Communications Biology

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53BP1 domains that are required for genome stability through heterochromatin maintenance and double strand break repair. Schematic diagram of 53BP1 protein domains that are important for DNA repair and heterochromatin maintenance functions of 53BP1. The oligomerization domain (OD) is required to bind to HP1α and for liquid-liquid phase separation (LLPS) at heterochromatin, which promotes genome stability. Many domains of 53BP1 are necessary for recruitment of 53BP1 to DNA double strand breaks (DSBs). LLPS liquid-liquid phase separation, HP1α heterochromatin protein 1 alpha, OD oligomerization domain, Tudor tandem tudor domain, UDR ubiquitin-dependent recruitment motif, NLS nuclear localization sequence, BRCT BRCA1 carboxy terminal domain, DSBs, DNA double strand breaks. Created with BioRender.com.
In this Comment, Naveen Tangudu and Katherine Aird discuss recent findings showing that 53BP1 regulates heterochromatin through liquid-liquid phase separation.
 
Many genetic disorders are a result of single or multiple genome abnormalities. A possible approach to circumvent genetic disorders is to use gene editing agents to correct these mistakes, but a major challenge remains in the mode of delivery of gene editing agents to different regions of the body. Banskota et al. present the use of engineered DNA-free virus-like particles (eVLPs) to deliver base editors to different organs in a mice model for improved outcomes, highlighting the potential of eVLPS to deliver base editors and as an efficient delivery mechanism, leveraging the advantages of viral and nonviral delivery methods.
 
Characterization of sorghum transformation. a-c Gene delivery capacity of ternary transformation system. Gene delivery was represented by the number of transgenic cells exhibiting YFP fluorescence on the surface of sorghum embryos. d-f Tissue culture response represented by callus proliferation after two weeks of Agrobacterium infection on the multi-purpose medium without selection. g-i YFP images of early-stage somatic embryo formation (indicated by the arrows) induced by morphogenic gene expression on the surface of immature scutella. j-l Shoot regeneration after four-week of maturation. m-o The pleiotropic impacts of morphogenic gene expression on single-copy QEs. a, d, g, j and m Tx430. b, e, h, k, and n Tx623. c, f, i, l and o Tx2752.
Somatic embryo formation and regeneration mediated by morphogenic gene excision-induced selection-activation system. Bright field and fluorescence images of early-stage somatic embryo formation (couple of examples indicated by the arrows) on the surface of immature scutella in Tx430 (a, b), Macia (c, d), Malisor 84-7 (e, f), Tegemeo (g, h). a, c, e, and g Bright field images. b, d, f, and h Fluorescence images. i Tx430 shoot regeneration after four-week maturation.
Targeted mutation and gene-dropout frequencies of different sorghum transformation systems.
For many important crops including sorghum, use of CRISPR/Cas technology is limited not only by the delivery of the gene-modification components into a plant cell, but also by the ability to regenerate a fertile plant from the engineered cell through tissue culture. Here, we report that Wuschel2 (Wus2) -enabled transformation increases not only the transformation efficiency, but also the CRISPR/Cas-targeted genome editing frequency in sorghum ( Sorghum bicolor L .). Using Agrobacterium -mediated transformation, we have demonstrated Wus2 -induced direct somatic embryo formation and regeneration, bypassing genotype-dependent callus formation and significantly shortening the tissue culture cycle time. This method also increased the regeneration capacity that resulted in higher transformation efficiency across different sorghum varieties. Subsequently, advanced excision systems and “altruistic” transformation technology have been developed to generate high-quality morphogenic gene-free and/or selectable marker-free sorghum events. Finally, we demonstrate up to 6.8-fold increase in CRISPR/Cas9-mediated gene dropout frequency using Wus2 -enabled transformation, compared to without Wus2 , across various targeted loci in different sorghum genotypes.
 
Chromatin occupancy pattern of TOX4 resembles that of Pol II. a Correlation plots for biological replicates of ChIP-seq (left) and CUT&Tag (middle) of TOX4, and for ChIP-seq versus CUT&Tag of TOX4 (right). b Annotation of TOX4 occupancy. Left: a Venn diagram showing overlap between TOX4 peaks identified by ChIP-seq and CUT&Tag, respectively, Right: TOX4 peak distribution across genomic features. c Normalized read distribution of TOX4 ChIP-seq (top), TOX4 CUT&Tag (middle) and Pol II CUT&Tag (bottom) within the SERBP1 locus in K562 cells. d Generation and characterization of a TOX4 knockout K562 cell line. Left: Schematic of TOX4 knockout strategy. Right: Characterization of the TOX4 KO K562 cell line by Western blot. e Comparison of proliferation of control and TOX4 KO cells by MTT assays. Statistical significance was determined with a two-sided Student's t-test; the centers and the error bars represent the mean and the SD of five independent experiments, respectively. NS: P ≥ 0.05. f Comparison of association of WDR82 or PP1γ with PNUTS by co-IP in control and TOX4 KO cells. g A Venn diagram showing overlaps between TOX4-bound genes and downregulated or upregulated genes of RNA-seq. h A volcano plot showing expression changes of TOX4-bound genes upon TOX4 loss. i A heatmap comparing expression of TOX4 direct targets in TOX4 KO versus control cells. j Pathway analysis results of downregulated genes upon TOX4 loss. k Meta-gene profiles and heatmaps of ChIP-seq (left) and CUT&Tag (right) each showing a positive correlation between TOX4 occupancies and mRNA levels of TOX4 direct targets in K562 cells. Genes were sorted according to TOX4 occupancy level detected by ChIP-seq (left) and CUT&Tag (right), respectively, in control cells. High: top 25% of the non-silent genes (mean TPM ≥ 1); Medium: non-silent genes between top 25% and bottom 25%; Low: bottom 25% of the non-silent genes or genes with mean TPM < 1.
TOX4 may facilitate Pol II CTD dephosphorylation. a Comparison of cellular levels of total, Ser-5 phosphorylated and Ser-2 phosphorylated Pol II by Western blot in control and TOX4 KO cells. Top: Western blot images, Bottom: A bar graph showing relative levels of Ser-5 phosphorylated Pol II and Ser-2 phosphorylated Pol II quantified by ImageJ in control and TOX4 KO cells. Pictures are representative of three independent experiments. Statistical significance was determined with a two-sided Student's t-test; the centers and the error bars represent the mean and the SD, respectively. *P < 0.05, **P < 0.01. b-d Correlation plots for biological replicates of CUT&Tag of total (b), Ser-5 phosphorylated (c), and Ser-2 phosphorylated (d) Pol II. e Normalized read distribution of CUT&Tag of total, Ser-5 phosphorylated and Ser-2 phosphorylated Pol II within the SERBP1 locus in TOX4 KO versus control cells. f-h Genome-wide meta-gene profiles and heatmaps of CUT&Tag comparing chromatin occupancies of total (f), Ser-5 phosphorylated (g), and Ser-2 phosphorylated (h) Pol II in TOX4 KO versus control (Ctrl) cells. A genome-wide meta-gene profile and a heatmap of TOX4 CUT&Tag in control cells are placed to the left of those of Pol II to facilitate comparison of TOX4 and Pol II occupancies. Genes were sorted by total Pol II CUT&Tag signal in control cells. i A correlation plot of CUT&Tag data of TOX4 and Pol II. j A Venn diagram showing overlap between TOX4-bound and Pol II-bound promoters. Statistical significance of the overlap between them was determined with a Hypergeometric test.
TOX4 facilitates promoter-proximal pause of Pol II. a Schematic representation describing the calculation of traveling ratio (TR) at each Pol IIbound gene. b A cumulative distribution plot comparing Pol II TRs of TOX4-bound genes in control and TOX4 KO cells. c A scatter plot comparing Pol II TRs of TOX4-bound genes in TOX4 KO versus control cells.
TOX4 may restrict early elongation but promote late productive elongation of Pol II. a Schematic representation of experimental design of the 4sUDRB-seq. Labeled RNA was extracted at 0, 10, and 20 min after DRB removal. b Density plots comparing Pol II elongation rates of 759 TOX4-bound genes in control and TOX4 KO cells, for which high confidence elongation rates from 0 to 10 min after DRB removal could be determined. c A bar graph categorizing Pol II elongation rate changes of the above-mentioned 759 TOX4-bound genes in TOX4 KO versus control cells. d-f Density plots comparing Pol II elongation rates from 0 to 20 min (d), 0 to 10 min (e), and 10 to 20 min (f) after DRB removal of 200 TOX4-bound genes in control and TOX4 KO cells, for which high confidence elongation rates from 0 to 10 min and 0 to 20 min could be determined. g A Sankey diagram visualizing Pol II elongation rate changes from 0 to 10 min and 10 to 20 min of 200 TOX4-bound genes described in d-f. Heights of rectangles and widths of "streams" between rectangles each are proportional to gene counts, which are shown either aside of the rectangles or inside of the "streams". h Normalized 4sUDRB-seq read distribution at 0, 10 and 20 min after DRB removal within the UVRAG locus in control and TOX4 KO cells. i A heatmap of standardized residuals of the chisquare test of independence between Pol II elongation rate changes from 0 to 10 min after DRB removal and Pol II TR changes upon TOX4 loss. Overlapping gene counts between categories are shown in the cells.
TOX4 may restrict early elongation by facilitating SPT5 Thr-806 dephosphorylation. a Comparison of cellular levels of SPT5 and p-SPT5 Thr-806 by Western blot in control and TOX4 KO cells. Top: Western blot images with nonspecific bands highlighted by a "*", Bottom: A bar graph showing relative levels of p-SPT5 Thr-806 quantified by ImageJ in control and TOX4 KO cells. Pictures are representative of four independent experiments. Statistical significance was determined with a two-sided Student's t-test; the centers and the error bars represent the mean and the SD, respectively. NS: P ≥ 0.05, *P < 0.05, **P < 0.01. b-d Correlation plots for biological replicates of CUT&Tag of SPT5 (b), p-SPT5 Thr-806 (c), and NELF-E (d). e Normalized CUT&Tag read distribution of Pol II, SPT5, p-SPT5 Thr806 and NELF-E within the SERBP1 locus in TOX4 KO versus control cells. f-h Genome-wide meta-gene profiles and heatmaps of CUT&Tag comparing chromatin occupancies of SPT5 (f), p-SPT5 Thr-806 (g), and NELF-E (h) in TOX4 KO versus control cells. Genes were sorted by total Pol II CUT&Tag signal in control cells.
TOX4 is one of the regulatory factors of PP1 phosphatases with poorly understood functions. Here we show that chromatin occupancy pattern of TOX4 resembles that of RNA polymerase II (Pol II), and its loss increases cellular level of C-terminal domain (CTD) phosphorylated Pol II but mainly decreases Pol II occupancy on promoters. In addition, elongation rate analyses by 4sUDRB-seq suggest that TOX4 restricts pause release and early elongation but promotes late elongation. Moreover, TT-seq analyses indicate that TOX4 loss mainly decreases transcriptional output. Mechanistically, TOX4 may restrict pause release through facilitating CTD serine 2 and DSIF dephosphorylation, and promote Pol II recycling and reinitiation through facilitating CTD serines 2 and 5 dephosphorylation. Furthermore, among the PP1 phosphatases, TOX4 preferentially binds PP1α and is capable of facilitating Pol II CTD dephosphorylation in vitro. These results lay the foundation for a better understanding of the role of TOX4 in transcriptional regulation.
 
