Journal: Nature chemical biology

Article Title: Diabetes reversal by inhibition of the low molecular weight tyrosine phosphatase

doi: 10.1038/nchembio.2344

Figure Lengend Snippet: (a) Surface representation of human LMPTP-A showing phosphate (P) non-covalently bound in the active-site. Residues are colored according to magnitude of shift in the HSQC 15 N- 1 H spectrum upon Compd. 18 titration (red>orange>green). Gray residues had negligible shifts or could not be assigned. (b) Crystal structure of bovine LMPTP W49Y/N50E bound to orthovanadate and Compd. 18 (cyan and blue sticks; Q=quinoline; Pip=piperidine; BN=benzonitrile; L=linker), with selected side-chains (yellow=carbon; red=oxygen; blue=nitrogen; pink=vanadium) and H-bonds/ionic interactions (dashed green/gray lines) shown. (c) Inhibition of phosphatase activity of LMPTP-A/mutants by Compd. 18 using 0.4 mM OMFP substrate. Mean±SD % activity is shown. Data is representative of 3 independent experiments. (d) Compd. 18 modeled into the crystal structure of phosphate-bound human LMPTP, based on an overlay with the bovine ternary complex crystal structure (RMSD=0.33 Å). Selected residues are colored by NMR shift as in (a) . Dashed red line depicts predicted clash between apical oxygen (“A”) of phosphate and Q. (e–f) Structural rationale for SAR data, with atoms at 66% of their true radii. (e) “Side” view of pocket, rotated ~90° about a horizontal axis. The molecular surface has been sliced through the active-site to reveal the tight fit of Q in the pocket. Atoms with a formal charge (±) are labeled. BN is highly polarized, as indicated (δ±); arrows labeled “S” indicate solvent exposure of ring substitutions. (f) “Top” view looking down at the active-site pocket filled by Q. Arrow above atom N1 locates the “saddle-point” at pocket exit.

Article Snippet: cDNAs encoding mouse and human LMPTP-A (protein reference sequences NP_067305.2 and NP_004291.1) and bovine LMPTP (protein reference sequence NP_776403.1) were codon-optimized for E. coli , synthesized, and cloned into the pGEX-4T vector using BamHI/EcoRI by Genscript.

Techniques: Titration, Inhibition, Activity Assay, Labeling, Solvent

Journal: Cell Reports Methods

Article Title: Approaches for identification of 5′ UTR mutations impacting translation and protein production from neurodevelopmental disorder genes

doi: 10.1016/j.crmeth.2025.101247

Figure Lengend Snippet: MPRA captures 5′ UTR variant effects at key levels of translation (A) Boxplots overlaid on violin plots comparing the distribution of PhastCons(left) and PhyloP(right) scores of 5′ UTRs of non-autism genes ( n = 13,511) and autism-linked (exome analysis, n = 83 and SFARI score 1–3, n = 37, 178, and 36) (Kruskal-Wallis ANOVA with pairwise Wilcoxon test ∗ p < 0.05, ∗∗ p < 0.01,∗∗∗∗ p < 0.0001). (B) Histogram showing distribution of 5′ UTR lengths for highest brain expression transcripts. Dashed line: 220 nt. (C) Plasmid illustration with locations of inserted 5′ UTR elements, spliced tdTomato reporter, barcode, and RT primer site (flanked by LoxP sites; FLEX). (D) Experimental schematic: for each replicate, total RNA and DNA were collected. A portion lysate was fractionated into 40S-, 80S-, and polysome-associated RNA and sequenced. (E) Histogram of 5′ UTR lengths for transcripts in MPRA. Dashed line: 220 nt. 38% are <220 nt. (F) PCA of MPRA count data showing replicates cluster by fraction type. (G) Correlograms of the three measures showing significant (∗∗∗ p < 0.001) positive or negative Pearson correlation coefficients (PCCs). Red lines on scatterplot indicate the smoothed conditional mean fit by a general additive model. (H) Per fraction Pearson correlation between 5′ UTR isoforms represented in our library in both full-length forms (short isoform, <220 nt) and in a truncated form (long isoform, >220 nt). Red line indicates linear fit. MPRA results show that full-length 5′ UTRs correlate reasonably with their truncated counterparts. (I) 5′ UTR sequences (from both Ref and Alt) display higher transcript abundance compared to randomly shuffled sequences (Shuf), yet no difference in 80S/40S enrichment or polysome/80S enrichment (Mann-Whitney U test: ∗∗ p < 0.01, ∗∗∗ p < 0.001).

