EMBO/soda-nel huggingface dataset:
Gut neuroendocrine signaling regulates synaptic assembly in C. elegans (2022)
Figure 2

Fig. 2 AIY presynaptic formation requires intestinal expressed nlp-40.

A Quantification of the fluorescence intensity of AIY GFP::RAB-3 in wild-type , egl-3(n589) and aex-5(sa23) mutants.

B A Schematic diagram describes the RNAi screen strategy to identify intestinal neuropeptides required for AIY synaptogenesis.

C Quantification of the fluorescence intensity of AIY GFP::RAB-3 for the indicated RNAi treatment. Candidates are intestinal expressed nlp or flp.

D Diagram of nlp-40 genomic structure. P1 to P4 indicates the predicted peptides encoded by nlp-40. The boxes and lines represent exons and introns. Black and gray indicate coding sequence and UTRs. The lines beneath indicate the deletion region.

E-I Confocal images of AIY labeled with GFP::RAB-3 in wild-type (E), nlp-40(tm4085) (F), nlp-40(vj3) (G), nlp-40(tm4085) (H) or nlp-40(vj3) (I) with a wild type nlp-40 transgene.

J Quantification of the fluorescence intensity of AIY GFP::RAB-3 for the indicated genotypes. Transgenic data are averaged from at least two independent lines.

K-L. Confocal images of AIY synaptic marker GFP::RAB-3 for the indicated intestinal-specific RNAi treatment.

M Quantification for (K) and (L).

Data information: The scale bar in (E) and (K) are 10μm and applies to (F-I) and (L), respectively. Asterisks indicate AIY soma. The AIY synaptic region was quantified for the synaptic intensity analysis. For (A, C, J and M), each dot represents one animal. Data were collected from at least three independent experiments. Statistics are based on one-way ANOVA followed by Dunnett's test (C and J) or two-tailed Student's t-test (A and M). **p<0.01, ****p<0.0001. Error bars represent SEM.
Gut neuroendocrine signaling regulates synaptic assembly in C. elegans (2022)
Figure 3

Fig. 3 Wnt signaling regulates AIY presynaptic formation by promoting nlp-40 expression.

A-C Quantification of the fluorescence intensity of AIY presynaptic marker GFP::RAB-3 (A, B) and nlp-40 mRNA level (C) for the indicated genotypes. The AIY synaptic region was quantified for the synaptic intensity analysis.

D-I Confocal images of the transcriptional nlp-40 reporter (Pnlp-40::GFP) in wild-type (D), cwn-2(ok895) (E), cfz-2(ok1201) (F), pop-1(hu9) (G), and control or sys-1 RNAi (H and I) animals at adult Day 1 stage.

J Quantification of the Pnlp-40::GFP fluorescence intensity for the indicated genotypes.

K-P Confocal images of an intestinal-specific reporter ERM-1::GFP in wild type (K), cwn-2(ok895) (L), cfz-2(ok1201) (M), pop-1(hu9) (N), and control or sys-1 RNAi (O and P) animals at the adult Day1 stage.

Q Quantification of the ERM-1::GFP fluorescence intensity for the indicated genotype.

Data information: The scale bar in (D) is 50μm and applies to (E-I, K-P). For (A, B, C, J and Q) ,each dot represents one animal. Data were collected from at least three independent experiments. Statistical analyses are based on one-way ANOVA followed by Dunnett's test (for mutants) and two-tailed student's t-test (for RNAi). **p<0.01, ***p<0.001, ****p<0.0001, ns: not significant as compared to WT or control RNAi (p>0.05). Error bars represent SEM.
Gut neuroendocrine signaling regulates synaptic assembly in C. elegans (2022)
Figure 4

Fig. 4 Neuropeptide/NLP-40 receptor GPCR/AEX-2 is required for AIY presynaptic formation.

A Diagram of aex-2 genomic structure. The boxes and lines represent exons and introns. Black and gray indicate coding sequence and UTRs. The white asterisk marks the location of the R232Q mutation site of sa3 allele.