Identified chemical hits reduced persister levels in E. coli MG1655 cultures. a-c Biphasic kill curves. Early stationary phase E. coli MG1655 cells, grown in LB, were treated with OFX (5 µg/ml) and the chemicals at indicated concentrations. Throughout treatment cells were plated for viable cell counts at indicated time points. Of note, the control data (OFX only) is the same in all three panels. TDZ Thioridazine; TFP Trifluoperazine; FPZ Fluphenazine; PPZ Perphenazine; CPZ Chlorpromazine; AMT Amitriptyline; HCP Hexachlorophene; PCP Pentachlorophenol; PT Potassium tellurite. n = 4. d Cell survival after quinolone and phenothiazine treatment. Cells (E. coli MG1655) treated with indicated quinolone and/or phenothiazines for 20 h were plated for viable cell counts. NOR: norfloxacin (0.8 µg/ml); MXF Moxifloxacin (0.8 µg/ml), LVX Levofloxacin (0.3 µg/ml); CIP Ciprofloxacin (0.2 µg/ml). n = 4. Statistical analysis was performed using one-way ANOVA with Dunnett's post-test, where *P < 0.05, ***P < 0.001, ****P < 0.0001. Data corresponding to each time point represent mean value ± standard deviation.
Phenothiazine drugs reduced persister levels of Gram-negative bacteria. Early stationary phase cells of P. aeruginosa, K. pneumonia, and A. baumannii strains (see Supplementary Fig. 1 for growth curves) were treated
Phenothiazine drugs perturb OFX-induced RecA expression during recovery. Early stationary phase E. coli MG1655 cells with P recA -gfp (grown in LB) were treated with (a) OFX or (b) OFX + TDZ for 20 h. After treatment, cells were washed and transferred to liquid LB medium for persister recovery. At designated time points during persister recovery, 10 µl cells samples were spotted on 1% agarose pads for phase contrast and fluorescent microscopy. A representative image is shown; all independent biological replicates have similar results. Green arrow: elongated cells with high GFP, blue arrow: dividing exponential phase cells with low GFP. n = 3. Scale bar: 25 µm.
Phenothiazine drugs reduced energy metabolism in E. coli MG1655. a Mass spectrometry analysis of TDZ-treated and control cells. E. coli MG1655 cells grown in LB were treated with 0.25 mM TDZ in early stationary phase (t = 5 h) for 20 h. After treatment, cells were collected and analyzed by mass spectrometry. Unsupervised hierarchical clustering was performed on the metabolic data. The data was standardized across all samples for each metabolite to obtain a mean value of 0 and a standard deviation of 1. Color codes show how a data point deviates from the mean value. Each column represents a biological replicate. n = 4. b, c Pathway enrichment maps. Metabolites of the TCA cycle and glycolysis from TDZ-treated cells were compared to those of untreated cells. The circle size is proportional to the ratio of the normalized intensities of metabolites between TDZ-treated and untreated cells. Blue (P ≤ 0.05) and red (P ≤ 0.05 for dark red; 0.05 < P < 0.10 for light red) colors represent the metabolites that are significantly upregulated (red) or downregulated (blue) in the treatment group compared to the control. Gray indicates that there was no significant difference between the groups. n = 4. d Relative amounts of carbon source metabolites in the treatment and control groups obtained from mass spectrometry analysis. n = 4. e RSG staining of phenothiazine-treated cells. Early stationary phase cells (t = 5 h) were treated with phenothiazines for 20 h and then stained with RSG prior to analysis by flow cytometry. The mean fluorescence intensity of each treatment group was normalized to that of the control (untreated) group. n = 6. f ATP levels in
Phenothiazine can transiently permeabilize the cell membrane by inhibiting membrane-bound metabolic proteins. a Phenothiazine treatment induced membrane permeabilization. E. coli MG1655 cells at early stationary phase (grown in LB) were treated with phenothiazine drugs at indicated concentrations. Following treatment, samples were collected and stained with PI at indicated time points for flow cytometry analysis. Live cells and ethanoltreated dead cells were used as negative and positive controls, respectively, to gate the PI negative (−) and PI positive (+) populations (see Supplementary Fig. 8b). n = 3. b Screening E. coli (K-12 BW25113) Keio Knockout Collection. Cells at early stationary phase (grown in LB) were treated with 0.25 mM TDZ for 1 h and then stained with PI for flow cytometry analysis to quantify PI (+) cell fractions (%). n = 1. c Phenothiazine-induced membrane permeabilization in E. coli MG1655 mutant strains. The mutant strains at early stationary phase were treated with 0.25 mM TDZ for 1 h and then stained with PI for flow cytometry analysis. n = 4. Statistical analysis was performed using one-way ANOVA with Dunnett's post-test. ****P < 0.0001. FSC-H: Forward scatter. Data corresponding to each time point represent mean value ± standard deviation.
Bacterial persister cells are temporarily tolerant to bactericidal antibiotics but are not necessarily dormant and may exhibit physiological activities leading to cell damage. Based on the link between fluoroquinolone-mediated SOS responses and persister cell recovery, we screened chemicals that target fluoroquinolone persisters. Metabolic inhibitors (e.g., phenothiazines) combined with ofloxacin (OFX) perturbed persister levels in metabolically active cell populations. When metabolically stimulated, intrinsically tolerant stationary phase cells also became OFX-sensitive in the presence of phenothiazines. The effects of phenothiazines on cell metabolism and physiology are highly pleiotropic: at sublethal concentrations, phenothiazines reduce cellular metabolic, transcriptional, and translational activities; impair cell repair and recovery mechanisms; transiently perturb membrane integrity; and disrupt proton motive force by dissipating the proton concentration gradient across the cell membrane. Screening a subset of mutant strains lacking membrane-bound proteins revealed the pleiotropic effects of phenothiazines potentially rely on their ability to inhibit a wide range of critical metabolic proteins. Altogether, our study further highlights the complex roles of metabolism in persister cell formation, survival and recovery, and suggests metabolic inhibitors such as phenothiazines can be selectively detrimental to persister cells.
 
T cells are critically important for host defense against infections. T cell activation is specific because signal initiation requires T cell receptor (TCR) recognition of foreign antigen peptides presented by major histocompatibility complexes (pMHC) on antigen presenting cells (APCs). Recent advances reveal that the TCR acts as a mechanoreceptor, but it remains unclear how pMHC/TCR engagement generates mechanical forces that are converted to intracellular signals. Here we propose a TCR Bending Mechanosignal (TBM) model, in which local bending of the T cell membrane on the nanometer scale allows sustained contact of relatively small pMHC/TCR complexes interspersed among large surface receptors and adhesion molecules on the opposing surfaces of T cells and APCs. Localized T cell membrane bending is suggested to increase accessibility of TCR signaling domains to phosphorylation, facilitate selective recognition of agonists that form catch bonds, and reduce noise signals associated with slip bonds.
 
XNAzymes specifically cleave individual members of the miR-17~92 / OncomiR-1 microRNA cluster
a Chemical structure of a 2’-deoxy-2’-fluoro-β-D-arabino nucleic acid (FANA) nucleotide and schematic showing the putative secondary structure of the FR6_1 RNA endonuclease XNAzyme composed of FANA (purple) bound to an RNA substrate (red). b Sequence similarity between mature microRNAs of the human miR-17 family; note that although alternative sequences have been reported, in this study we use those from ref. ⁴² as proof of concept. Variants of the FR6_1 XNAzyme engineered to cleave (c–f) miR-17 (“Fz_miR_17”) or (g–j) miR-20a (“Fz_miR_20a”): (c, g) putative secondary structures, (d–f, h–j) urea-PAGE gels and graphs showing (d, h) RNA substrate cleavage, (e, i) catalytic rate constant (kobs) and (f, j) XNAzyme specificity of pseudo first-order single-turnover reactions between cognate miRNA substrates and XNAzymes for (d, h) 15 h, (f, j) 5 h, or the times indicated, under quasi-physiological conditions (37 °C, 1 mM Mg²⁺, pH 7.4) (d, e, h, i: 1 μM substrate, 5 μM enzyme. f, j 0.25 μM substrate, 1.25 μM enzyme). Black arrows indicate site of substrate cleavage. (⁻OH) indicates RNA substrates subjected to partial alkaline hydrolysis. Black circles and error bars represent mean and standard error (SEM) for n = 3 independent experiments, red crosses represent individual data points.
An XNAzyme specifically cleaves microRNA miR-21 and outperforms analogous DNAzyme and ribozyme catalysts
a Schematic showing the putative secondary structure of a variant of the FR6_1 XNAzyme, “Fz_miR_21B”, engineered to cleave human miR-21. b–d Urea-PAGE gels and graphs showing (b) substrate cleavage, (c) catalytic rate constant (kobs) and (d) XNAzyme specificity of pseudo first-order single-turnover reactions between (0.5 μM) miR-21 RNA substrate and (2.5 μM) XNAzyme Fz_miR_21B for (b, d) 15 h, or (c) a time series, under quasi-physiological conditions (37 °C, 1 mM Mg²⁺, pH 7.4). (⁻OH) indicates RNA substrates subjected to partial alkaline hydrolysis. Black circles and error bars represent mean and standard error (SEM) for n = 3 independent experiments, red crosses represent individual data points. e–g Schematics showing the putative secondary structure analogous DNAzymes (e) “miR21dz1”⁴⁸ and (f) “RNV541B”⁴⁹, and (g) ribozyme “miR21rz1_TLRB”⁴⁸ (see Supplementary Figs. 3 and 4). h–j Graphs showing pseudo first-order single-turnover reactions between (0.5 μM) miR-21 RNA substrate and (2.5 μM) (h) miR21dz1 (black squares), (i) RNV541B (black triangles) or (j) miR21rz1_TLRB (black diamonds), under quasi-physiological conditions (37 °C, 1 mM Mg²⁺, pH 7.4). Nucleic acid chemistry is indicated by colour: FANA = purple or (to highlight mutated positions) magenta, RNA = orange (substrate) or green (catalyst), DNA = cyan. Black arrows indicate site of substrate cleavage.
An XNAzyme specifically cleaves non-coding Y RNA hY5
a Schematic showing the putative secondary structure of a variant of the FR6_1 XNAzyme, “Fz_hY5_4B”, engineered to cleave human non-coding Y RNA hY5. FANA residues are indicated in purple or (to highlight mutated positions) magenta. Substrate hY5 RNA is shown in cyan. Black arrow indicates site of substrate cleavage. b Graph showing catalytic rate constant (kobs) of a pseudo first-order single-turnover reaction between (0.5 μM) hY5 RNA substrate and (2.5 μM) XNAzyme Fz_hY5_4B under quasi-physiological conditions (37 °C, 1 mM Mg²⁺, pH 7.4). Black circles and error bars represent mean and standard error (SEM) for n = 3 independent experiments, red crosses represent individual data points. c Sequence similarity of human Y RNAs at the target site of XNAzyme Fz_hY5_4B. d Schematics and urea-PAGE gels showing specificity of XNAzyme Fz_hY5_4B in pseudo first-order single-turnover reactions between (0.5 μM) Y RNA substrates and (2.5 μM) XNAzyme Fz_hY5_4B under quasi-physiological conditions (24 h, 37 °C, 1 mM Mg²⁺, pH 7.4). Note that unglycosylated versions of the Y RNAs were used in this study as the site and chemical nature of glycosylation linkage to these RNAs is currently unknown⁷⁰.
A catalytic XNA nanostructure has improved biostability and targets multiple microRNAs
a Schematics showing design of a three-component fully-FANA tetrahedron (“TFz_miR3”) presenting three different anti-miR XNAzymes (Fz_miR_17, Fz_miR_20a and Fz_miR_21B) along single-stranded edges (strand hybridisation is designed to be mediated by base pairing indicated by black lines; red lines indicate residues mediating microRNA substrate binding). b Native PAGE gel showing self-assembly of the compete TFz_miR3 tetrahedron (lanes 1-3: individual TFz_miR components, lanes 4-6: combinations of two TFz_miR components, lane 7: all three TFz_miR components). Mwt = NEB low molecular weight ladder (dsDNA). c Native PAGE gels showing crude or spin filter purified TFz_miR3 (following 1 h at 37 °C). d Urea-PAGE gels and graph showing stability of the assembled and purified TFz_miR3 (red squares) or a single-stranded XNAzyme (“ssFz”)(Fz_miR_21B)(black circles) in 50% human serum at 37 °C. e Urea-PAGE gels showing activity of (2.5 μM) assembled and purified TFz_miR3 or the appropriate single-stranded XNAzyme (Fz_miR_17, Fz_miR_20a or Fz_miR_21B) in pseudo first-order single-turnover reactions with (0.5 μM) cognate RNA substrates or an untargeted control miRNA (miR-93) under quasi-physiological conditions (24 h, 37 °C, 1 mM Mg²⁺, pH 7.4).
  • Maria J. DondeMaria J. Donde
  • Adam M. RochussenAdam M. Rochussen
  • Saksham KapoorSaksham Kapoor
  • Alexander I. TaylorAlexander I. Taylor
Non-coding RNAs (ncRNAs) offer a wealth of therapeutic targets for a range of diseases. However, secondary structures and high similarity within sequence families make specific knockdown challenging. Here, we engineer a series of artificial oligonucleotide enzymes (XNAzymes) composed of 2’-deoxy-2’-fluoro-β-D-arabino nucleic acid (FANA) that specifically or preferentially cleave individual ncRNA family members under quasi-physiological conditions, including members of the classic microRNA cluster miR-17~92 (oncomiR-1) and the Y RNA hY5. We demonstrate self-assembly of three anti-miR XNAzymes into a biostable catalytic XNA nanostructure, which targets the cancer-associated microRNAs miR-17, miR-20a and miR-21. Our results provide a starting point for the development of XNAzymes as a platform technology for precision knockdown of specific non-coding RNAs, with the potential to reduce off-target effects compared with other nucleic acid technologies. Engineered RNA endonuclease XNAzymes can specifically target individual disease-associated non-coding RNAs (ncRNAs) in vitro with potential as platform technology for precision detection or knockdown of individual microRNAs or longer ncRNAs.
 
