of wells as well as the varying levels of resistance in the supplied strains. NR includes both plates that could not be read due to issues with the control wells and problems with individual drugs such as skip wells. (C) The distribution of all dilutions measured by the BashTheBug volunteers. (D) A heatmap showing the concordance between the Expert +AMyGDA dataset and the classifications made by individual BashTheBug volunteers. Only cells with gt 0.1 % are labelled. (E) Two example drug images where both the Expert and AMyGDA assessed the MIC as being a dilution of 5 whilst a single volunteer decided no growth could be seen in the image. (F) Two example drug images where both the laboratory scientist and a volunteer agreed that the MIC was a dilution of 5. (G) Two example drug images where the laboratory scientist decided there was no growth in any of the wells, whilst a single volunteer decided there was growth in the first four wells. The online version of this article includes the following figure supplement(s) for figure 2: Figure supplement 1. Heatmap showing how all the individual BashTheBug classifications (n=214,164) compare to the set of dilutions where the measurement made by the laboratory scientist using the Thermo Fisher Vizion instrument and a mirrored box after 14 days incubation concur (n=9402) (A).
Taking the mean of 17 classifications is ≥95% reproducible whilst applying either the median or mode is ≥90% accurate. (A) Only calculating the mean of 17 classifications achieves an essential agreement ≥95% for reproducibility International Standards Organization, 2007, followed by the median and the mode. (B) Heatmaps of the consensus formed via the mean, median or mode after 14 days incubation. Only drug images from the Expert + AMyGDA dataset are included. (C) The essential agreement between a consensus dilution Figure 3 continued on next page
The reproducibility and accuracy of the consensus MICs varies by drug. Consensus MICs were arrived at by taking the median of 17 classifications after 14 days incubation. The essential and exact agreements are drawn as red and green bars, respectively. For the former the minimum thresholds required are 95% and 90% for the reproducibility and accuracy, respectively (International Standards Organization, 2007). See (Figure 5-figure supplement 1) for the other consensus methods. The online version of this article includes the following figure supplement(s) for figure 5: Figure supplement 1. The reproducibility and accuracy after 14 days incubation of the 13 antibiotics on the UKMYC5 plate.
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.
ctbp-1 mutant AIAs retain multiple aspects of their AIA gene expression profile. (A-B) Expression of (A) M4 markers egl-17, dbl-1, ser-7.b, and flp-21 and (B) AIA markers gcy-28.d, ins-1, ttx-3, cho-1, and mgl-1 in wild-type (left image) and ctbp-1(n4784) (right image) L4 larval worms. Arrow, M4 neuron. Circles, AIAs. Scale bar, 5 μm. Images are oriented such that left corresponds to anterior, top to dorsal. Quantification of reporter expression in Figure 2-figure supplement 1A-B.
A suppressor screen reveals egl-13 and ttx-3 as ctbp-1 genetic interactors. (A) Schematic of ctbp-1 suppressor screen design. ctbp-1 mutant worms carrying nIs175[P ceh-28 ::gfp] were mutagenized with ethyl methanesulfonate (EMS), and their F2 progeny were screened for continued nIs175 expression in M4 and loss of expression in the AIA neurons (red circle). (B) Gene diagram of the egl-13a isoform. Arrows (above), point mutations. Line (below), deletion. Scale bar (bottom right), 1 kb. (C) Gene diagram of the ttx-3a isoform. Arrows (above), point mutations. Scale bar, 1 kb. (D) Percentage Figure 6 continued on next page
EGL-13 controls aspects of AIA gene expression. (A-C) Expression of markers for AIA misexpressed genes (A) nEx3081[P acbp-6 ::gfp], (B) otIs123[P sra-11 ::gfp], or (C) ivEx138[P glr-2 ::gfp] in egl-13(n5937) ctbp-1(n4784) double mutants at the (top) L1 and (bottom) L4 larval stages. Arrow, M4 neuron. Circle, AIAs. Scale bar, 10 μm. (D) Percentage of egl-13(n5937) ctbp-1(n4784) double mutants expressing the indicated reporter in the AIA neurons at the L1 and L4 larval stages. Mean ± SEM. n ≥ 50 worms scored per strain, three biological replicates. The ctbp-1 allele used for all panels of this figure was n4784. All strains in A-D contain nIs348[P ceh-28 ::mCherry]. Images are oriented such that left corresponds to anterior, top to dorsal. The online version of this article includes the following source data for figure 8: Source data 1. Source data for Figure 8.
