Life Science Alliance

Interleukin-33 (IL-33) is a key cytokine in mast cell mediated immunity, promoting inflammatory cytokine production without inducing degranulation. Here, we compared IL-33 induced proteomic responses across three mast cell culture systems, Foetal Liver derived Mast Cells (FLMCs), Bone Marrow derived Mast Cells (BMMCs), and Peritoneal Mast Cells (PMCs), using quantitative data-independent acquisition mass spectrometry. Although baseline proteomes were largely conserved across all mast cell types, clear differences were observed between culture systems. PMCs exhibited a more mature phenotype, characterised by higher abundance of granule-associated proteins and lower levels of proteins involved in metabolism and translation. In contrast, FLMCs and BMMCs displayed higher levels of biosynthetic and metabolic machinery, consistent with a less differentiated state. IL-33 stimulation induced a conserved proteomic programme across all mast cell types, enriched for inflammatory signalling pathways, cytokine production, and enzymes involved in prostaglandin and biogenic amine biosynthesis. Pathway analysis demonstrated robust activation of nuclear factor {kappa}B (NF{kappa}B) associated signalling, with a relative enrichment of components linked to non-canonical NF{kappa}B signalling and tumour necrosis factor (TNF) receptor associated pathways. Mechanistically, IL-33 driven proteomic remodelling was strongly regulated by mitogen-activated protein kinase (MAPK) signalling. p38 MAPK emerged as the dominant regulator of the IL-33 response, with ERK1/2 contributing to a subset of induced proteins. These pathways differentially regulated key effector outputs, including IL-6, IL-9, IL-1 family cytokines, and enzymes required for prostaglandin, serotonin, and histamine biosynthesis. Together, these data define conserved IL-33 dependent inflammatory programmes across mast cell differentiation states and demonstrate how MAPK signalling pathways shape the composition of mast cell effector responses.

The endoplasmic reticulum (ER) and mitochondria maintain a dynamic structural partnership essential for pancreatic {beta}-cell homeostasis, yet the high-resolution 3D remodeling of these networks under stress conditions remains poorly defined. We employed Focused Ion Beam Scanning Electron Microscopy (FIB-SEM) to perform 3D reconstructions of INS1E cells subjected to mitochondrial respiratory chain inhibition, uncoupling, and exogenous oxidative stress. Quantitative analysis revealed that mitochondrial dysfunction induces profound ultrastructural transitions, characterized by significant luminal swelling of the ER, expansion of the perinuclear space, and mitochondrial diameter enlargement. 3D volume imaging identified a coordinated fragmentation of both ER and mitochondrial networks into discrete, spatially separated structures--a phenomenon distinct from the reticular morphology observed in control cells. The similarity between respiratory inhibition- and H2O2-induced phenotypes, together with preservation of ER structure following mitochondrial uncoupling, suggests a potential contribution of reactive oxygen species to the observed remodeling process. Despite this extensive organelle breakdown, interorganelle membrane contact sites were not only preserved but expanded under stress conditions. We further provide a quantitative description of nuclear envelope-mitochondria contact sites (NAMs), demonstrating their selective remodeling during mitochondrial dysfunction. Our findings provide a high-resolution structural framework for organelle remodeling in {beta}-cells, demonstrating that membrane contact sites are actively preserved and reorganized despite profound organelle fragmentation. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=110 SRC="FIGDIR/small/727587v1_ufig1.gif" ALT="Figure 1"> View larger version (36K): org.highwire.dtl.DTLVardef@4f63b5org.highwire.dtl.DTLVardef@1b3dc8org.highwire.dtl.DTLVardef@7527fcorg.highwire.dtl.DTLVardef@1944f2c_HPS_FORMAT_FIGEXP M_FIG C_FIG

The long noncoding RNA ZFAS1 plays a role in cell proliferation and has been linked to cancer development and prognosis. However, the ZFAS1 locus is predicted to encode over 40 ZFAS1 splice variants, which remain largely uncharacterized. To shed light on the role of ZFAS1 in hepatocyte models, we examined the transcriptome-wide effects of separately targeting three exons present in representative ZFAS1 variants with gapmer antisense oligonucleotides. Evidence of cross-exon compensatory regulation was obtained by qRT-PCR. Although targeting resulted in a subset of concerted transcript perturbations-indicating specificity and shared functionalities-the overall effects were predominantly non-redundant. Overrepresentation analyses revealed that proximal exon and distal exon targeting affected cell cycle and transcription regulation, respectively. Strikingly, interrogation of the entire transcriptome with gene set enrichment analysis identified a shared subset of pathways related to cell-cycle control and translation, which were affected antagonistically in an exon-dependent manner. Whereas targeting the proximal exon was predicted to broadly compromise cell-cycle and translational functions, targeting distal exons produced contrasting effects on these processes. Together, these findings demonstrate that the arrangement of ZFAS1 exons can markedly modulate its function.