Article Snippet: Full-length sequence for UTRs was obtained and reference and variant entire UTR sequence was ordered from Twist Biosciences with BamHI and and BglII/AgeI restriction sites for five case variants whose shorter constructs increased (three, one each in SMC5, ZFYVE9, and ZNF311), decreased (NSD2), and had no significant change (NR4A2.2), in the dual-luciferase assay ( ).

Techniques: Variant Assay, Expressing, Plasmid Preparation, MANN-WHITNEY

Journal: Cell Reports Methods

Article Title: Approaches for identification of 5′ UTR mutations impacting translation and protein production from neurodevelopmental disorder genes

doi: 10.1016/j.crmeth.2025.101247

Figure Lengend Snippet: The impact of UTR elements on measures of translation in cellulo (A) uORFs decrease transcript abundance, increase the 80S/40S ratio, and increase polysome/80S enrichment, though with a potentially bimodal distribution indicating that impact of uORFs varies by sequence (Mann-Whitney U test: ∗ p < 0.05, ∗∗∗∗ p < 0.0001). (B) 5′ UTR GC content and predicted folding (minimum free energy, MFE) correlate with RNA abundance and polysome/80S enrichment. Red line indicates linear fit, and R represents Pearson’s correlation. (C–E) Synthetic controls demonstrate the sensitivity of different MPRA measurements to changes in secondary structure or uORFs (Mann-Whitney U tests). (F) Dual luciferase reporter assay of stem-loop- and uORF-containing 5′ UTR sequences results in decreased protein expression ( n = 4 per construct, Mann-Whitney U test: ∗ p < 0.05).

Article Snippet: Full-length sequence for UTRs was obtained and reference and variant entire UTR sequence was ordered from Twist Biosciences with BamHI and and BglII/AgeI restriction sites for five case variants whose shorter constructs increased (three, one each in SMC5, ZFYVE9, and ZNF311), decreased (NSD2), and had no significant change (NR4A2.2), in the dual-luciferase assay ( ).

Techniques: Sequencing, MANN-WHITNEY, Luciferase, Reporter Assay, Expressing, Construct

Journal: Cell Reports Methods

Article Title: Approaches for identification of 5′ UTR mutations impacting translation and protein production from neurodevelopmental disorder genes

doi: 10.1016/j.crmeth.2025.101247

Figure Lengend Snippet: The functional effects of 5′ UTR variants primarily impact translation (A–C) Volcano plots of allelic effects across 1,507 5′ UTRs mutations (purple points q value <0.05, see ). (D and E) Violin/boxplots of allelic effects of elements changing the number of uORFs (Mann-Whitney U test). (F and G) Violin/boxplots of variants that are significant on MPRA measures (purple) have greater absolute changes in folding free energy (Mann-Whitney U test).

Article Snippet: Full-length sequence for UTRs was obtained and reference and variant entire UTR sequence was ordered from Twist Biosciences with BamHI and and BglII/AgeI restriction sites for five case variants whose shorter constructs increased (three, one each in SMC5, ZFYVE9, and ZNF311), decreased (NSD2), and had no significant change (NR4A2.2), in the dual-luciferase assay ( ).