B-K Confocal images of AIY presynaptic site labeled with GFP::RAB-3 in wild type(B), nlp-40(tm4085)(C), aex-2(sa3)(D), nlp-40(tm4085); aex-2(sa3)

(E) over-expression NLP-40 in aex-2(sa3) mutants(F) and aex-2(sa3) with a wild-type aex-2 transgene driven by the endogenous promoter (Paex-2) (G), neuron-specific promoter (Prab-3) (H) , AIY-specific promoter (Pttx-3) (I) intestinal-specific promoter (Pges-1) (J) or muscle-specific promoter (Pmyo-3)(K).

L-M Quantification of the fluorescence intensity of AIY GFP::RAB-3 for the indicated genotypes. Each dot represents one animal. Data were collected from at least three independent experiments. Transgenic data are averaged from at least two independent lines. tg: Paex-2::aex-2, Pttx-3::aex-2 or Pges-1::aex-2 transgene, +: wild type ; -: mutant or without tg. The AIY synaptic region was quantified for the synaptic intensity analysis.

N-Q' Confocal images of Paex-2::mNeonGreen of whole animal with bright field (N), the head region labeled with Paex-2::mNeonGreen and Pttx-3::mCherry with green channel (O), red channel (P) and the merged channels (Q). (O'-Q') are the cross sections corresponding to the dashed line sites.

Data information: The scale bar in (B) and (O) are 10μm, and applies to (C-K) and (P-Q') respectively; the scale bar in (N) is 50μm. Asterisks indicate AIY soma. Statistical analyses are based on one-way ANOVA followed by Dunnett's test. ns: not significance (p>0.05), ****p<0.0001. Error bars represent SEM.
Gut neuroendocrine signaling regulates synaptic assembly in C. elegans (2022)
Figure 6

Fig. 6 Synaptic formation in the nerve ring requires Wnt-NLP-40 neuropeptide signaling.

A A cartoon diagram of the C. elegans head. Green marks the presynaptic region in the nerve ring.

B-G Confocal images of Prab-3::GFP::RAB-3 in the nerve region corresponding to the dashed box in (A) of wild type (B), cwn-2(ok895) (C), cfz-2(ok1201) (D), pop-1(hu9) (E), nlp-40(tm4085) (F) and aex-2(sa3) (G) mutants.

H-I Quantification of the GFP::RAB-3 intensity in the nerve ring synaptic region for the indicated genotypes. Each dot represents one animal. Data were collected from at least three independent experiments. Transgenic data are averaged from at least two independent lines.

Data information: The scale bar in (B) is 10μm and applies to (C-G). Statistical analyses are based on one-way ANOVA followed by Dunnett's test (H and I) and two-tailed Student's t-test (cwn-2 vs cwn-2;tg, nlp-40 vs nlp-40;tg, aex-2 vs aex-2;tg). ****p<0.0001. Error bars represent SEM.
The 18S ribosomal RNA m6A methyltransferase Mettl5 is required for normal walking behavior in Drosophila (2020)
Figure 1

Figure 1. Mettl5 regulates m6A level in total RNA.

(A) List of human methyltransferases and their orthologs in Drosophila melanogaster. Empty cells indicate that no ortholog could be identified (see Methods for details).

(B, C) LC-MS/MS measurements of m6A levels in total RNA (B) or in poly(A)+ RNA (C) upon KD of predicted methyltransferases in Drosophila S2R+ cells. m6A abundance in total RNA is significantly reduced when Mettl5 is depleted, while its depletion has no effect on m6A level in mRNA. As expected, the KD of Mettl3 and Mettl14 reduce m6A levels in mRNA. Bar chart represents the mean ± standard deviation of three technical measurements from three biological replicates. *P < 0.05, **P < 0.01, ***P < 0.001 (two-tailed t-test).
The 18S ribosomal RNA m6A methyltransferase Mettl5 is required for normal walking behavior in Drosophila (2020)
Figure 2

Figure 2. Phylogenetic and expression analyses of Mettl5.