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-13L exhibits conserved homodimerization and heterodimerization in C. elegans. (A, B) Sequence alignment of the C2A domain between worm UNC-13L and rat Munc13-1, and the zinc finger (ZF) domain between worm UNC-10 and mouse RIM1. The conserved residues in the C2A domain (K49) and the ZF domain (K77, K79) that disrupt homodimer and heterodimer formation are indicated by stars. (C) Separation of purified UNC-13L C2A monomer and homodimer by gel filtration. The wild-type C2A forms a homodimer that runs at 12.57 mL (blue trace, corresponding to an apparent Figure 1 continued on next page
Tonic release is reduced by disrupting C2A/RIM heterodimerization in low Ca 2+. (A) Representative traces of mEPSCs and mIPSCs recorded at 0 mM Ca 2+ from indicated genotypes. (B, C) Averaged frequency and amplitude of the mEPSCs and mIPSCs. (D) Representative traces of mEPSCs and mIPSCs recorded at 1 mM Ca 2+ from the same genotypes in (A). (E, F) Quantification of the mEPSC and mIPSC frequency and amplitude from the genotypes in (D). Data are mean ± SEM (##, p < 0.01, ###, p < 0.001, n.s., non-significant when compared to UNC-13L rescue; ***, p < 0.001 when Figure 2 continued on next page
Effects of homodimerization and heterodimerization on evoked neurotransmitter release. Electronic stimulus and hypertonic sucrose-evoked EPSCs were recorded from the body wall muscle of adult animals in 1 mM Ca 2+. (A) Example traces of the evoked EPSCs from indicated genotypes. (BD) Quantification of the amplitude, charge transfer and 20-80% risetime of the evoked EPSCs in (A). Example traces and averaged charge transfer of the sucrose-evoked EPSCs from the same genotypes in (A) are shown in (E) and (F). (G) Quantification of the probability of SV release (P vr ). Data are Figure 3 continued on next page
Synaptic depression and recovery are unaltered by disrupting C2A/RIM heterodimerization. Synaptic depression and recovery were investigated by applying a train (1 Hz or 5 Hz) or a paired light stimulus onto the ventral nerve cord of the adult worms with expression of ChIEF in cholinergic motor neurons. (A, C) Example traces of 1 Hz and 5 Hz light train stimulus-evoked EPSCs (black, wild-type; red, unc-10(K77/79E) mutants). (B, D) Quantification of synaptic depression by normalizing the EPSC amplitude (EPSC i ) to the first EPSC amplitude (EPSC 1 ). (E) Evoked EPSCs Figure 5 continued on next page
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.
Preparation of AUDIT Sum-Scores for two-part latent growth mixture model. Upper row: data from original scale (Sum Score), zeros are shown in black and indicate non-drinking individuals. Middle row: Transformation of data into consumer and non-consumer without fine-grading of alcohol use scores. Bottom row: Alcohol use score (AUDIT Sum-Score) for individuals who drink at all. Note that to enhance readability of the figure, sum-scales (upper and bottom row) are truncated at a score of 20. DOI: https://doi.org/10.7554/eLife.44056.003
Two-part latent growth mixture model. c = continuous, d = discrete, BL = baseline, FU = follow up, I = intercept, S = slope, TBV = total brain volume, MRI site was not a single indicator as depicted for reasons of simplicity, but consisted of 9-1 separate indicators dummy coding the different scanners used. DOI: https://doi.org/10.7554/eLife.44056.006
Brain regions showing a significant regression path from brain voxel to the latent slope of alcohol use score increase over time. The higher the grey matter volume the larger the slope increase. DOI: https://doi.org/10.7554/eLife.44056.007
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.
Interactions between PIF7 derivatives and 14-3-3 proteins. (a) Interactions between PIF7, PIF7(2A) or PIF7(2D) and 14-3-3 λ /or 14-3-3 κ in a yeast two-hybrid assay. Each yeast clone containing the pGADT7 (AD), or AD-PIF7(2A), or AD-PIF7 or AD-PIF7 (2D) together with pGBKT7 (BD) or BD-14-3-3 λ or BD-14-3-3 κ was grown on transformation selection (SD-L-T) or interaction selection (SD-L-T-H+3AT) plates. Dilution of the inoculation is shown at the top of the picture. Yeast growth on SD-L-T-H+3AT indicates a positive protein-protein interaction. (b) Interaction between PIF7 derivatives and 14-3-3 λ or 14-3-3 κ detected by BiFC. nYFP-PIF7, nYFP-PIF7(2A), nYFP-PIF7 or nYFP-PIF7(5A) and 14-3-3 λ-cYFP or 14-3-3 κ-cYFP constructs were co-transformed into tobacco leaf cells. YFP fluorescence images were obtained using a confocal microscope. White scale bar represents 75 μm. (c) Interaction between PIF7, PIF7(2A) or PIF7(2D) and 14-3-3s as detected by co-immunoprecipitation. Anti-FLAG M2 agarose beads were used to precipitate PIF7-Flash, PIF7(2A)-Flash or PIF7(2D)-Flash from overexpression plants grown under white light. Western blots were performed using anti-Myc and anti-14-3-3s antibodies as indicated in the 'Materials and methods'. The following figure supplement is available for figure 3: Figure supplement 1. Potential phosphorylation sites on PIF7. Figure supplement 2. Expression of 14-3-3 proteins and PIF7, and quantification of YFP signals in Figure 3b. Figure supplement 3. Interactions between PIF7, PIF7(2A) or PIF7(2D) and 14-3-3 λ or 14-3-3 κ in BiFC and in a pull-down assay. 