Techniques: Functional Assay, MANN-WHITNEY

Journal: Cell Reports Methods

Article Title: Approaches for identification of 5′ UTR mutations impacting translation and protein production from neurodevelopmental disorder genes

doi: 10.1016/j.crmeth.2025.101247

Figure Lengend Snippet: 5′ UTR variants from autism probands alter translation in the native genomic context (A) Examples of polysome/80S enrichments altered by the alternate allele from the MPRA ( ∗∗∗ q value < 0.001, ∗∗∗∗ q value < 0.0001; for full results). (B) LCLs from these patients were subjected to polysome fractionation followed by targeted sequencing of the variant loci to quantify relative Ref and Alt allele abundance. (C) Differences in the relative abundance of each allele between total RNA and DNA (AEI) were tested. Variants in MKRN1 , SMC5 , and C12orf57 had an effect on transcript abundance. (D) ATI showed variants in SMC5 , MKRN1 , and USP48 had translational effects ( n = 2, significance of change in Alt allele abundance between fractions assessed by negative binomial generalized linear model fit; see STAR Methods and ; see also A). (E) Measurement of the relative allele abundance in 80S and 40S RNA fractions from proband-derived LCLs reveals an effect of proband variants in NSD2 and C12orf57. (F) Highly robust allelic effects in polysome/total RNA enrichment are found for proband variants in SMC5 and MKRN1. (G–I) Western blotting of MKRN1, NSD2, and SMC5 in proband-derived cell lines (light blue) alongside cell lines from matched sibling control (dark blue) and cell lines from unrelated control individuals (gray) ( n = 8 technical replicates per sample in G and I, n = 4 technical replicates per sample in H, Wilcoxon signed-rank test in G and I; Mann-Whitney U test in H; ∗ p < 0.05, ∗∗ p < 0.01).

Article Snippet: Full-length sequence for UTRs was obtained and reference and variant entire UTR sequence was ordered from Twist Biosciences with BamHI and and BglII/AgeI restriction sites for five case variants whose shorter constructs increased (three, one each in SMC5, ZFYVE9, and ZNF311), decreased (NSD2), and had no significant change (NR4A2.2), in the dual-luciferase assay ( ).

Techniques: Fractionation, Sequencing, Variant Assay, Derivative Assay, Western Blot, Control, MANN-WHITNEY

Journal: Cell Reports Methods

Article Title: Approaches for identification of 5′ UTR mutations impacting translation and protein production from neurodevelopmental disorder genes

doi: 10.1016/j.crmeth.2025.101247

Figure Lengend Snippet: A Vglut2-Cre system allows for neuron-specific measures of translation in vivo (A) Illustration of experimental design: the same 5′ UTR libraries were packaged into AAV and delivered into the perinatal mouse brain, collected at P21, and fractionated as above. LoxP sites enable a Cre-dependent inversion of a primer site (top rows), resulting in mRNAs with a unique RT primer site in excitatory neurons. (B) Neuronal and non-neuronal cell types in the lateral cortex display expression of the AAV-delivered tdTomato reporter gene while eGFP expression in Vglut2-positive Cre-expressing neurons partially overlaps the transduced cell population (scale bars: 100 μm). (C) The rate of barcode recovery with 20 or more UMI counts varied by the sequenced fraction and the Cre state of the library. The 80S RNA libraries prepared with the Cre OFF RT primer had particularly low barcode recovery rates. (D) All pairwise comparisons within each fraction type show a generally high correlation (Pearson’s correlation coefficient > 0.9) between replicates with Cre OFF 80S RNA and Cre ON DNA libraries being the exceptions. (E–G) Reporter transcript abundance [log 2 (total RNA/DNA)] (C) measurements averaged across barcodes, and animals show a high correlation across Cre ON and Cre OFF libraries. Measures of polysome/80S (D) or polysome/total RNA (E) enrichment span narrower ranges than transcript abundance in both cellular contexts and show significantly lower correlations. Red line indicates linear fit, and R represents Pearson’s correlation. (H–J) Synthetic controls demonstrate the sensitivity of different MPRA measurements to changes in secondary structure or uORFs in vivo . Compared to a sequence without secondary structure, a synthetic 5′ UTR containing a 15-bp stem loop increases overall transcript abundance and 80S occupancy relative to polysome-associated fractions. A 5′ UTR containing a short uORF proximal to the reporter start codon has no measurable effect on transcript abundance compared to similar sequence with a disrupted uAUG; however, the uORF slightly increases polysome enrichment relative to the 80S-associated and total RNA-associated fraction (Mann-Whitney U test).