Abstract/SummaryGross chromosomal rearrangements are a hallmark of many diseases and cancers. The study of their biogenesis and the mechanisms underlying their formation is greatly facilitated by the availability of genetic reporter assays in model organisms. We present here a novel GCR assay developed in fission yeast, a highly relevant model for understanding genome instability related to human biology. The reporter employs canavanine counter-selection to detect GCRs within a chromosomal context. Using this assay, we identified natural hotspots for GCRs, including inverted long terminal repeats (IR-LTRs). Structural analysis of GCR events showed that IR-LTR-induced GCRs mainly result in either terminal deletions with adjacent inverted duplications or repair via long-range break-induced replication (BIR). Deleting IR-LTRs reduces the GCR rate and reveals another hotspot driven by BIR between homeologous aldo/keto reductase genes on opposite arms of chromosome I. This is the first evidence that BIR can occur in S. pombe on long tracks reaching up to 600 kb. Besides highlighting genome rearrangement hotspots, the assay also identifies regulators of genome instability in fission yeast. Loss of Nup132, a component of the nuclear pore complex, increases IR-LTRs-induced GCRs, while the budding yeast homolog Nup133 has no effect on the stability of a structurally similar IR. In contrast, disrupting djc9, which encodes a conserved histone H3-H4 binding protein, decreases GCR rates. Overall, this sensitive GCR assay enables the identification of factors that control spontaneous and fragile motif-induced chromosomal instability, including those conserved in humans but lost through evolution in other organisms.

In Caenorhabditis elegans embryos, the nuclear transport receptor IMB-2 (Importin Beta Family-2, a karyopherin {beta}2) preferentially localizes to the nuclear envelope along with its encoding mRNA. This suggests that imb-2 mRNA is locally translated at the nuclear envelope. To test whether imb-2s two putative human orthologs, Transportin 1 (TNPO1) and Transportin 2 (TNPO2), exhibited similar mRNA localization and local translation, we performed smiFISH and microscopy in U2OS, HeLa, and human pluripotent stem cells. Neither human TNPO1 nor TNPO2 mRNA localized to the nuclear envelope in any tested human cell type. However, the human TNPO1 protein and the C. elegans IMB-2 protein both localized to the nucleus and its periphery. This suggests that despite their shared functional roles and high amino acid sequence identities (52% and 51%, respectively), these karyopherins differed in their translational dynamics.

Telomere-led rapid prophase chromosome movements (RPMs) during meiotic prophase are critical for homologous chromosome pairing and proper meiotic progression. These movements are generated by the cytoskeleton and are transmitted to the telomeres via the LINC complex, yet the cytoplasmic components that generate these forces remain poorly defined. Among candidates of microtubule-associated motor proteins in mouse primary spermatocytes, we confirmed KIF5B as a specific interactor of the KASH5-LINC complex. Total internal reflection fluorescence microscopy and microtubule sedimentation assays performed with purified recombinant proteins suggest a direct interaction between KASH5 and KIF5B on microtubules, enhanced by MAP7, a known KIF5B-recruiting and activating cofactor. Mapping the KIF5B-binding surface of KASH5 revealed that KASH5 N-terminal EF-hand domains mediate the interaction. Further, in vivo KIF5B-KASH5 interaction and KIF5B role in RPMs are evidenced as (1) KIF5B is recruited by KASH5-SUN1 to the nuclear envelope in two different cultured somatic cell models, (2) KIF5B is telomere-associated and colocalizes with KASH5, and microtubules associated with the nuclear envelope in mouse spermatocytes, and (3) chemical inhibition of KIF5B reduces telomere-led chromosome motions. Altogether, our findings identify the KIF5B kinesin as a previously unrecognized component of the force-generating machinery that drives chromosome movement during meiotic prophase I, acting through KASH5 as a specific nuclear membrane adaptor.

IntroductionImpaired motile cilia function contributes to many respiratory disorders, but therapies targeting this cellular defect are currently lacking. Personalized airway epithelial models combined with quantitative, complementary ciliary assays can pave the way for the development of such therapies. However, existing airway epithelial cultures often show variable ciliogenesis, and ciliary function is frequently assessed using a single assay that does not capture the phenotypic heterogeneity of ciliary dysfunction. Here, we established a personalized, multi-assay in vitro platform using human nasal epithelial cells (HNECs) to assess ciliary function and therapeutic response, using primary ciliary dyskinesia (PCD) as a model disease. MethodsHNECs from 8 healthy individuals and 13 individuals with PCD carrying distinct disease-associated variants were obtained by nasal brushing. Cells were differentiated under optimized conditions, including {gamma}-secretase/Notch and BMP pathway inhibitors and a low liquid-liquid interface, to generate highly ciliated 2D epithelial cultures. Ciliary function was assessed using ciliary beat frequency, bead transport, and apical-out nasal organoid rotation assays. Therapeutic rescue was assessed in HNECs harboring DNAI1 alterations using DNAI1 mRNA-loaded lipid nanoparticles. ResultsOptimized differentiation yielded reproducibly multiciliated HNEC cultures. The multi-assay platform distinguished healthy from PCD-derived HNECs and revealed individual- and genotype-specific patterns of ciliary dysfunction not captured by a single assay. Basolateral administration of DNAI1 mRNA-loaded lipid nanoparticles resulted in partial, dose-dependent recovery of ciliary function in DNAI1-deficient HNECs. ConclusionThis study establishes a standardized, individual-specific multi-assay nasal epithelial platform for functional phenotyping of motile cilia and preclinical evaluation of emerging therapies, with demonstrated utility in PCD.