  • Fatimah MatalkahFatimah Matalkah
  • Bohye JeongBohye Jeong
  • Macie SheridanMacie Sheridan
  • [...]
  • Peter StoilovPeter Stoilov
The Musashi proteins, MSI1 and MSI2, are conserved RNA binding proteins with a role in the maintenance and renewal of stem cells. Contrasting with this role, terminally differentiated photoreceptor cells express high levels of MSI1 and MSI2, pointing to a role for the two proteins in vision. Combined knockout of Msi1 and Msi2 in mature photoreceptor cells abrogated the retinal response to light and caused photoreceptor cell death. In photoreceptor cells the Musashi proteins perform distinct nuclear and cytoplasmic functions. In the nucleus, the Musashi proteins promote splicing of photoreceptor-specific alternative exons. Surprisingly, conserved photoreceptor-specific alternative exons in genes critical for vision proved to be dispensable, raising questions about the selective pressures that lead to their conservation. In the cytoplasm MSI1 and MSI2 activate protein expression. Loss of Msi1 and Msi2 lead to reduction in the levels of multiple proteins including proteins required for vision and photoreceptor survival. The requirement for MSI1 and MSI2 in terminally differentiated photoreceptors alongside their role in stem cells shows that, depending on cellular context, these two proteins can control processes ranging from cell proliferation to sensory perception.
 
Anthocyanins, a major class of flavonoids, are important pigments of grape berries. Despite the recent discovery of the genetic cause underlying the loss of color, the metabolomic and molecular responses are unknown. Anthocyanin quantification among diverse berry color muscadines suggests that all genotypes could produce adequate anthocyanin quantities, irrespective of berry color. Transcriptome profiling of contrasting color muscadine genotypes proposes a potential deficiency that occurs within the anthocyanin transport and/or degradation mechanisms and might cause unpigmented berries. Genome-wide association studies highlighted a region on chromosome-4, comprising several genes encoding glutathione S-transferases involved in anthocyanin transport. Sequence comparison among genotypes reveals the presence of two GST4b alleles that differ by substituting the conserved amino acid residue Pro 171-to-Leu. Molecular dynamics simulations demonstrate that GST4b2-Leu 171 encodes an inactive protein due to modifications within the H-binding site. Population gen-otyping suggests the recessive inheritance of the unpigmented trait with a GST4b2/2 homozygous. A model defining colorless muscadines' response to the mutation stimulus, avoiding the impact of trapped anthocyanins within the cytoplasm is established.
 
  • Esin KarahanEsin Karahan
  • Luke TaitLuke Tait
  • Ruoguang SiRuoguang Si
  • [...]
  • Jiaxiang ZhangJiaxiang Zhang
Humans differ from each other in a wide range of biometrics, but to what extent brain connectivity varies between individuals remains largely unknown. By combining diffusion-weighted imaging (DWI) and magnetoencephalography (MEG), this study characterizes the inter-subject variability (ISV) of multimodal brain connectivity. Structural connectivity is characterized by higher ISV in association cortices including the core multiple-demand network and lower ISV in the sensorimotor cortex. MEG ISV exhibits frequency-dependent signatures, and the extent of MEG ISV is consistent with that of structural connectivity ISV in selective macroscopic cortical clusters. Across the cortex, the ISVs of structural connectivity and beta-band MEG functional connectivity are negatively associated with cortical myelin content indexed by the quantitative T1 relaxation rate measured by high-resolution 7 T MRI. Furthermore, MEG ISV from alpha to gamma bands relates to the hindrance and restriction of the white-matter tissue estimated by DWI microstructural models. Our findings depict the inter-relationship between the ISV of brain connectivity from multiple modalities, and highlight the role of tissue microstructure underpinning the ISV.
 
  • Panjiao LinPanjiao Lin
  • Benjamin T. GillardBenjamin T. Gillard
  • Audrys G. PaužaAudrys G. Pauža
  • [...]
  • David MurphyDavid Murphy
Water conservation is vital for life in the desert. The dromedary camel (Camelus dromedarius) produces low volumes of highly concentrated urine, more so when water is scarce, to conserve body water. Two hormones, arginine vasopressin and oxytocin, both produced in the supraoptic nucleus, the core hypothalamic osmoregulatory control centre, are vital for this adaptive process, but the mechanisms that enable the camel supraoptic nucleus to cope with osmotic stress are not known. To investigate the central control of water homeostasis in the camel, we first build three dimensional models of the camel supraoptic nucleus based on the expression of the vasopressin and oxytocin mRNAs in order to facilitate sampling. We then compare the transcriptomes of the supraoptic nucleus under control and water deprived conditions and identified genes that change in expression due to hyperosmotic stress. By comparing camel and rat datasets, we have identified common elements of the water deprivation transcriptomic response network, as well as elements, such as extracellular matrix remodelling and upregulation of angiotensinogen expression, that appear to be unique to the dromedary camel and that may be essential adaptations necessary for life in the desert. Comparative transcriptomic analyses between dromedary camels and rats provide insight into water homeostasis gene responses in animals under water stress conditions.
 
Widespread use of unregulated acoustic technologies in maritime industries raises concerns about effects on acoustically sensitive marine fauna worldwide. Anthropogenic noise can disrupt behavior and may cause short- to long-term disturbance with possible population-level consequences, particularly for animals with a limited geographic range. Ultrasonic antifouling devices are commercially available, installed globally on a variety of vessel types, and are marketed as an environmentally-friendly method for biofouling control. Here we show that they can be an acoustic disturbance to marine wildlife, as seasonal operation of these hull-mounted systems by tourist vessels in the marine protected area of Guadalupe Island, México resulted in the reduced presence of a potentially resident population of Cuvier’s beaked whales (Ziphius cavirostris). Human activities are rapidly altering soundscapes on local and global scales, and these findings highlight the need to identify key noise sources and assess their impacts on marine life to effectively manage oceanic ecosystems.
 
Highly unsaturated fatty acids (HUFAs) are fatty acids with more than three double bonds in the molecule. Mammalian testes contain very high levels of omega-6 HUFAs compared with other tissues. However, the metabolic and biological significance of these HUFAs in the mammalian testis is poorly understood. Here we show that Leydig cells vigorously synthesize omega-6 HUFAs to facilitate male sex hormone production. In the testis, FADS2 (Fatty acid desaturase 2), the rate-limiting enzyme for HUFA biosynthesis, is highly expressed in Leydig cells. In this study, pharmacological and genetic inhibition of FADS2 drastically reduces the production of omega-6 HUFAs and male steroid hormones in Leydig cells; this reduction is significantly rescued by supplementation with omega-6 HUFAs. Mechanistically, hormone- sensitive lipase (HSL; also called LIPE), a lipase that supplies free cholesterol for steroid hormone production, preferentially hydrolyzes HUFA-containing cholesteryl esters as sub- strates. Taken together, our results demonstrate that Leydig cells highly express FADS2 to facilitate male steroid hormone production by accumulating omega-6 HUFA-containing cholesteryl esters, which serve as preferred substrates for HSL. These findings unveil a previously unrecognized importance of omega-6 HUFAs in the mammalian male reproductive system.
 
The deficient metabolic conversion of P5C to glutamic acid leads to aberrant reactivity
a Deficiency of P5C dehydrogenase leads to the accumulation of P5C and glutamate 5-semialdehyde. b Reaction of P5C with PLP leads to the deactivation of the latter. c Previously proposed biomarkers for HPII⁶. d Biomarkers of P5C with 1,3-dicarbonyl compounds were identified in this study.
Collision-induced dissociation (CID) MS/MS spectra of features A and C isolated from HPII plasma samples
a CID MS/MS spectra of feature A (m/z 172.0964). b CID MS/MS spectra of feature C (m/z 174.0765).
Confirmation of the structural assignment of feature A and B with synthetic reference standards
Comparison of the experimental IR spectra of a feature A (black trace) and reference standard 4cis (blue line) with theoretical IR spectrum of 4cis (orange trace), b feature A (black trace) and reference standard 4trans (blue line) with theoretical IR spectrum of 4trans (orange trace). Molecular structures corresponding to the protonated reference standards are inlayed in each panel.
Synthesis of the standards used for the confirmation of the biomarkers
Biomarkers 4-5 were synthesized via (a) an incubation of malonic acid or acetoacetic acid with P5C and b the mixture of diasteroeisomers of 4 was synthesized via N-acyliminium ion chemistry.
Biomarkers identified for HPII
a Proposed molecular structure of the original detected reaction product of P5C with acetoacetic acid⁶. b Previously characterized adduct of P5C with PLP. c Biomarkers identified in this work. d Pathway of formation of the detected biomarkers.
Hyperprolinemia type II (HPII) is an inborn error of metabolism due to genetic variants in ALDH4A1, leading to a deficiency in Δ-1-pyrroline-5-carboxylate (P5C) dehydrogenase. This leads to an accumulation of toxic levels of P5C, an intermediate in proline catabolism. The accumulating P5C spontaneously reacts with, and inactivates, pyridoxal 5’-phosphate, a crucial cofactor for many enzymatic processes, which is thought to be the pathophysiological mechanism for HPII. Here, we describe the use of a combination of LC-QTOF untargeted metabolomics, NMR spectroscopy and infrared ion spectroscopy (IRIS) to identify and characterize biomarkers for HPII that result of the spontaneous reaction of P5C with malonic acid and acetoacetic acid. We show that these biomarkers can differentiate between HPI, caused by a deficiency of proline oxidase activity, and HPII. The elucidation of their molecular structures yields insights into the disease pathophysiology of HPII.
 
Strokes cause lesions that damage brain tissue, disrupt normal brain activity patterns and can lead to impairments in motor function. Although modulation of cortical activity is central to stimulation-based rehabilitative therapies, aberrant and adaptive patterns of brain activity after stroke have not yet been fully characterized. Here, we apply a brain dynamics analysis approach to study longitudinal brain activity patterns in individuals with ischemic pontine stroke. We first found 4 commonly occurring brain states largely characterized by high amplitude activations in the visual, frontoparietal, default mode, and motor networks. Stroke subjects spent less time in the frontoparietal state compared to controls. For individuals with dominant-hand CST damage, more time spent in the frontoparietal state from 1 week to 3-6 months post-stroke was associated with better motor recovery over the same time period, an association which was independent of baseline impairment. Furthermore, the amount of time spent in brain states was linked empirically to functional connectivity. This work suggests that when the dominant-hand CST is compromised in stroke, resting state configurations may include increased activation of the frontoparietal network, which may facilitate compensatory neural pathways that support recovery of motor function when traditional motor circuits of the dominant-hemisphere are compromised.
 
Detection of nucleic acid amplification has typically required sophisticated laboratory instrumentation, but as the amplification techniques have moved away from the lab, complementary detection techniques have been implemented to facilitate point-of-care, field, and even at-home applications. Simple visual detection approaches have been widely used for isothermal amplification methods, but have generally displayed weak color changes or been highly sensitive to sample and atmospheric effects. Here we describe the use of pyridylazophenol dyes and binding to manganese ion to produce a strong visible color that changes in response to nucleic acid amplification. This detection approach is easily quantitated with absorbance, rapidly and clearly visible by eye, robust to sample effects, and notably compatible with both isothermal and PCR amplification. Nucleic acid amplification and molecular diagnostic methods are being used in an increasing number of novel applications and settings, and the ability to reliably and sensitively detect them without the need for additional instrumentation will enable even more access to these powerful techniques.
 
An efficient strategy for cancer therapy is presented, in which a tumor mass is initially pretreated with calcium hydroxide, then treated with Taxotere (TXT). In this regard, an advanced delivery system based on iron oxide nanoparticles has been designed. The surface of nanoparticles was functionalized with sortilin (SORT-1, a human IgG1 monoclonal antibody) that specifically encodes caov-4 ovarian cancerous cells. Plasmonic heating of the incorporated gold nanoparticles in polyvinyl alcohol (PVA) has been exploited to control the release process of TXT. The in vitro, ex vivo and in vivo experiments have exhibited high efficacy of a seven-day pretreatment by Ca(OH)2 plus 14 days treatment program by Ca(OH)2@Fe3O4/PVA/Au-SORT nano-therapeutics, where more penetration ratio resulted in tumor growth inhibition by ca. 78.3%. As a result, due to showing high values of the anti-tumor properties and biosafety, the presented pretreatment strategy is suggested for more effective treatment on the aged tumors.
 