Article Snippet: Full-length sequence for UTRs was obtained and reference and variant entire UTR sequence was ordered from Twist Biosciences with BamHI and and BglII/AgeI restriction sites for five case variants whose shorter constructs increased (three, one each in SMC5, ZFYVE9, and ZNF311), decreased (NSD2), and had no significant change (NR4A2.2), in the dual-luciferase assay ( ).

Techniques: In Vivo, Expressing, Sequencing, MANN-WHITNEY

Journal: Cell Reports Methods

Article Title: Approaches for identification of 5′ UTR mutations impacting translation and protein production from neurodevelopmental disorder genes

doi: 10.1016/j.crmeth.2025.101247

Figure Lengend Snippet: The impact of UTR elements on measures of translation in excitatory neurons in vivo (A) Comparison of transcript abundance and translation measures between assays performed in HEK and cortical neurons. Red line indicates linear fit, and R represents Pearson’s correlation. (B) Violin/boxplots comparing 5′ UTRs with and without uORFs on MPRA measures in neurons (Mann-Whitney U test: ∗∗∗∗ p < 0.0001). (C) Scatterplots show GC content, and predicted folding free energy of transcript 5′ UTRs strongly predicts transcript abundance but show little correlation with translation measures. Red line indicates linear fit, and R represents Pearson’s correlation. (D and E) Volcano plots showing allelic effects in glutamatergic neurons for transcript abundance and polysome/80S (purple, q value < 0.05).

Article Snippet: Full-length sequence for UTRs was obtained and reference and variant entire UTR sequence was ordered from Twist Biosciences with BamHI and and BglII/AgeI restriction sites for five case variants whose shorter constructs increased (three, one each in SMC5, ZFYVE9, and ZNF311), decreased (NSD2), and had no significant change (NR4A2.2), in the dual-luciferase assay ( ).

Techniques: In Vivo, Comparison, MANN-WHITNEY

Journal: Frontiers in Plant Science

Article Title: Parallel Microbial Ecology of Pasteuria and Nematode Species in Scottish Soils

doi: 10.3389/fpls.2019.01763

Figure Lengend Snippet: Maximum likelihood phylogeny and pairwise sequence identity heatmap of Pasteuria spp. taxonomic reference sequences used in primer design and Pasteuria spp. ZOTU taxonomic assignment.

Article Snippet: The pairwise percentage identity of reference Pasteuria spp. sequences in the aligned region of the 16S rRNA gene ranged from a low of 84.7% ( P. ramosa vs P. usgae ) to a high of 99.3% ( P. penetrans to Pasteuria HcP) with an average of 95% identity between all reference sequences.

Techniques: Sequencing

Journal: Frontiers in Plant Science

Article Title: Parallel Microbial Ecology of Pasteuria and Nematode Species in Scottish Soils

doi: 10.3389/fpls.2019.01763

Figure Lengend Snippet: Map of distribution of the most abundant Pasteuria spp. ZOTUs across both NSIS2 (circles) and ESFN (triangles) datasets. Plot points are colored by ZOTU and sized as a function of the total number of merged read pairs recovered for that ZOTU µl −1 of product added to the final pool from the corresponding sample.

Article Snippet: The pairwise percentage identity of reference Pasteuria spp. sequences in the aligned region of the 16S rRNA gene ranged from a low of 84.7% ( P. ramosa vs P. usgae ) to a high of 99.3% ( P. penetrans to Pasteuria HcP) with an average of 95% identity between all reference sequences.