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 interactions between Lem8 and 14-3-3ζ. (A) Interactions between Lem8 and 14-3-3ζ detected by yeast two-hybrid assay. Yeast strains harboring the indicated constructs were streaked on Leu − and Trp − medium to select for plasmids (left) or on Leu − , Trp − , Ade − , and His − medium to assess the interactions (right). Images were acquired after 3-day incubation at 30°C. (B) Lem8 and 14-3-3ζ form a protein complex in mammalian cells. Total lysates of HEK293T cells transfected with the indicated plasmid combinations were immunoprecipitated with a Flag-specific antibody (left panels) or GFP-specific antibodies (right panels), and the precipitates were probed with both Flag and GFP antibodies. Similar results were obtained from at least three independent experiments and the data shown here were from one representative experiment. (C) Lem8 directly interacts with 14-3-3ζ. GST-14-3-3ζ was incubated with His 6 -Lem8 or His 6 -Lem8 C280S , and the potential protein complex was captured by glutathione beads for 1 hr at 4°C. After extensive washing, bound proteins were solubilized with sodium dodecyl sulfate (SDS) loading buffer, and proteins were detected by Coomassie brilliant blue staining after being resolved by SDS/polyacrylamide gel electrophoresis PAGE. Similar results were obtained from at least three independent experiments and the data shown here were from one representative experiment. (D) Interactions between 14-3-3ζ and Lem8 deletion mutants. Lysates of 293T cells expressing Flag-14-3-3ζ and each of the HA-tagged deletion Lem8 were subjected to immunoprecipitation with the anti-HA antibody and the presence of 14-3-3ζ in the precipitates was probed with the Flag-specific antibody. Similar results were obtained from at least three independent experiments and the data shown here were from one representative experiment.
Autoprocessed Lem8 retains the cysteine protease activity. (A) The autoprocessed form of Lem8 cleaves Phldb2 in cells. HA-Phldb2-Flag was coexpressed in HEK293T cells with Lem8 or the indicated truncation mutants including the self-processed form, Lem8 ∆C52 . Twenty-four hours after transfection, the samples were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and probed by a HAspecific antibody and a Flag-specific antibody. Tubulin was used as a loading control. Results shown were one representative from three independent experiments with similar results. (B) Lem8 ∆C52 causes redistribution of GFP-Phldb2. Truncations of Lem8, including Lem8 ∆N25 , Lem8 ∆C52 , and Lem8 ∆C100 fused to mCherry was individually expressed in HEK293T cells with GFP-Phldb2. Twenty-four hours after transfection, the fluorescence images were acquired with a Zeiss LSM 880 confocal microscope. The percentage of cells with membrane Phldb2 was calculated in Phldb2-and Lem8-positive cells (right panel). Bar, 10 μm. (C) The interaction between Lem8 ∆C52 and 14-3-3ζ. Total lysates of HEK293T cells transfected with indicated plasmid combinations were immunoprecipitated with antibodies specific for HA (left panel) or Flag (right), and the precipitates were probed with both HA and Flag antibodies. Similar results were obtained from at least three independent experiments and the data shown here were from one representative experiment. (D) Lem8 △C52 directly interacts with 14-3-3ζ. Mixtures containing GST-14-3-3ζ and His 6 -Lem8 ∆C52 were incubated with glutathione beads for 1 hr at 4°C. After washing, samples resolved by SDS/PAGE were detected by Coomassie brilliant blue staining. Results shown were one representative from three independent experiments with similar results. (E) The cleavage of Phldb2 by Lem8 ∆C52 requires 14-3-3ζ. Purified HA-Phldb2-Flag from HEK293T was incubated with His 6 -Lem8 ∆C52 in reactions with or without His 6 -14-3-3ζ. Total proteins of all samples were resolved with SDS-PAGE, and probed by immunoblotting with antibody specific for HA, Flag, and His 6 , respectively. Results shown were one representative from three independent experiments with similar results.
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.
Top-cited authors
Dari Kimanius
  • Stockholm University
Björn O. Forsberg
  • Stockholm University
Takanori Nakane
  • University of Cambridge
Jin-Wu Nam
  • Hanyang University
Wim Hagen
  • European Molecular Biology Laboratory