Unconjugated bilirubin (UCB) confers Th17-cells immunosuppressive features by activating aryl-hydrocarbon-receptor, a modulator of toxin and adaptive immune responses. In Crohn’s disease, Th17-cells fail to acquire regulatory properties in response to UCB, remaining at an inflammatory/pathogenic state. Here we show that UCB modulates Th17-cell metabolism by limiting glycolysis and through downregulation of glycolysis-related genes, namely phosphoglycerate-kinase-1 (PGK1) and aldolase-A (ALDOA). Th17-cells of Crohn’s disease patients display heightened PGK1 and ALDOA and defective response to UCB. Silencing of PGK1 or ALDOA restores Th17-cell response to UCB, as reflected by increase in immunoregulatory markers like FOXP3, IL-10 and CD39. In vivo, PGK1 and ALDOA silencing enhances UCB salutary effects in trinitro-benzene-sulfonic-acid-induced colitis in NOD/scid/gamma humanized mice where control over disease activity and enhanced immunoregulatory phenotypes are achieved. PGK1 and/or ALDOA blockade might have therapeutic effects in Crohn’s disease by favoring acquisition of regulatory properties by Th17-cells along with control over their pathogenic potential.
 
FADS2 is highly expressed in Leydig cells
a The biosynthetic pathway of HUFAs. b Tissues from FADS2+/+ (WT) and FADS2−/− (KO) mice were analyzed by immunoblotting using an anti-mouse FADS2 antibody. β-actin was used as a loading control. c Immunohistochemical analysis of testis sections from 12-week-old FADS2+/+ and FADS2−/− mice. Green, FADS2; Magenta, HSD3B1; blue, DAPI. Scale bar, 50 µm.
HUFAs are reduced in FADS2−/− liver and testis
Quantification of HUFA species of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) in livers (a–d) and testes (e–h) from FADS2+/+ (WT) and FADS2−/− (KO) mice. The values of each molecular species are expressed as a percentage relative to WT. n = 3 (a–d) and n = 4 (e–h) for each group. Data shown are the mean ± SEM. The detailed lipidomics data are shown in Supplementary Fig. 3. ARA arachidonic acid, DHA docosahexaenoic acid, DPA n-6 docosapentaenoic acid.
Omega-6 HUFAs are required for steroid hormone production in MA-10 cells
a Immunoblotting using anti-mouse FADS2 antibody of samples from testes and Leydig cells from FADS2+/+ (WT) and FADS2−/− (KO) mice and MA-10 cells. β-actin was used as a loading control. b–d Quantification of HUFA species in MA-10 cells. The values of each molecular species were expressed as a percentage relative to CTL. n = 3 for each group. The detailed lipidomics data are shown in Supplementary Fig. 4. e Reduced steroid hormone production in MA-10 cells treated with SC-26196. Steroid hormones were extracted from the supernatant. n = 4 for each group. Significance is based on unpaired two-tailed t-test with Welch’s correction. f Steroid hormone production in MA-10 cells supplemented with HUFAs. Steroid hormones were extracted from the supernatant. n = 4 for each group. Significance is based on Dunnett’s multiple comparisons test. Data shown are the mean ± SEM. Steroid hormone levels in the cell pellets are shown in Supplementary Fig. 4f. CTL control, hCG human chorionic gonadotropin, ARA arachidonic acid, EPA eicosapentaenoic acid, DHA docosahexaenoic acid, DPA n-6 docosapentaenoic acid.
Omega-6 HUFAs are required for steroid hormone production in Leydig cells
a–c Quantification of HUFA species in Leydig cells from FADS2+/+ (WT) and FADS2−/− (KO) mice. n = 3 for each group. The detailed lipidomics data are shown in Supplementary Fig. 5. d Reduced steroid hormone production in Leydig cells from FADS2−/− mice. Steroid hormones were extracted from the supernatant. n = 4 for each group. Significance is based on unpaired two-tailed t-test with Welch’s correction. e–g Steroid hormone production in Leydig cells from FADS2−/− mice supplemented with ARA (e), DPAn-6 (f), or DHA (g). n = 4 for each group. Significance is based on Tukey’s multiple comparisons test. Data shown are the mean ± SEM. Steroid hormone levels in the cell pellets were shown in Supplementary Fig. 5f–h. DHT dihydrotestosterone, hCG human chorionic gonadotropin, ARA arachidonic acid, DHA docosahexaenoic acid, DPA n-6 docosapentaenoic acid.
HUFA-containing cholesteryl ester species are preferred substrates for HSL
a The lipase activity of recombinant mouse hormone-sensitive lipase (mHSL). HEK293T cells were transfected with empty vector (mock) or mHSL-expressing vector (mHSL) and the cell lysate (1 mg/mL) was incubated with each cholesterol ester species (100 μM) for 30 min at 37 °C. FFA, free fatty acid. n = 3 for each group. b HSL-dependent FFA liberation upon human chorionic gonadotropin (hCG) stimulation in MA-10 cells. Triacsin C was pretreated to block the conversion of FFAs to fatty acyl-CoAs. The FFA levels insensitive to HSL-IN-1 were subtracted to evaluate HSL-dependent FFA production. n = 3 for each group. Significance is based on unpaired two-tailed t-test with Welch’s correction. Data shown are the mean ± SEM. The detailed data are shown in Supplementary Fig. 13.
Highly unsaturated fatty acids (HUFAs) are fatty acids with more than three double bonds in the molecule. Mammalian testes contain very high levels of omega-6 HUFAs compared with other tissues. However, the metabolic and biological significance of these HUFAs in the mammalian testis is poorly understood. Here we show that Leydig cells vigorously synthesize omega-6 HUFAs to facilitate male sex hormone production. In the testis, FADS2 (Fatty acid desaturase 2), the rate-limiting enzyme for HUFA biosynthesis, is highly expressed in Leydig cells. In this study, pharmacological and genetic inhibition of FADS2 drastically reduces the production of omega-6 HUFAs and male steroid hormones in Leydig cells; this reduction is significantly rescued by supplementation with omega-6 HUFAs. Mechanistically, hormone-sensitive lipase (HSL; also called LIPE), a lipase that supplies free cholesterol for steroid hormone production, preferentially hydrolyzes HUFA-containing cholesteryl esters as sub-strates. Taken together, our results demonstrate that Leydig cells highly express FADS2 to facilitate male steroid hormone production by accumulating omega-6 HUFA-containing cholesteryl esters, which serve as preferred substrates for HSL. These findings unveil a previously unrecognized importance of omega-6 HUFAs in the mammalian male reproductive system.
 
Despite the clear potential of livestock models of human functional variants to provide important insights into the biological mechanisms driving human diseases and traits, their use to date has been limited. Generating such models via genome editing is costly and time consuming, and it is unclear which variants will have conserved effects across species. In this study we address these issues by studying naturally occurring livestock models of human functional variants. We show that orthologues of over 1.6 million human variants are already segregating in domesticated mammalian species, including several hundred previously directly linked to human traits and diseases. Models of variants linked to particular phenotypes, including metabolomic disorders and height, are preferentially shared across species, meaning studying the genetic basis of these phenotypes is particularly tractable in livestock. Using machine learning we demonstrate it is possible to identify human variants that are more likely to have an existing livestock orthologue, and, importantly, we show that the effects of functional variants are often conserved in livestock, acting on orthologous genes with the same direction of effect. Consequently, this work demonstrates the substantial potential of naturally occurring livestock carriers of orthologues of human functional variants to disentangle their functional impacts.
 
Wearing a face mask has become essential to contain the spread of COVID-19 and has become mandatory when collecting fMRI data at most research institutions. Here, we investigate the effects of wearing a surgical mask on fMRI data in n = 37 healthy participants. Activations during finger tapping, emotional face matching, working memory tasks, and rest were examined. Preliminary fMRI analyses show that despite the different mask states, resting-state signals and task activations were relatively similar. Resting-state functional connectivity showed negligible attenuation patterns in mask-on compared with mask-off. Task-based ROI analysis also demonstrated no significant difference between the two mask states under each contrast investigated. Notwithstanding the overall insignificant effects, these results indicate that wearing a face mask during fMRI has little to no significant effect on resting-state and task activations.
 
Illustration of time-delay embedded Hidden Markov Model (TDE-HMM) approach applied on MEG data
MEG data preprocessing (Independent component analysis (ICA) for artifact removal, source reconstruction, parcellation, leakage correction, and sign disambiguation—see Methods). We used a linearly constrained minimum variance beamformer to extract a continuous time series for nodes of the dynamic pain connectome based on anatomical 3D T1 scans of each participant. Spontaneous cortical activity transiently organizes into frequency-specific phase-coupling networks. Principal component analysis was applied for dimensionality reduction, and HMM inference was then used to identify the state time-courses (state probability) and the state parameters in HCs and those with neuropathic pain. Each state was characterized as having its own distinct spatial, temporal, and spectral properties. Individual power maps and phase-coupling patterns (networks) were estimated for active states and illustrated by their temporal features (i.e., fractional occupancy, interval times, switching rates, and lifetimes). States were spatially defined and spectrally resolved according to the main frequency bands (i.e., delta/theta, alpha, beta, and low gamma).
Brain states identified using an HMM show functional networks of spectral power and coherence in the DPC
a Brain regions with significant power and phase-coupling are represented across a wideband frequency range (1–30 Hz) for the 12 estimated states in n = 40 healthy controls. Node spectral power is relative to the temporal average, and node size is in relation to the mean power (Z score) across states (blue and red colors reflect power that is lower or higher than the average over states, respectively). Edges between nodes show functional connectivity, and only significant and high-valued connections are shown. Significant coherences were mainly found between active nodes in each state. The same 12 states were found in the patients cohort, and the nodes’ repartition was similar across a wideband frequency range. b Spectral power averaged across all brain regions in brain states as a function of frequency (green: delta/theta, blue: alpha, and red: beta). c Spectral profiles of the DMN (post. DMN: blue; ant. DMN: purple), SMN (green), and visual (yellow) states, in terms of power averaged across brain regions, in comparison to the grand average (black line); Standard deviations are represented. *P < 0.001; permutation P value, 5000 bootstrap samples. DMN Default mode network, ANP ascending nociceptive pathway, OP-insular (operculo-insular), SMN sensorimotor, DPC dynamic pain connectome.
Individuals with neuropathic pain had higher alpha power in the right ANP state and higher beta band power in the SMN state
Brain nodes in neuropathic pain (NP) showing significant local power increased (red nodes) and decreased (blue nodes) in the a right Ascending nociceptive pathway (ANP: higher alpha power in the right thalamus, primary somatosensory (S1), and motor cortex (M1), posterior insula (pINS), temporo-parietal junction (TPJ) and lower power in the dorsolateral prefrontal cortex (DLPFC), medial prefrontal cortex (mPFC), and left anterior insula (aINS) and b sensorimotor (SMN: S1 and TPJ, but had lower power in frontal regions including the DLPFC, mPFC, dorso-medial prefrontal cortex (dmPFC), subgenual anterior cingulate cortex (sgACC), midcingulate cortex (MCC) and sensorimotor area (SMA) state compared with healthy controls (HCs) are depicted. The effect size (Cohen’s d) between the average power of n = 40 healthy controls and n = 40 individuals with neuropathic pain is shown in the Gardner-Altman estimation plots. 5000 bootstrap samples were taken; the confidence interval is bias-corrected and accelerated. Each dot on the brain maps represents a brain region, and its size is in relation to the difference in spectral power between the neuropathic pain and control groups (i.e., proportional to the effect size, larger dot means a stronger difference).
Coherence changes in individuals with neuropathic pain compared to HC group in specific frequency ranges
The brain maps show connections (in blue) associated with significant changes between neuropathic pain and healthy control groups in one brain state; each dot (black) represents one brain region. In the circular coherence plots, blue and red/yellow colors reflect respectively coherence that is lower and higher in neuropathic pain than in the control group. In neuropathic pain patients, we found that the coherence was a higher in the salience (SN; left: S1, pINS, aINS, thalamus, caudate, mPFC, MCC, sgACC; right: DLPFC, M1, medial temporal lobe (MTL), pINS, S1, posterior cingulate cortex (PCC), Precuneus, occipital lobe) state in the alpha band; and b in the sensorimotor (SMN; dmPFC, mPFC, sgACC, MTL, pallidum, putamen, caudate Left: occi, TPJ, S1, M1, DLPFC, right: amygdala, S2) state in the beta band, c whereas coherence was lower in the dorsal attention state (sgACC, MCC; right: aINS, DLPFC, pallidum, putamen) in the delta/theta compared with healthy control. *P < 0.01, corrected 5000 permutations, network-based statistic method.
Brain states temporal features: chronnectome in healthy controls and disturbances associated with neuropathic pain
a Distribution of state life times, b distribution of interval times between state visits, and c fractional occupancies (proportion of time spent in each state) are depicted for the n = 40 healthy controls group. The two states related to the default mode network (DMN) have distinct temporal features compared with the other states. The posterior DMN state had higher lifetimes, and both posterior and anterior DMN states had higher interval times, but the proportion of time spent in the posterior DMN state was lower compared to the other states (P < 0.01). In n = 40 participants with neuropathic pain, d the maximum fractional occupancy was lower, and e the sensorimotor (SMN) was more frequently active compared with n = 40 healthy controls. f The proportion of time spent was higher in the SMN state and lower in the dorsal attention state compared to healthy controls. Standard deviations are shown. *P < 0.01; permutation P value, 5000 bootstrap samples. DMN Default mode network, SMN sensorimotor, HC healthy control, NP neuropathic pain.
Neuronal populations in the brain are engaged in a temporally coordinated manner at rest. Here we show that spontaneous transitions between large-scale resting-state networks are altered in chronic neuropathic pain. We applied an approach based on the Hidden Markov Model to magnetoencephalography data to describe how the brain moves from one activity state to another. This identified 12 fast transient (~80 ms) brain states including the sensorimotor, ascending nociceptive pathway, salience, visual, and default mode networks. Compared to healthy controls, we found that people with neuropathic pain exhibited abnormal alpha power in the right ascending nociceptive pathway state, but higher power and coherence in the sensorimotor network state in the beta band, and shorter time intervals between visits of the sensorimotor network, indicating more active time in this state. Conversely, the neuropathic pain group showed lower coherence and spent less time in the frontal attentional state. Therefore, this study reveals a temporal imbalance and dysregulation of spectral frequency-specific brain microstates in patients with neuropathic pain. These findings can potentially impact the development of a mechanism-based therapeutic approach by identifying brain targets to stimulate using neuromodulation to modify abnormal activity and to restore effective neuronal synchrony between brain states.
 