(A) Phylogenetic tree of the alignment of representative Mettl5 orthologs from selected species (see Materials and Methods for details). Prokaryotic sequences from archaea (Pyrococcus, Bathyarchaeota) and bacteria (Acidobacteriales) are included as outliers.

(B) Multiple sequence alignment of Mettl5 orthologs showing conservation of the NPPF motif.

(C) Subcellular localization of Mettl5-FLAG (purple) and Trmt112-eGFP (green) expressed under the control of their own promoter in S2R+ cells. 63X magnified merge of immunofluorescently-labelled S2R+ cells. Scale bar: 4.47 μm.

(D) Developmental expression of Mettl5 transcript assayed by RT-qPCR analysis. The figure shows mean ± standard deviation of three technical measurements from three biological replicates.

(E) In situ hybridization of Mettl5 transcript at different embryonic stages. The central nervous system (CNS) is highlighted in the schematics. Data retrieved from FlyExpress 7. (http://www.flyexpress.net/search.php?type=image&search=FBgn0036856).
The 18S ribosomal RNA m6A methyltransferase Mettl5 is required for normal walking behavior in Drosophila (2020)
Figure 3

Figure 3. Drosophila Mettl5 is required for m6A deposition on 18S rRNA.

(A) Representation of the two Mettl5 mutant alleles generated in this work, consisting either of a two amino acid deletion upstream of the methyltransferase domain (top, Mettl5∆2AA), or of a frameshift mutation leading to a premature stop codon (bottom, Mettl5fs).

(B) LC-MS/MS measurements of m6A levels in total RNA of WT and Mettl5 mutant flies. Bars represent mean ± standard deviation of measurements of three biological replicates. ***P<0.001 (two-tailed t-test).

(C) Purified 18S rRNA analyzed for its m6A content by quantitative HPLC. The 18S rRNA was extracted from 40S subunits isolated on sucrose gradients. The calibration control is a commercial source of m6A (in grey). m6A elutes at 48 min.

(D) Pre-rRNA processing in Drosophila: four mature rRNAs (the small ribosomal subunit 18S, and the large ribosomal subunit 5.8S, 2S, and 28Sa and 28Sb) are produced by sequential RNA cleavage following two alternative pathways, as depicted. Processing sites are indicated (1 to 6). The major pre-rRNA intermediates (a, b, c, and d) are highlighted.

(E) Mature rRNA analysis on ethidium-stained denaturing agarose gels. The same amounts of total RNA extracted from the indicated flies, and from S2R+ cells depleted or not of Mettl5, were loaded. The 28S/18S ratio was established by densitometry.

(F) Analysis of 18S rRNA maturation in WT and mutant flies (Mettl5fs). Total RNA extracted from the indicated animals was resolved on denaturing agarose gels and processed for northern blotting with specific probes (complementary to ITS1 or ITS2 sequences). The pre-rRNAs detected accumulate to normal levels indicating that processing is unaffected.
The 18S ribosomal RNA m6A methyltransferase Mettl5 is required for normal walking behavior in Drosophila (2020)
Figure 4

Figure 4. Mettl5 interacts with Trmt112 to install m6A on 18S rRNA

(A) Mettl5 partners identified as significantly-enriched candidates in mass spectrometric analysis of Mettl5-FLAG-Myc affinity purifications (ribosomal proteins not listed for simplicity). Measurements of 2 biological replicates.

(B) Western blot validation of the co-immunoprecipitated proteins Mettl5-FLAG-Myc and Trmt112, bearing either a N-terminal HA tag (top) or a C-terminal GFP tag (bottom), in S2R+ cells. The arrows point at the indicated proteins. HA-Trmt12 migrates below the antibody light chains (top). Trmt112-eGFP migrates above the heavy chains indicated by an asterisk (bottom).

(C) Trmt112 depletion reduces m6A levels in total RNA from Drosophila S2R+ cells. Mean ± standard deviation of three technical measurements from three biological replicates. *P < 0.05 (two-tailed t-test).