Techniques:

Journal: Frontiers in Plant Science

Article Title: Parallel Microbial Ecology of Pasteuria and Nematode Species in Scottish Soils

doi: 10.3389/fpls.2019.01763

Figure Lengend Snippet: NDMS ordination plot of NSIS2 Pasteuria spp. community composition. Points are colored by soil horizon where L = litter; H = humus; O = peaty material formed under wet, anaerobic conditions; and A = mineral horizon formed at or near the surface showing accumulation and incorporation of organic matter. Points are sized by the total number of merged read pairs µl −1 of PCR product added to the final pool from the corresponding sample. Stress = 0.245235.

Article Snippet: The pairwise percentage identity of reference Pasteuria spp. sequences in the aligned region of the 16S rRNA gene ranged from a low of 84.7% ( P. ramosa vs P. usgae ) to a high of 99.3% ( P. penetrans to Pasteuria HcP) with an average of 95% identity between all reference sequences.

Techniques:

Journal: Frontiers in Plant Science

Article Title: Parallel Microbial Ecology of Pasteuria and Nematode Species in Scottish Soils

doi: 10.3389/fpls.2019.01763

Figure Lengend Snippet: Environmental variables with a statistically significant relationship to Pasteuria spp. community ordination before or after Benjamini-Hochberg correction.

Article Snippet: The pairwise percentage identity of reference Pasteuria spp. sequences in the aligned region of the 16S rRNA gene ranged from a low of 84.7% ( P. ramosa vs P. usgae ) to a high of 99.3% ( P. penetrans to Pasteuria HcP) with an average of 95% identity between all reference sequences.

Techniques:

Journal: Frontiers in Plant Science

Article Title: Parallel Microbial Ecology of Pasteuria and Nematode Species in Scottish Soils

doi: 10.3389/fpls.2019.01763

Figure Lengend Snippet: Spearman’s rank correlation of soil properties with statistically significant correlation to Pasteuria spp. community composition.

Article Snippet: The pairwise percentage identity of reference Pasteuria spp. sequences in the aligned region of the 16S rRNA gene ranged from a low of 84.7% ( P. ramosa vs P. usgae ) to a high of 99.3% ( P. penetrans to Pasteuria HcP) with an average of 95% identity between all reference sequences.

Techniques:

Journal: Frontiers in Plant Science

Article Title: Parallel Microbial Ecology of Pasteuria and Nematode Species in Scottish Soils

doi: 10.3389/fpls.2019.01763

Figure Lengend Snippet: The top three most abundant Pasteuria spp. ZOTUs in the ESFN dataset and respective metazoan ZOTU Spearman’s rank correlations which were statistically significant after Benjamini-Hochberg correction.

Article Snippet: The pairwise percentage identity of reference Pasteuria spp. sequences in the aligned region of the 16S rRNA gene ranged from a low of 84.7% ( P. ramosa vs P. usgae ) to a high of 99.3% ( P. penetrans to Pasteuria HcP) with an average of 95% identity between all reference sequences.

Techniques:

Journal: Frontiers in Plant Science

Article Title: Parallel Microbial Ecology of Pasteuria and Nematode Species in Scottish Soils

doi: 10.3389/fpls.2019.01763

Figure Lengend Snippet: (A) 400x magnification Pratylenchus spp. recovered from ESFN soil with Pasteuria spp. endospore attached (position indicated by red arrow). (B) 1000x magnification of Pratylenchus spp. recovered from ESFN soil with Pasteuria spp. endospore attached (position indicated by red arrow). (C) 1000x magnification of free living non-parasitic nematode recovered from ESFN soil with several Pasteuria spp. endospores attached (positions indicated by red arrows). (D) 1000x magnification of fluorescence image of Pratylenchus spp. recovered from ESFN soil with Pasteuria spp. endospore attached (position indicated by blue arrow), showing anti- Pasteuria penetrans antibody recognition.

Article Snippet: The pairwise percentage identity of reference Pasteuria spp. sequences in the aligned region of the 16S rRNA gene ranged from a low of 84.7% ( P. ramosa vs P. usgae ) to a high of 99.3% ( P. penetrans to Pasteuria HcP) with an average of 95% identity between all reference sequences.

Techniques: Fluorescence