Non-alcoholic fatty liver disease (NAFLD) is associated with hepatic metabolism dysfunction. However, the mechanistic role of miR204 in the development of NAFLD is unknown. We investigate the functional significance of miR204 in the evolution of NAFLD. IDH2 KO mice feed a normal diet (ND) or HFD increased body weight, epididymal fat-pad weight, lipid droplet in liver, blood parameter and inflammation compared to WT mice fed a ND or HFD. Moreover, the expression of miR204 is increased in mice with IDH2 deficiency. Increased miR204 by IDH2 deficiency regulates carnitine palmitoyltransferase 1a (cpt1a) synthesis, which inhibits fatty acid β-oxidation. Inhibition of miR204 prevents the disassembly of two fatty acid-related genes by activating CPT1a expression, which decreases lipid droplet in liver, inflammatory cytokines, epididymal fat pad weight, blood parameters. Increased miR204 by IDH2 deficiency promotes the pathogenesis of HFD-induced NAFLD by regulating hepatic fatty acid metabolism and inflammation.
 
Clockwise rotation of the primitive heart tube, a process regulated by restricted left-sided Nodal signaling, is the first morphological manifestation of left-right asymmetry. How Nodal regulates cell behaviors to drive asymmetric morphogenesis remains poorly understood. Here, using high-resolution live imaging of zebrafish embryos, we simultaneously visualized cellular dynamics underlying early heart morphogenesis and resulting changes in tissue shape, to identify two key cell behaviors: cell rearrangement and cell shape change, which convert initially flat heart primordia into a tube through convergent extension. Interestingly, left cells were more active in these behaviors than right cells, driving more rapid convergence of the left primordium, and thereby rotating the heart tube. Loss of Nodal signaling abolished the asymmetric cell behaviors as well as the asymmetric convergence of the left and right heart primordia. Collectively, our results demonstrate that Nodal signaling regulates the magnitude of morphological changes by acting on basic cellular behaviors underlying heart tube formation, driving asymmetric deformation and rotation of the heart tube.
 
Encephalization slopes estimated for carnivoran taxonomic groups
Slopes were estimated for a carnivoran families and b the two suborders. Values on the allometries of encephalization indicate slope characterizing the encephalization for each taxonomic group. Black doted slope represents the correlation between brain volume and body mass in terrestrial carnivorans using a phylogenetic generalized least squares regression on dataset 1 (N = 174), with R2 and p-values associated.
Phylogeny of the Carnivora order and illustrations of representative species
The phylogenetic relationships of the carnivoran species used in this study, derived from Slater and Friscia (2019) associated with illustrations of a representative species of each family, its estimated brain volume, and the relative brain size (RBS). 3D crania represent different specimens from our dataset. They were digitized using a white light fringe surface scanner and a laser surface scanning. The silhouettes were drawn by the authors (M.M) and are available on the Phylopic website http://www.phylopic.org/.
Patterns of encephalization among carnivoran taxonomic groups
Calculated Phylogenetic relative brain size estimated for a families and b the two suborders. Grey dotted lines indicate the expected relative brain size under an allometric model (i.e., brain size is entirely explained by body mass and explained the entire variation observed for the dependent variable). Boxes represent the third and first quartiles and bold line represents the median.
Phylogeny of the 174 species of terrestrial carnivorans studied showing the repartition of their relative brain size and the estimation of its evolutionary rate and shifts at the ancestral nodes
Coloured dots represent the evolution rates of encephalization computed according to our phylogenetic Ridge Regression. Arrows represent the significant shifts of the evolution rate for the relative brain size. Taxonomic groups highlighted in red boxes display a significant increase in the rate of encephalization whereas the blue box indicate a significant decrease. The silhouettes were drawn by the authors (M.M) and are available on the Phylopic website http://www.phylopic.org/.
Correlation between the different predictors used in this study
Correlation matrix of the ecological, environmental, social, and life-trait history predictors tested in this study with associated values and p-values. Negatively correlated variables are shown in blue while those positively correlated are coloured in red. The statistical significance of each correlation is signified by asterisks whereas size of the circle is defined by the correlation strength. Asterisks indicate the level of significance (*0.05; **0.01 and ***0.001 respectively).
The reasons why some animals have developed larger brains has long been a subject of debate. Yet, it remains unclear which selective pressures may favour the encephalization and how it may act during evolution at different taxonomic scales. Here we studied the patterns and tempo of brain evolution within the order Carnivora and present large-scale comparative analysis of the effect of ecological, environmental, social, and physiological variables on relative brain size in a sample of 174 extant carnivoran species. We found a complex pattern of brain size change between carnivoran families with differences in both the rate and diversity of encephalization. Our findings suggest that during carnivorans' evolution, a trade-off have occurred between the cognitive advantages of acquiring a relatively large brain allowing to adapt to specific environments, and the metabolic costs of the brain which may constitute a disadvantage when facing the need to colonize new environments.
 
Long-read sequencing technology enable better characterization of structural variants (SVs). To adapt the technology to population-scale analyses, one critical issue is to obtain sufficient amount of high-molecular-weight genomic DNA. Here, we propose utilizing activated T lymphocytes, which can be established efficiently in a biobank to stably supply high-grade genomic DNA sufficiently. We conducted nanopore sequencing of 333 individuals constituting 111 trios with high-coverage long-read sequencing data (depth 22.2x, N50 of 25.8 kb) and identified 74,201 SVs. Our trio-based analysis revealed that more than 95% of the SVs were concordant with Mendelian inheritance. We also identified SVs associated with clinical phenotypes, all of which appear to be stably transmitted from parents to offspring. Our data provide a catalog of SVs in the general Japanese population, and the applied approach using the activated T-lymphocyte resource will contribute to biobank-based human genetic studies focusing on SVs at the population scale.
 
Rhabdomyosarcoma, the most common pediatric sarcoma, has no effective treatment for the pleomorphic subtype. Still, what triggers transformation into this aggressive phenotype remains poorly understood. Here we used Ptch1+/−/ETV7TG/+/− mice with enhanced incidence of rhabdomyosarcoma to generate a model of pleomorphic rhabdomyosarcoma driven by haploinsufficiency of the lysosomal sialidase neuraminidase 1. These tumors share mostly features of embryonal and some of alveolar rhabdomyosarcoma. Mechanistically, we show that the transforming pathway is increased lysosomal exocytosis downstream of reduced neuraminidase 1, exemplified by the redistribution of the lysosomal associated membrane protein 1 at the plasma membrane of tumor and stromal cells. Here we exploit this unique feature for single cell analysis and define heterogeneous populations of exocytic, only partially differentiated cells that force tumors to pleomorphism and promote a fibrotic microenvironment. These data together with the identification of an adipogenic signature shared by human rhabdomyosarcoma, and likely fueling the tumor’s metabolism, make this model of pleomorphic rhabdomyosarcoma ideal for diagnostic and therapeutic studies. A mouse model of pleomorphic rhabdomyosarcoma, an aggressive and treatment-resistant form of pediatric sarcoma, provides an ideal tool for future diagnostic and therapeutic studies.
 
In Duchenne muscular dystrophy, dystrophin loss leads to chronic muscle damage, dysregulation of repair, fibro-fatty replacement, and weakness. We develop methodology to efficiently isolate individual nuclei from minute quantities of frozen skeletal muscle, allowing single nuclei sequencing of irreplaceable archival samples and from very small samples. We apply this method to identify cell and gene expression dynamics within human DMD and mdx mouse muscle, characterizing effects of dystrophin rescue by exon skipping therapy at single nuclei resolution. DMD exon 23 skipping events are directly observed and increased in myonuclei from treated mice. We describe partial rescue of type IIa and IIx myofibers, expansion of an MDSC-like myeloid population, recovery of repair/remodeling M2-macrophage, and repression of inflammatory POSTN1 + fibroblasts in response to exon skipping and partial dystrophin restoration. Use of this method enables exploration of cellular and transcriptomic mechanisms of dystrophin loss and repair within an intact muscle environment. Our initial findings will scaffold our future work to more directly examine muscular dystrophies and putative recovery pathways.
 
Hybrid cells derived through fertilization or somatic cell fusion recognize and separate chromosomes of different origins. The underlying mechanisms are unknown but could prevent aneuploidy and tumor formation. Here, we acutely induce fusion between Drosophila neural stem cells (neuroblasts; NBs) and differentiating ganglion mother cells (GMCs) in vivo to define how epigenetically distinct chromatin is recognized and segregated. We find that NB-GMC hybrid cells align both endogenous (neuroblast-origin) and ectopic (GMC-origin) chromosomes at the metaphase plate through centrosome derived dual-spindles. Physical separation of endogenous and ectopic chromatin is achieved through asymmetric, microtubule-dependent chromatin retention in interphase and physical boundaries imposed by nuclear envelopes. The chromatin separation mechanisms described here could apply to the first zygotic division in insects, arthropods, and vertebrates or potentially inform biased chromatid segregation in stem cells.
 
Alternative splicing is an RNA processing mechanism involved in skeletal muscle development and pathology. Muscular diseases exhibit splicing alterations and changes in mechanobiology leading us to investigate the interconnection between mechanical forces and RNA processing. We performed deep RNA-sequencing after stretching muscle cells. First, we uncovered transcriptional changes in genes encoding proteins involved in muscle function and transcription. Second, we observed that numerous mechanosensitive genes were part of the MAPK pathway which was activated in response to stretching. Third, we revealed that stretching skeletal muscle cells increased the proportion of alternatively spliced cassette exons and their inclusion. Fourth, we demonstrated that the serine and arginine-rich proteins exhibited stronger transcriptional changes than other RNA-binding proteins and that SRSF4 phosphorylation is mechanosensitive. Identifying SRSF4 as a mechanosensitive RNA-binding protein that might contribute to crosstalk between mechanotransduction, transcription, and splicing could potentially reveal novel insights into muscular diseases, particularly those with unknown etiologies.
 
TMEM16F is a Ca²⁺-activated phospholipid scramblase in the TMEM16 family of membrane proteins. Unlike other TMEM16s exhibiting a membrane-exposed hydrophilic groove that serves as a translocation pathway for lipids, the experimentally determined structures of TMEM16F shows the groove in a closed conformation even under conditions of maximal scramblase activity. It is currently unknown if/how TMEM16F groove can open for lipid scrambling. Here we describe the analysis of ~400 µs all-atom molecular dynamics (MD) simulations of the TMEM16F revealing an allosteric mechanism leading to an open-groove, lipid scrambling competent state of the protein. The groove opens into a continuous hydrophilic conduit that is highly similar in structure to that seen in other activated scramblases. The allosteric pathway connects this opening to an observed destabilization of the Ca²⁺ ion bound at the distal site near the dimer interface, to the dynamics of specific protein regions that produces the open-groove state to scramble phospholipids.
 
The twin-arginine translocation (Tat) system transports folded proteins across bacterial and plastid energy transducing membranes. Ion leaks are generally considered to be mitigated by the creation and destruction of the translocation conduit in a cargo-dependent manner, a mechanism that enables tight sealing around a wide range of cargo shapes and sizes. In contrast to the variable stoichiometry of the active translocon, the oligomerization state of the receptor complex is considered more consistently stable but has proved stubbornly difficult to establish. Here, using a single molecule photobleaching analysis of individual inverted membrane vesicles, we demonstrate that Tat receptor complexes are tetrameric in native membranes with respect to both TatB and TatC. This establishes a maximal diameter for a resting state closed pore. A large percentage of Tat-deficient vesicles explains the typically low transport efficiencies observed. This individual reaction chamber approach will facilitate examination of the effects of stochastically distributed molecules.
 