(D) The sequences of D. melanogaster Mettl5 and Trmt112 were modelled in the experimentally-determined atomic resolution structure of the human METTL5-TRM112 complex (based on PDB model 6H2V), revealing high conservation and formation of a parallel ß-zipper involving main chain atoms at the complex interface. Mettl5 and Trmt112 are coloured light brown and blue, respectively. The NPPF signature, known to coordinate planar nitrogen groups to be methylated, is shown as sticks. The S-adenosyl-methionine (SAM) is shown in stick representation (yellow) with the methyl group to be transferred to the 18S rRNA represented as a black sphere. Important secondary structure elements, including ß3 on Mettl5 and ß4 on Trmt112, are indicated.
The 18S ribosomal RNA m6A methyltransferase Mettl5 is required for normal walking behavior in Drosophila (2020)
Figure 5

Figure 5. Mettl5 is required for fly orientation

(A) Cartoon depicting the experiment with the main components of the setup showing the arena in white, the visual landmarks as black stripes and the camera to record the fly (left, from Colomb et al, 2012) and cartoon showing the normal behavior of a WT fly in the arena. The black rectangles represent the visual landmarks and the red arrow the main trajectory undertaken by WT flies relative to the position of the landmarks (right).

(B) Representative trajectories of WT and Mettl5 mutant flies analysed by the Buridan's Paradigm. The blue lines indicate when the fly stops and changes direction.

(C) Orientation evaluated with the help of the Buridan's paradigm for Mettl5 mutant flies. Orientation was measured as the angular deviation from the straight path needed to move from one landmark to another in the arena. Number of flies tested per genotype: 30. Mean ± standard deviation. Shapiro-Wilk test was used to test for normal distribution in each group. Normally distributed groups were tested by t-test. Due to multiple comparison Bonferroni correction was applied. (p<0.05 = *; p<0.0001=****).
Decreased blood vessel density and endothelial cell subset dynamics during ageing of the endocrine system (2020)
Figure 1

Figure 1. 3D atlas of vascular-related cells and molecules in young and aged endocrine glands

A Representative 3D tile scan images of young adrenal gland, ovary, pituitary gland, testis, pancreas and thyroid gland stained with the antibodies indicated in the panel. Scale bars are 200 µm. The white and dashed lines represent the outlines of each organ.

B Representative 3D images at single-cell resolution level of adrenal gland, ovary, pituitary gland, testis, pancreas and thyroid gland. Insets (*) indicate higher magnification images of regions with single-cell resolution and surface rendering for all markers. Scale bars for tile scan 3D images are 200 µm, for single-cell resolution insets are 5 µm. The dashed lines represent the outlines of each organ.

C Exemplar image illustrates the cell-cell interactome analysis with the distance estimates of CD68+ macrophages to blood vessels (Endoglin) in cortex, medulla and the transition zone (TZ) of an aged adrenal gland. The colour code indicates the distance which is very close in violet and very far in red. The scale bar is 50 µm. The white and dashed lines represent the outline of the transition zone.

D Illustration of the EC interactome in adrenal glands and changes in the interactome with ageing. Cells ≤18 μm of distance from the blood vessels were considered to be within the interactome of blood vessels. Analysis of endothelium in the different region showed that they have distinct marker expressions. CD102pos / VCAM-1lo / Vimentinneg, CD102neg / VCAM-1hi / Vimentinneg and CD102neg / VCAM-1hi / Vimentinpos ECs were located in cortex, transition and medulla regions respectively in young and aged adrenal glands. Their interactions in young and aged adrenal glands with PDGFRβ+ and NG2+ pericytes, PDGFRα+ mesenchymal cells, α-SMA+ pericytes, Desmin+ stromal cells, Decorin+ stromal cells, CXCR4+ stromal cells, FSP1+ fibroblasts, CD34+ hematopoietic cells, F4/80+ macrophages and CD68+ macrophages are shown. Interaction with Decorin expressing stromal cells is only observed in ageing.

E Schematic illustration of the study design with 3D spatial proteomic data and cell-cell interactome database strategy, analysis of age-dependent changes and validation of protein expression followed by functional analysis.