Phylogenetic tree showing the timelines of divergence (MYA: million years ago) and lineage of rats (Rattus norvegicus), squirrels (Sciurus carolinensis) and marmosets (Callithrix jacchus)
Based on ref. 49.
Region of interest (ROI) locations and fingerprint plots
a ROI locations for rats. Top, seed ROIs (all in the right hemisphere) are shown in red, and 8 ROIs for the fingerprint analysis are shown in blue and overlaid on coronal slices of anatomical MR images. Bottom, fingerprint spider plots for frontal seeds with the posterior cingulate cortex (PCC), posterior parietal cortex (PPC), the primary somatosensory cortex (S1), the primary motor cortex (M1), insular cortex (Ins), striatum (Str), pulvinar (Pul), and superior colliculus (SC). b Same as (a), but for squirrels. c Same as (a), but for marmosets. Target region abbreviations: Frontal association cortex (FrA); lateral orbital cortex (LO); medial orbital cortex (MO); frontal cortex, area 3 (Fr3); secondary motor cortex (M2); frontal areas 1–6 (F1–F6); area 6 of cortex, ventral, part a (6 VA); area 6 of cortex, dorsorostral part (6DR); area 8a or cortex, dorsal part (8AD); area 8a of cortex, ventral part (8AV); area 45 of cortex;⁴⁶ area 47 of cortex;⁴⁸ area 46 of cortex, dorsal part (46D); area 46 of cortex, ventral part (46 V).
Similarity of interareal functional connectivity patterns between the species
a Comparison of interareal functional connectivity patterns between rats and marmosets. For each species, cosine similarity values are plotted in matrix form. A high cosine similarity value suggests that the connectivity values are more comparable than a lower cosine similarity value. Significant differences are marked by a white asterisk within the similarity matrix. b Same as (a), but for comparing squirrels with marmosets. c Same as (a), but for comparing rats with squirrels. Seed region abbreviations: Frontal association cortex (FrA); lateral orbital cortex (LO); medial orbital cortex (MO); frontal cortex, area 3 (Fr3); secondary motor cortex (M2); frontal areas 1–6 (F1–F6); area 6 of cortex, ventral, part a (6 VA); area 6 of cortex, dorsorostral part (6DR); area 8a or cortex, dorsal part (8AD); area 8a of cortex, ventral part (8AV); area 45 of cortex; area 47 of cortex; area 46 of cortex, dorsal part (46D); area 46 of cortex, ventral part (46 V).
Similarities in interareal functional connectivity of squirrel region F3 and marmoset area 47L
a Fingerprint for squirrel region F3 and marmoset area 47 L shows a similar interareal functional connectivity pattern. Note that for each species, the fingerprints are normalized between 0 and 1 to allow for pattern comparability: A 0 value does not necessarily mean that there was no activation in a region, but just that the region had the lowest relative value within that fingerprint. b Photograph of ex vivo squirrel and marmoset brains and the location of squirrel region F3 and marmoset area 47 L. c Anatomically annotated surface rendering and functional connectivity map of squirrel region F3 with the rest of the brain and overlaid on coronal slices of anatomical MR images. d Anatomically annotated surface rendering and functional connectivity map of marmoset area 47 L with the rest of the brain overlaid on coronal MR atlas slices.
Robust frontoparietal connectivity is a defining feature of primate cortical organization. Whether mammals outside the primate order, such as rodents, possess similar frontoparietal functional connectivity organization is a controversial topic. Previous work has primarily focused on comparing mice and rats to primates. However, as these rodents are nocturnal and terrestrial, they rely much less on visual input than primates. Here, we investigated the functional cortical organization of grey squirrels which are diurnal and arboreal, thereby better resembling primate ecology. We used ultra-high field resting-state fMRI data to compute and compare the functional connectivity patterns of frontal regions in grey squirrels (Sciurus carolinensis), rats (Rattus norvegicus), and marmosets (Callithrix jacchus). We utilized a fingerprinting analysis to compare interareal patterns of functional connectivity from seeds across frontal cortex in all three species. The results show that grey squirrels, but not rats, possess a frontoparietal connectivity organization that resembles the connectivity pattern of marmoset lateral prefrontal cortical areas. Since grey squirrels and marmosets have acquired an arboreal way of life but show no common arboreal ancestor, the expansion of the visual system and the formation of a frontoparietal connectivity architecture might reflect convergent evolution driven by similar ecological niches in primates and tree squirrels. Comparisons of frontoparietal connectivity between marmosets, rats and grey squirrels suggest the formation of a common frontoparietal network architecture among arboreal species (grey squirrels and marmosets) that might reflect convergent evolution.
 
SPACA6 is a sperm-expressed surface protein that is critical for gamete fusion during mammalian sexual reproduction. Despite this fundamental role, little is known about how SPACA6 specifically functions. We elucidated the crystal structure of the SPACA6 ectodomain at 2.2-Å resolution, revealing a two-domain protein containing a four-helix bundle and Ig-like β-sandwich connected via a quasi-flexible linker. This structure is reminiscent of IZUMO1, another gamete fusion-associated protein, making SPACA6 and IZUMO1 founding members of a superfamily of fertilization-associated proteins, herein dubbed the IST superfamily. The IST superfamily is defined structurally by its distorted four-helix bundle and a pair of disulfide-bonded CXXC motifs. A structure-based search of the AlphaFold human proteome identified more protein members to this superfamily; remarkably, many of these proteins are linked to gamete fusion. The SPACA6 structure and its connection to other IST-superfamily members provide a missing link in our knowledge of mammalian gamete fusion.
 
Rising temperatures can lead to the occurrence of a large-scale climatic event, such as the melting of Greenland ice sheet, weakening the AMOC and further increasing dissimilarities between current and future climate. The impacts of such an event are still poorly assessed. Here, we evaluate those impacts across megadiverse countries on 21,146 species of tetrapods and vascular plants using the pessimistic climate change scenario (RCP 8.5) and four different scenarios of Greenland's ice sheet melting. We show that RCP 8.5 emission scenario would lead to a widespread reduction in species' geographic ranges (28-48%), which is projected to be magnified (58-99%) with any added contribution from the melting of Greenland. Also, declines in the potential geographical extent of species hotspots (12-89%) and alterations of species composition (19-91%) will be intensified. These results imply that the influence of a strong and rapid Greenland ice sheet melting, resulting in a large AMOC weakening, can lead to a faster collapse of biodiversity across the globe.
 
Transgenic animals expressing fluorescent proteins are widely used to label specific cells and proteins. By using a split Cre recombinase fused with mCherry-binding nanobodies or designed ankyrin repeat proteins, we created Cre recombinase dependent on red fluorescent protein (RFP) (Cre-DOR). Functional binding units for monomeric RFPs are different from those for polymeric RFPs. We confirmed selective target RFP-dependent gene expression in the mouse cerebral cortex using stereotaxic injection of adeno-associated virus vectors. In estrogen receptor-beta (Esr2)-mRFP1 mice and gastrin-releasing peptide receptor (Grpr)-mRFP1 rats, we confirmed that Cre-DOR can be used for selective tracing of the neural projection from RFP-expressing specific neurons. Cellular localization of RFPs affects recombination efficiency of Cre-DOR, and light and chemical-induced nuclear translocation of an RFP-fused protein can modulate Cre-DOR efficiency. Our results provide a method for manipulating gene expression in specific cells expressing RFPs and expand the repertory of nanobody-based genetic tools.
 
Topoisomerase I (TOP1) controls the topological state of DNA during DNA replication, and its dysfunction due to treatment with an inhibitor, such as camptothecin (CPT), causes replication arrest and cell death. Although CPT has excellent cytotoxicity, it has the disadvantage of instability under physiological conditions. Therefore, new types of TOP1 inhibitor have attracted particular attention. Here, we characterised the effect of a non-camptothecin inhibitor, Genz-644282 (Genz). First, we found that treatment with Genz showed cytotoxicity by introducing double-strand breaks (DSBs), which was suppressed by co-treatment with aphidicolin. Genz-induced DSB formation required the functions of TOP1. Next, we explored the advantages of Genz over CPT and found it was effective against CPT-resistant TOP1 carrying either N722S or N722A mutation. The effect of Genz was also confirmed at the cellular level using a CPT-resistant cell line carrying N722S mutation in the TOP1 gene. Moreover, we found arginine residue 364 plays a crucial role for the binding of Genz. Because tyrosine residue 723 is the active centre for DNA cleavage and re-ligation by TOP1, asparagine residue 722 plays crucial roles in the accessibility of the drug. Here, we discuss the mechanism of action of Genz on TOP1 inhibition.
 
The monomeric catalytic domain (residues 1–199) of SARS-CoV-2 main protease (MPro¹⁻¹⁹⁹) fused to 25 amino acids of its flanking nsp4 region mediates its autoprocessing at the nsp4-MPro¹⁻¹⁹⁹ junction. We report the catalytic activity and the dissociation constants of MPro¹⁻¹⁹⁹ and its analogs with the covalent inhibitors GC373 and nirmatrelvir (NMV), and the estimated monomer-dimer equilibrium constants of these complexes. Mass spectrometry indicates the presence of the accumulated adduct of NMV bound to MProWT and MPro¹⁻¹⁹⁹ and not of GC373. A room temperature crystal structure reveals a native-like fold of the catalytic domain with an unwound oxyanion loop (E state). In contrast, the structure of a covalent complex of the catalytic domain-GC373 or NMV shows an oxyanion loop conformation (E* state) resembling the full-length mature dimer. These results suggest that the E-E* equilibrium modulates autoprocessing of the main protease when converting from a monomeric polyprotein precursor to the mature dimer.
 
Workflow of FVC
Taking the VCF and BAM files as input, FVC uses the feature construction module to build three types of features related to sequence content, sequencing experiment, and bioinformatic analysis process. If a pre-trained model is already built for the specific pipeline, the variants can be immediately classified as true or false using the filtering module. Otherwise, the pre-trained model can be built using the data construction and supervised learning modules of FVC.
The performance of different filtering methods when applied to SNV or INDEL variants
The SNV and INDEL variants used as testing data are derived from whole-genome sequencing datasets (HG001, HG003, HG004, and HG006) at 30× coverage. The performance of different methods is assessed on the SNV and INDEL variants identified by a GATK HaplotypeCaller; b Mutect2; c Varscan2; and d DeepVariant. The performance is assessed by using the leave-one-individual-out cross-validation method. The shaded area indicates the 95% confidence intervals (n = 4 biologically independent samples). FVC consistently achieves the highest AUC score when applied to both SNV and INDEL variants.
The performance of different filtering methods when applied to high-frequency variants or low-frequency variants
The high-frequency variants and low-frequency variants used as testing data are derived from whole-genome sequencing datasets (HG001, HG003, HG004, and HG006) at 30× coverage and identified by GATK, Varscan2, Mutect2, and DeepVariant, separately. Variant calls with variant allelic frequency (VAF) of more than 20% are defined as high-frequency variants. The other variant calls are defined as low-frequency variant calls. The performance of the filtering methods is assessed on the a high frequency variants using MCC; b high frequency variants using OFO; c low frequency variants using MCC; and d low frequency variants using OFO. The circle indicates the metric score achieved by FVC when applied to each specified testing data. The error bar indicates the 95% confidence intervals (n = 4 biologically independent samples). Asterisk denotes the significance of the comparison using a one-sided paired T-test (*p < 0.05, **p < 0.001), where the null hypothesis is that the FVC performs no better than the compared method. FVC consistently shows log OFO < 0 when applied to high-frequency and low-frequency variants.
The performance of different filtering methods when applied to hard-to-detect or easy-to-detect variants
The easy-to-detect and hard-to-detect variant calls used as testing data are derived from the whole-genome sequencing datasets (HG001, HG003, HG004, and HG006) at 30× coverage and identified by GATK, Varscan2, Mutect2, and DeepVariant, separately. The easy-to-detect variants are defined as the variants that are consistently and correctly classified by all the unsupervised filtering methods (Frequency, Hard-Filter, and VQSR). The other variants are defined as hard-to-detect variants. The performance of the filtering methods is assessed on the a hard-to-detect variants using MCC; b hard-to-detect variants using OFO; c easy-to-detect variants using MCC (Frequency, Hard-Filter, and VQSR consistently scored MCC = 1); and d easy-to-detect variants using OFO (Frequency, Hard-Filter, and VQSR consistently scored logOFO = −∞). The circle indicates the metric score achieved by the filtering method when applied to each specified testing data. The error bar indicates the 95% confidence intervals (n = 4 biologically independent samples). Asterisk denotes the significance of the comparison using a one-sided paired T-test (*p < 0.05, **p < 0.001), where the null hypothesis is that the FVC performs no better than the compared method.
The performance of different filtering methods when applied to coding or non-coding variants
The coding and non-coding variant calls used as testing data are derived from whole-genome sequencing data (HG001, HG003, HG004, and HG006) at 30× coverage and identified by GATK, Varscan2, Mutect2, and DeepVariant, separately. The different filtering methods are separately assessed on the a coding variants using MCC; b coding variants measured using OFO; c non-coding variants measured using MCC; and d non-coding variants using OFO. FVC achieves the highest MCC and the lowest log OFO when applied to both types of variants identified by GATK HaplotypeCaller, Varscan2, Mutect2, and DeepVariant. The circle indicates the metric score achieved by the filtering method when applied to each specified testing data. The error bar indicates the 95% confidence intervals (n = 4 biologically independent samples). Asterisk denotes the significance of the comparison using a one-sided paired T-test (*p < 0.05, **p < 0.001), where the null hypothesis is that the FVC performs no better than the compared method.
The quality control of variants from whole-genome sequencing data is vital in clinical diagnosis and human genetics research. However, current filtering methods (Frequency, Hard-Filter, VQSR, GARFIELD, and VEF) were developed to be utilized on particular variant callers and have certain limitations. Especially, the number of eliminated true variants far exceeds the number of removed false variants using these methods. Here, we present an adaptive method for quality control on genetic variants from different analysis pipelines, and validate it on the variants generated from four popular variant callers (GATK HaplotypeCaller, Mutect2, Varscan2, and DeepVariant). FVC consistently exhibited the best performance. It removed far more false variants than the current state-of-the-art filtering methods and recalled ~51-99% true variants filtered out by the other methods. Once trained, FVC can be conveniently integrated into a user-specific variant calling pipeline.
 
Outline of the protocols followed in the study of maternal aggression behaviour
a In Experiment 1, repeated aggression, females were randomly assigned into two groups: dams (n = 8) and pup-sensitised females (n = 8) (not represented). On postpartum days (PPD) 4–6, both groups of females were exposed to a different intruder male each day. b In Experiment 2, effect of DREADD-inhibition of MeA in aggression, females had stereotaxic injections of DREADD-AAV in the MeA. Then, they were mated and randomly assigned to two groups. After parturition, for behavioural testing, we followed an intragroup design. Group 1 received i.p. injection of CNO in PPD4 and Veh in PPD5, and Group 2 received Veh injection in PPD4 and CNO in PPD5. Then, 30 min after i.p. injections, females were exposed to a different male intruder each day. In parallel, we run two control groups of females, without DREADD injection, and the same drug regime, to discard any effect of CNO treatment (Supplementary information). CNO clozapine N-oxide, Veh vehicle.
Lactating females displayed increasing levels of maternal aggression across testing days, whereas pup-sensitised females did not develop intruder-directed aggression
a Total duration of attacks averaged across testing days was significantly higher in dams than in pup-sensitised virgins. a’ In lactating females, repeated testing leads to an increase in the total duration of attacks. a” Attacks were negligible in pup-sensitised females across testing days. b The latency to attack was significantly shorter in lactating females than in pup-sensitised virgin females. b’ In lactating females, the latency to the first attack was significantly shorter on the second day of testing than on the first. b” Since attacks were negligible in pup-sensitised females, latency to attack did not change across testing. c, d Social investigation was significantly higher in pup-sensitised females than in lactating dams, and did not significantly change across testing days, except for a trend towards decreased anogenital investigation in lactating dams (c’). Data are represented as mean ± SEM. **p ≤ 0.01, *p < 0.05, #p = 0.058.
Injection sites in the MeA showing the rostro-caudal extent of the DREADD infections
Photomicrographs showing the biggest (a, b) and the smallest (c, d) injection included in the analysis. Scale bar a, b: 500 µm and c, d: 100 µm. e, f Schematic reconstruction of coronal brain sections showing the extent of DREADD infections. Different coloured outlines represent the biggest (light pink) and the smallest (green) injection. The brain drawings are modified from ref. ⁶¹. ACo anterior cortical amygdaloid nucleus, BMA anterior basomedial amygdaloid nucleus, BSTIA bed nucleus of the stria terminalis intraamygdaloid division, CeM medial part of the central amygdaloid nucleus, ic internal capsule, MeAa anterior medial amygdaloid nucleus, MeApv posteroventral medial amygdaloid nucleus, opt optic tract (opt), MeApd posterodorsal medial amygdaloid nucleus, SI substantia innominate.
Inhibition of MeA did not block maternal aggression but prevented its increase during the second day of testing
In Group 1, injected with CNO in PPD4 and VEH in PPD5, the ANOVA showed significant differences between PPD4 and PPD5, with an increase in the total duration of aggression during the second session of testing (a) and a trend towards a decrease in latency to attack on the second day of testing (c). For Group 2, injected with Veh in PPD4 and CNO in PPD5, the ANOVA did not reveal significant differences between studied days in the total amount of time (b) or the latency to attack (d). Data are represented as mean ± SEM; *p ≤ 0.05, #p = 0.051. CNO clozapine N-oxide, Veh vehicle.
Representation of the social brain network (SBN) nuclei involved in aggression in dam mice related to MeA projections
Projections in red represent circuits with direct experimental evidence for the corresponding behaviour. AOB accessory olfactory bulb, BST bed nucleus of the stria terminalis, MeA medial amygdaloid nucleus, MOB main olfactory bulb, PAG periaqueductal grey, VMH ventromedial hypothalamic nucleus.
Virgin female laboratory mice readily express pup care when co-housed with dams and pups. However, pup-sensitized virgins fail to express intruder-directed aggression on a single session of testing. To study whether repeated testing would affect the onset and dynamics of maternal or intruder-directed aggression, we tested dams and their accompanying virgins from postpartum day 4 to 6. Repeated testing led to escalated aggression towards male intruders in dams, but virgins never developed aggression. In dams, inhibition of the medial amygdala using DREADD (designer receptors exclusively activated by designer drugs) vectors carrying the hM4Di receptor blocked the expected increase in maternal aggression on the second testing day. Our data support that the onset of maternal aggression is linked to physiological changes occurring during motherhood, and that medial amygdala, a key centre integrating vomeronasal, olfactory and hormonal information, enables the expression of escalated aggression induced by repeated testing. Future studies selectively targeting specific neuronal populations of the medial amygdala are needed to allow a deeper understanding of the control of experience-dependent aggression increase, a phenomenon leading to the high aggression levels found in violent behaviours.
 
Overview of MyxoEE-3 treatments and time-points examined here for latent evolution of fruiting body morphology
a MyxoEE-3 initiation and cycling scheme (adapted from ref. ¹⁰⁴). Ancestral subclones (collectively 'Anc') were isolated, grown in liquid, and used to start MyxoEE-3 populations (see Supp. Table 1). During each evolutionary cycle, populations (yellow circles) were allowed to swarm for 2 weeks at 32 °C and 90% rH before a small rectangle at the furthest point from the swarm center was cut and transferred upside down to the center of a new plate. Populations examined here underwent either 18 or 40 such cycles according to the specific treatment set (TS). b Overview of MyoxEE-3 treatments relevant to this study subdivided into two treatment sets (TS)—abiotic and biotic. TS-abiotic populations (yellow circles) evolved on either CTT hard agar (CTT-HA, dark orange plates) or CTT soft agar (CTT-SA, beige plates) for a total of 40 cycles. TS-biotic populations evolved on either CTT-HA only (same as CTT-HA in TS-abiotic) or on CTT-HA (dark orange plates) or TPM-HA (light blue plates) with lawns of either Bacillus subtilis (white area) or Escherichia coli (light yellow area). The TS-biotic populations were examined after 18 MyxoEE-3 cycles. c Scheme representing the distinctive experimental selective and inductive environments that characterize this study. Populations evolved in a range of different environments (selective environments) (panel b), where the abiotic (TS-abiotic) and biotic (TS-biotic) sources of selection acted on motility and/or predation during vegetative growth. Importantly, while exposed to selection during MyxoEE-3, transferred populations did not undergo fruiting body formation, such that development per se was hidden from the selection. Post-evolution development was subsequently induced on starvation plates (inductive environment) in all MyxoEE-3 terminal populations independently to study the latent evolution of fruiting body morphology.
Surface stiffness shapes developmental LPE
a Representative developmental phenotypes of the ancestor (Anc) and TS-abiotic evolved populations from CTT-HA (Population 3, P3; see Supp. Table 1) and CTT-SA (P39) (scale bar = 2 mm). b PCA based on four morphological traits showing the overall treatment-level phenotypic differentiation among the CTT-HA (dark blue) and CTT-SA (light blue) TS-abiotic treatments (MyxoEE-3 cycle 40) and their ancestor (black). Large circles represent average morphospace localization (centroids) obtained from three independent biological replicates (small circles, n = 3). Ellipses represent 95% confidence regions, while percentage values on the x and y axis report the variation explained by the principal components PC1 and PC2, respectively (Supp. Fig. 1A). c Mean values ± SEM of each analyzed morphological trait relative to the ancestral levels (Anc, black-horizontal line in the graphs) (n = 3). d Mean values ± SEM of log10-transformed spore counts (n = 4). In both c and d, red and black circles indicate significant (p < 0.05) and non-significant (NS) differences from Anc levels, respectively. (Significance was estimated for both c and d with one-way ANOVA followed by two-tailed Tukey tests. p values of all comparisons of evolved treatments with Anc, as well as all pairwise comparisons between evolved treatments, are reported in Data Fig. 2).
Deterministic and stochastic evolution of latent reaction norms
Reaction norms of four morphological traits across three agar concentrations (0.5, 1.0, and 1.5%, x-axis in both panels). a Reaction norms across ancestral clones (black circles and lines) and treatment-level average trait values (CTT-HA and CTT-SA evolved populations, dark and light blue circles and lines, respectively). n = 3 independent replicates (gray circles). b Reaction norms for each individual Anc sub-clone (c1–c6) and each TS-abiotic evolved population across the three biological replicates (n = 3). Labels at the end of each reaction norm indicate the identity of the ancestral clone or evolved population. In both a and b, large circles and error bars indicate the mean ± SEM, respectively. (Significance of variables’ contribution and their interaction were calculated for both a and b with two-way ANOVA. Employed models and results of all comparisons are reported in Data Fig. 3).
Prey presence and identity shape developmental LPE
a Developmental phenotypes of representative TS-biotic evolved populations from each selective environment and Anc (CTT-HA = P1; B. subtilis-CTT = P99; E. coli-CTT = P93; B. subtilis-TPM = P133; E. coli-TPM = P127). b PCA of overall morphological divergence across all five TS-B evolutionary treatments (MyxoEE-3 cycle 18) and the Anc subclones. Large circles represent average morphospace localization (centroids) obtained from three independent biological replicates (small circles, n = 3), while ellipses represent a 95% confidence region. Percentage values on the x and y axis report the variation explained by the two principal components, PC1 and PC2, respectively (Supp. Fig. 5B). c Mean values ± SEM of individual developmental traits relative to the ancestral (Anc) levels (black-horizontal line in each graph) (n = 3). d Mean values ± SEM of log10-transformed spore counts obtained after five days of starvation (n = 3). In both c and d, red and black circles indicate significant (p < 0.05) and non-significant (NS) differences from Anc levels, respectively. (Significance was calculated for both c and d with one-way ANOVA followed by two-tailed Tukey tests. p values of all comparisons of evolved treatments with Anc, as well as all pairwise comparisons between evolved treatments, are reported in Data Fig. 4).
Deterministic limitation of stochastic latent-phenotype diversification by MyxoEE-3 selective environments
a, c Morphological diversity among replicate TS-abiotic (a) and TS-biotic (c) populations evolved in the same selective environment compared to diversity among Anc subclones. b, d Variance of single morphological traits distributions calculated across TS-abiotic (b) and TS-biotic (d) evolved populations and across the Anc subclones. In all cases, large circles represent mean values ± SEM of three independent biological replicates (gray circles, n = 3), while red and black circles indicate significant (p < 0.05) and non-significant (NS) differences from Anc levels, respectively. (Significance was calculated in all cases with one-way ANOVA followed by two-tailed Tukey tests. p values of all comparisons of evolved treatments with Anc, as well as all pairwise comparisons between evolved treatments, are reported in Data Fig. 5).
Ecological causes of developmental evolution, for example from predation, remain much investigated, but the potential importance of latent phenotypes in eco-evo-devo has received little attention. Using the predatory bacterium Myxococcus xanthus, which undergoes aggregative fruiting body development upon starvation, we tested whether adaptation to distinct growth environments that do not induce development latently alters developmental phenotypes under starvation conditions that do induce development. In an evolution experiment named MyxoEE-3, growing M. xanthus populations swarmed across agar surfaces while adapting to conditions varying at factors such as surface stiffness or prey identity. Such ecological variation during growth was found to greatly impact the latent evolution of development, including fruiting body morphology, the degree of morphological trait correlation, reaction norms, degrees of developmental plasticity and stochastic diversification. For example, some prey environments promoted retention of developmental proficiency whereas others led to its systematic loss. Our results have implications for understanding evolutionary interactions among predation, development and motility in myxobacterial life cycles, and, more broadly, how ecology can profoundly shape the evolution of developmental systems latently rather than by direct selection on developmental features. An evolution experiment with the predatory bacterium Myxococcus xanthus reveals the potential for ecological factors to impact the evolution of multicellular morphology latently, rather than via direct selection.
 