Data information: Nuclei: DAPI or TO-PRO-3 as indicated.
Decreased blood vessel density and endothelial cell subset dynamics during ageing of the endocrine system (2020)
Figure 2

Figure 2. Age-associated vascular modulations in mouse endocrine glands

A Young and aged adrenal gland immunolabelled with HSPG2, NG2 and Podocalyxin. Higher magnification insets show cortex (Co) and medulla (Me). Combined box and whiskers, and scatter plot with quantification of vessel density in young and aged adrenal glands.

B Young and aged ovary immunolabelled with Sca-1, PDGFRβ and Emcn. Higher magnification insets show cortex and medulla. Combo plot with quantification of vessel density in young and aged ovary.

C Young (10-weeks-old) and aged pituitary gland immunolabelled with ESM-1, Collagen IV and Emcn. Insets with higher magnifications of pars distalis and pars nervosa regions. Combo plot shows quantification of vessel density.

D Young and aged testis immunolabelled with Tie-2 and HSPG2. Higher magnifications of cortex and medulla. Combo plot shows quantification of vessel density.

E Young and aged pancreas immunolabelled with SM22α and Endoglin. Insets representative of higher magnifications of regions in young and aged pancreas. Combo plot shows quantification of vessel density.

F Young and aged thyroid gland immunolabelled with CXCR4, Laminin and Emcn. Combo plot shows quantification of vessel density in young and aged thyroid glands. Insets: periphery and centre of the thyroid gland.

Data information: The white and dashed lines in each panel represent the outlines of each organ. (n=5), P-values and two-tailed unpaired t-tests. ns: not significant; **: P < 0.01; ***: P < 0.001. Nuclei: TO-PRO-3. For the Combo plots, the boxes represent mean ± s.d., line in the box is the median, and the lower and upper lines show the minimum and the maximum of the values. The line on the right side of these combined plots represents the sample distribution. Co: Cortex; Me: Medulla; Pe: Periphery; Ct: Center; Pd: Pars Distalis; Pn: Pars Nervosa. Scale bars for tile scan 3D images 200 µm, for insets 50 µm.
Decreased blood vessel density and endothelial cell subset dynamics during ageing of the endocrine system (2020)
Figure 3

Figure 3. Light-sheet imaging of whole cleared endocrine glands from young and aged mice

A Whole-endocrine gland imaging of young and aged adrenal glands immunolabelled with α-SMA and Emcn acquired on a light-sheet microscope. Asterisks indicate higher magnifications of regions in adrenal glands from young and aged mice. Combined box and whiskers, and scatter plots with quantifications of vessel density and numbers of arteries.

B Whole-endocrine gland light-sheet imaging-based 3D images of young and aged ovaries stained with α-SMA and Emcn. Asterisks represent higher magnifications of regions in ovaries from young and aged mice. Combo plots show quantifications of vessel density and numbers of arteries in young and aged ovary.

C 3D images of whole cleared young and aged testes stained with α-SMA and Emcn. Asterisks indicate higher magnifications of indicated regions. Combo plots show quantifications of vessel density and numbers of arteries.

D Light-sheet imaging of whole cleared young and aged pancreases immunolabelled with α-SMA and Emcn. Asterisks indicate higher magnifications of regions. Combo plots with quantifications of vessel density and numbers of arteries in young and aged pancreas.

E 3D images acquired on a light sheet microscope of whole cleared young and aged thyroid glands stained with α-SMA and Emcn. Asterisks indicate higher magnifications of regions. Combo plots show quantifications of vessel density and numbers of arteries in young and aged thyroid glands.

F Heatmap of 3D spatial proteomic data showing qualitative analysis of differences in cell type markers and matrix expressions between young and aged murine endocrine glands. The colour code based on immunolabelling intensities indicates very high expression in red, high expression in yellow, medium in light green, low in dark green and no expression in grey.

Data information: The white and dashed lines in panel A-E represent the outlines of each organ. (n=4), P-values, and two-tailed unpaired t-tests for all the above panels. ns: not significant; *: P < 0.05; **: P < 0.01. For the Combo plots, the boxes represent mean ± s.d., line in the box is the median, and the lower and upper lines show the minimum and the maximum of the values. The line on the right side of the combo plots represents the sample distribution. Scale bars are 200 µm for whole-organ 3D images and 50 µm for higher magnification insets.
Decreased blood vessel density and endothelial cell subset dynamics during ageing of the endocrine system (2020)
Figure 4

Figure 4. Vascular and perivascular alterations in the ageing of human endocrine glands

A 3D images (left) with α-SMA and CD31 immunostaining in young and aged human adrenal gland. Middle panel images surface rendered for α-SMA, Vimentin and CD31. Arrowheads indicate arteries. Combo plots with quantifications of the number of arteries and vessel density.

B 3D images of young and aged human ovary immunolabelled with α-SMA, Vimentin and CD31. Images in the middle panel with a surface rendered for all the channels. Arrowheads indicate arteries. Combo plots with quantifications of the number of arteries and vessel density.

C Representative 3D images (left) of young and aged human testis with SM22α and Endoglin immunostaining. Middle panel images surface rendered for all the channels. Arrowheads indicate arteries. Combo plots show quantifications of the number of arteries and vessel density.

D 3D images from young and aged human pancreas staining with α-SMA, Vimentin and Endoglin. 3D images (middle) with a surface rendering for all the channels. Arrowheads indicate arteries. Combined box and whiskers, and scatter plots with the quantifications of the number of arteries and vessel density.

E Representative 3D images (left) show α-SMA and CD31 immunostaining in young and aged human thyroid gland. Middle panel images surface rendered for α-SMA, Vimentin and CD31. Arrowheads indicate arteries. Combo plots with quantifications of the number of arteries and vessel density.

F qPCR analysis of Efnb2, Cdh5 and Kdr expression (normalized to Actb) in aged human endocrine glands compared to young glands.

Data information: (n=4), P-values, and two-tailed unpaired t-tests for all the above panels. ns: not significant; *: P < 0.05; **: P < 0.01; ****: P < 0.0001. Human samples from donors at ages below 20 years and over 70 years were chosen for young and aged group sets, respectively. Nuclei: DAPI. For the Combo plots, the boxes represent mean ± s.d., line in the box is the median, and the lower and upper lines show the minimum and the maximum of the values. The line on the right side of the combo plots represents the sample distribution. Scale bars are 80 µm for 3D images and 10 µm for higher magnification single-cell resolution 3D images.
Host, Viral, and Environmental Transcriptome Profiles of the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) (2020)
Figure 6:

Host transcriptome responses and risk to SARS-CoV-2.(top row) Samples were quantified by a range of viral detection methods, including LAMP (Quantifluor), RNA-seq (log10 SARS-CoV-2 % of reads), and qRT-PCR (Ct values) to create a three-tier range of viral load for the positive samples (right) compared to the clinically-annotated negative samples (class, red or grey). (bottom) The differentially expressed genes of COVID+ patients compared to COVID-patients showed up-regulated (orange) genes as well as down-regulated (purple) genes. (b) Up-regulated genes, with boxplots across all samples, include IFI6, ACE2, SHFL, HERC6, IFI27, and IFIT1, based on data from (c), which is the total set of DEGs. The full set is shown in an intersecting heat map, with a core set of up-regulated genes (orange) distinct form the set of down-regulated genes (purple), compared to genes that are not significantly differently expressed (grey) in any comparison (DESeq2, q-value <0.01, |logFC| >0.58). (d) Significantly different gene ontology (GO) pathways between COVID-POS and COVID-NEG patients include interferon response and host response to infection, as well as some ion and heme transport mechanisms. (e)Survival curves for requiring mechanical respiration (identified by intubation procedure notes) (e) and mortality (f). Patients with a history of ACE inhibitor exposure were more likely to require intubation (HR=2.63 95%CI 2.01-3.43, p=1.22E-12; (e) and less likely to survive (HR=1.68 95%CI: 1.22-2.31, p=1.42E-03; (f). Because several individuals were intubated shortly before they first tested positive for SARS-CoV-2 infection, each first positive test was set back by seven days to account for testing delays.