The smallest phytoplankton species are key actors in oceans biogeochemical cycling and their abundance and distribution are affected with global environmental changes. Among them, algae of the Pelagophyceae class encompass coastal species causative of harmful algal blooms while others are cosmopolitan and abundant. The lack of genomic reference in this lineage is a main limitation to study its ecological importance. Here, we analysed Pelagomonas calceolata relative abundance, ecological niche and potential for the adaptation in all oceans using a complete chromosome-scale assembled genome sequence. Our results show that P. calceolata is one of the most abundant eukaryotic species in the oceans with a relative abundance favoured by high temperature, low-light and iron-poor conditions. Climate change projections based on its relative abundance suggest an extension of the P. calceolata habitat toward the poles at the end of this century. Finally, we observed a specific gene repertoire and expression level variations potentially explaining its ecological success in low-iron and low-nitrate environments. Collectively, these findings reveal the ecological importance of P. calceolata and lay the foundation for a global scale analysis of the adaptation and acclimation strategies of this small phytoplankton in a changing environment. Genomic inference reveals potential climate change-driven range expansion of the phytoplankton species Pelagomonas calceolata.
 
Geographic pattern of bank vole haemoglobin in Britain
Records of HbS are indicated by blue filling of the circles, those of HbF by red. The three sites where both HbS and HbF were detected are filled in half blue and half red. The background heat map shows the variation in mean summer temperature represented by the WorldClim variable BIO10⁵² (see text for details). The figure was created using ArcMap (v.10.8) and the Esri world countries dataset (www.esri.com).
Predicted climate suitability for HbS and HbF
Panels a-c represent HbS, panels d-f represent HbF. a, d Present climate. b, c, e, f Optimistic (b, e) and pessimistic (c, f) future climate warming scenarios for 2070 (see text for details). The figure was created using ArcMap (v.10.8) and the Esri world countries dataset (www.esri.com).
Relative climate suitability for HbS and HbF
Blue colour indicates areas where climate is predicted to favour HbS over HbF, red colour indicates areas where climate is predicted to favour HbF over HbS. a Present climate. b, c Optimistic (b) and pessimistic (c) future climate scenarios. The figure was created using ArcMap (v.10.8) and the Esri world countries dataset (www.esri.com).
The most likely pathway for many species to survive future climate change is by pre-existing trait variation providing a fitness advantage under the new climate. Here we evaluate the potential role of haemoglobin (Hb) variation in bank voles under future climate change. We model gene-climate relationships for two functionally distinct Hb types, HbS and HbF, which have a north-south distribution in Britain presenting an unusually tractable system linking genetic variation in physiology to geographical and temporal variation in climate. Projections to future climatic conditions suggest a change in relative climatic suitability that would result in HbS being displaced by HbF in northern Britain. This would facilitate local adaptation to future climate—without Hb displacement, populations in northern Britain would likely be suboptimally adapted because their Hb would not match local climatic conditions. Our study shows how pre-existing physiological differences can influence the adaptive capacity of species to climate change.
 
Leydig cells in fetal testes play crucial roles in masculinizing fetuses through androgen production. Gene knockout studies have revealed that growth factors are implicated in fetal Leydig cell (FLC) differentiation, but little is known about the mechanisms regulating this process. We investigate this issue by characterizing FLC progenitor cells using single-cell RNA sequencing. The sequence datasets suggest that thymosin β10 (Tmsb10) is transiently upregulated in the progenitors. While studying the function of Tmsb10, we reveal that platelet-derived growth factor (PDGF) regulates ciliogenesis through the RAS/ERK and PI3K/AKT pathways, and thereby promotes desert hedgehog (DHH)-dependent FLC differentiation. Tmsb10 expressed in the progenitor cells induces their differentiation into FLCs by suppressing the RAS/ERK pathway. Through characterizing the transiently expressed Tmsb10 in the FLC progenitors, this study unveils the molecular process of FLC differentiation and shows that it is cooperatively induced by DHH and PDGF.
 
Tree lycopsids prospered in the Late Devonian and constituted a major part of the Late Paleozoic forest ecosystem that deeply impacted the Earth’s climate. However, the fertile organs of these early tree lycopsids display low morphological disparity, which has hampered further knowledge about their ecological habit. Here, we report Omprelostrobus gigas gen. et sp. nov. from the Upper Devonian (Famennian) Wutong Formation at Changxing, Zhejiang, China. The collection includes aerial axes, strobili and associated roots. The strobili are the largest among coeval lycopsids to our knowledge, and are divided into proximal and distal portions by dimorphic sporophylls with differentiated laminae and probable strong photosynthetic capacity. The associated but not attached roots displaying multiple isotomous branches lack rootlets and typical rootlet scars. The varied strobili sizes of early tree lycopsids were relatively independent of their body plan, but the large strobili could suggest increased reproductive investment to overcome the disadvantages of the disturbed flooded habitat.
 
CF-BD localization and its effect on ovary development
a, b CF-BD localization in the rectum and ovary of newly emerged female and mature female. The red signals indicate CF-BD. Scale bars are 500 μm. c, d CF-BD localization in the rectum and ovary of mature female with histological sections and staining. The red signals indicate CF-BD. Scale bars are 100 μm. e CF-BD identification in ovaries of mature females with nested PCR. PCR amplification product size of CF-BD is 371 bp. Lane 1–19: ovary samples, lane 20: negative control. f, g Box-and-whisker plots show ovary width and length of female injected with CF-BD and control. h, i Box-and-whisker plots show ovary width and length of female injected with streptomycin and control. In box-and-whisker plots, where the boxes encompass the first to the third quartiles, inside the box the horizontal line shows the median, and the whiskers are the maximum and minimum observation. Different letters above the error bars indicate significant differences at the 0.05 level analyzed by ANOVA followed by Tukey’s test. Detail information for sample sizes and statistics can be found in Supplementary Data 2.
Oviposition attraction of CF-BD to B. dorsalis
a Device used to test oviposition preference. b Box-and-whisker plots show oviposition attraction of mango and guava purees mixed with CF-BD at different times. Data was analyzed by Wilcoxon matched-pairs signed rank test. “NS” no significance, **P < 0.01, ***P < 0.001. c, d Volatile identification in mango and guava purees added with CF-BD. The black arrows indicate 3-HA. e Box-and-whisker plots show 3-HA content comparison between CF-BD added guava puree and control. Data was analyzed by paired sample student t-test. **P < 0.01. f Box-and-whisker plots show egg proportions comparison between purees and purees supplemented with 3-HA. Different letters above the error bars indicate significant differences at the 0.05 level with the Kendall nonparametric test. In box-and-whisker plots, where the boxes encompass the first to the third quartiles, inside the box the horizontal line shows the median, and the whiskers are the maximum and minimum observation. Detail information for sample sizes and statistics can be found in Supplementary Data 2.
Olfactory trap effect of CF-BD and 3-HA
a A transparent cage used to capture the female flies. b Box-and-whisker plots show olfactory attraction of mango and guava purees added with CF-BD at different times. Data was analyzed by Wilcoxon matched-pairs signed rank test. “NS” no significant difference. c Box-and-whisker plots show female number comparison between purees and purees supplemented with 3-HA. The same letters above the error bars indicate no significant differences at the 0.05 level with the Kendall nonparametric test. d Attraction effect of purees added with CF-BD to females. Fly numbers are shown in the bars. Data was analyzed by chi-square test. “NS” no significance. e, f Attraction effect of mango and guava purees added with 3-HA to females. Fly numbers are shown in the bars. Data was analyzed by chi-square test. “NS” no significant difference. In box-and-whisker plots, where the boxes encompass the first to the third quartiles, inside the box the horizontal line shows the median, and the whiskers are the maximum and minimum observation. Detail information for sample sizes and statistics can be found in Supplementary Data 2.
Function of ovipositors as receptors for 3-HA and olfactory gene identification
a Sensilla scanning on the ovipositor. CA: coeloconic sensilla. Scale bars are 5 μm. b Box-and-whisker plots show EAG response of the ovipositors to 3-HA. Different letters above the error bars indicate significant differences at the 0.05 level by ANOVA followed by Tukey’s test. c Expression patterns of the identified olfactory genes in ovipositors at different developmental times. d–h Box-and-whisker plots show relative expression of olfactory genes in antennae and ovipositors. An: antennae. Different letters above the error bars indicate significant differences at the 0.05 level by ANOVA followed by Tukey’s test. Data in bar plots show mean values ± SEM. In box-and-whisker plots, where the boxes encompass the first to the third quartiles, inside the box the horizontal line shows the median, and the whiskers are the maximum and minimum observation. Detail information for sample sizes and statistics can be found in Supplementary Data 2.
Olfactory gene function verification
a Binding abilities of 1-NPN to Obp56d and Obp56d-2. b Competitive binding ability of 3-HA to 1-NPN. c Box-and-whisker plots show oviposition preference of females for 3-HA with Obp genes knocked down. Different letters above the error bars indicate significant differences at the 0.05 level by Kruskal–Wallis test followed by Dunn’s test. d Box-and-whisker plots show ovipositor EAG response to 3-HA (10 mg/ml) with both Obp56d and Obp56d-2 being knocked down. Different letters above the error bars indicate significant differences at the 0.05 level by ANOVA followed by Tukey’s test. Data in bar plots show mean values ± SEM. e Box-and-whisker plots show oviposition preference of females for 3-HA with OR7a-2 knocked down. The same letters above the error bars indicate no significant differences at the 0.05 level by ANOVA followed by Tukey’s test. f Box-and-whisker plots show oviposition preference of females for 3-HA with IR25a knockdown. The same letters above the error bars indicate no significant differences at the 0.05 level by ANOVA followed by Tukey’s test. In box-and-whisker plots, where the boxes encompass the first to the third quartiles, inside the box the horizontal line shows the median, and the whiskers are the maximum and minimum observation. Detail information for sample sizes and statistics can be found in Supplementary Data 2.
Gut bacteria play important roles in insect life cycle, and various routes can be used by insects to effectively transmit their gut bacteria. However, it is unclear if the gut bacteria can spread by actively attracting their insect hosts, and the recognition mechanisms of host insects are poorly understood. Here, we explore chemical interactions between Bactrocera dorsalis and its gut bacterium Citrobacter sp. (CF-BD). We found that CF-BD could affect the development of host ovaries and could be vertically transmitted via host oviposition. CF-BD could attract B. dorsalis to lay eggs by producing 3-hexenyl acetate (3-HA) in fruits that were hosts of B. dorsalis. Furthermore, we found that B. dorsalis could directly recognize CF-BD in fruits with their ovipositors in which olfactory genes were expressed to bind 3-HA. This work reports an important mechanism concerning the active spread of gut bacteria in their host insects.
 
Crucial conventional patch-clamp approaches to investigate cellular electrophysiology suffer from low-throughput and require considerable experimenter expertise. Automated patch-clamp (APC) approaches are more experimenter independent and offer high-throughput, but by design are predominantly limited to assays containing small, homogenous cells. In order to enable high-throughput APC assays on larger cells such as native cardiomyocytes isolated from mammalian hearts, we employed a fixed-well APC plate format. A broad range of detailed electrophysiological parameters including action potential, L-type calcium current and basal inward rectifier current were reliably acquired from isolated swine atrial and ventricular cardiomyocytes using APC. Effective pharmacological modulation also indicated that this technique is applicable for drug screening using native cardiomyocyte material. Furthermore, sequential acquisition of multiple parameters from a single cell was successful in a high throughput format, substantially increasing data richness and quantity per experimental run. When appropriately expanded, these protocols will provide a foundation for effective mechanistic and phenotyping studies of human cardiac electrophysiology. Utilizing scarce biopsy samples, regular high throughput characterization of primary cardiomyocytes using APC will facilitate drug development initiatives and personalized treatment strategies for a multitude of cardiac diseases.
 
Ple. Miss Joaquim ‘Agnes’
a The flower of Ple. Miss Joaquim ‘Agnes’ in front view with a dorsal sepal (ds), two lateral petals (lp), labellum (la), a modified petal, two lateral sepals (ls) and the gynostemium (gy), which is enlarged in the white-bordered inset image. b Terete leaves of Ple. hookeriana (PH), Ple. Teres (PT) and the hybrid Ple. Miss Joaquim ‘Agnes’ (PMJ), from left to right.