Abstract/SummaryRetroelements, including retrotransposons, endogenous retroviruses, and their fragments, as well as rare co-opted or domesticated retroelements, can contribute to neurodegenerative disorders and aging through modulation of gene expression and induction of neuroinflammation. Paternally Expressed Gene 10 (PEG10) is a retroelement-derived human gene that has recently been identified as a putative driver of Amyotrophic Lateral Sclerosis (ALS) and Angelmans Syndrome. PEG10 has been reported to bind nucleic acid and undergoes a complex self-processing pathway that results in gene expression changes when the protein accumulates in cells. Here, we report that PEG10 has selectivity for binding U/G-rich RNAs and influences widespread gene expression changes. PEG10 overexpression mimics the loss of TDP-43 in broad changes to gene expression, including dysregulation of mRNA splicing pathways. Specific changes to mRNA splicing were largely unique between TDP-43 knockdown and PEG10 overexpression, as classic TDP-43 targets including STMN2 were not altered by PEG10. Instead, we identified a unique role for PEG10 in regulating splicing of neuregulin 3 (NRG3), a ligand for the neuronal receptor ERBB4. In SH-SY5Y cells and in human neurons overexpressing PEG10, NRG3 protein levels were decreased along cellular processes, suggesting that these cells are less competent at signaling through the NRG3/ERBB4 axis. Using human patient data, we observed similar changes to NRG3 splicing in UBQLN2-mediated ALS, where PEG10 is accumulated, as well as in some cases of sporadic ALS. In conclusion, the retroelement-derived gene PEG10 plays an unexpected role in regulating splicing of neuronal transcripts, which mimics some of the transcript changes observed in human ALS patient samples. Ultimately, this work has implications for the study of PEG10, and mRNA splicing in neurological diseases associated with elevated PEG10 abundance. HighlightsO_LIPEG10 NC expression influences abundance of transcripts implicated in ALS C_LIO_LIPEG10 NC expression leads to an exon skipping event in neuregulin 3 (NRG3) C_LIO_LINRG3 expression is decreased along dendrites of PEG10 NC expressing human neurons C_LIO_LIExpression of PEG10 NC mimics changes observed in human ALS C_LI Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/727000v1_ufig1.gif" ALT="Figure 1"> View larger version (56K): org.highwire.dtl.DTLVardef@1a957d2org.highwire.dtl.DTLVardef@c4b15corg.highwire.dtl.DTLVardef@15825faorg.highwire.dtl.DTLVardef@25533d_HPS_FORMAT_FIGEXP M_FIG C_FIG

BackgroundThe increase in human lifespan without a proportional increase in healthspan imposes a substantial burden on individuals and society. Exceptionally long-lived individuals and members of long-lived families exhibit compression of multi-morbidity. Genetics, and in particular rare protein-altering variants, appear to play an important role in their longevity. MethodsIn this study, we employed a targeted pathway approach to provide functional evidence of the significance of rare variants in the insulin/insulin-like growth factor 1 signalling - mechanistic target of rapamycin (IIS/mTOR) signalling pathway identified in long-lived individuals. To this end, we used CRISPR/Cas9 to introduce these rare genetic variants into mouse embryonic stem cells (mESCs). We subsequently assessed several functional readouts that have previously been associated with lifespan regulation in model organisms and/or IIS/mTOR and mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) signalling pathway activity. ResultsFunctional characterisation revealed that the variants exhibit both shared and distinct effects on the signalling pathways. Principal component analysis of omics-based datasets showed that the variants clustered into two groups, a distribution that corresponds with the grouping observed for a subset of functional readouts. All variant mESC lines exhibited a downregulation in IIS/mTOR and MAPK/ERK signalling pathway activity as well as an increase in Foxo3 expression and FOXO3 binding activity. We identified alterations in lipid and mitochondrial metabolism, including a reduction in mitochondrial DNA levels, which were mostly shared among all variants. All variant mESC lines exhibited a signature implying increased pluripotency. The effects on stress resistance and growth rate diverged between the two variant groups, with partially opposing effects. Group 1 demonstrated a reduced growth rate and increased resistance to a subset of stressors, while Group 2 demonstrated an increased growth rate and reduced resistance to a subset of stressors. ConclusionsHere, we provide evidence that rare genetic variants in the IIS/mTOR and MAPK/ERK signalling pathways identified in long-lived human individuals result in shared functional effects associated with longevity in model organisms. These insights can serve as a foundation to better understand the role of rare variants in the insulin signalling network in the regulation of human longevity. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=68 SRC="FIGDIR/small/728260v1_ufig1.gif" ALT="Figure 1"> View larger version (18K): org.highwire.dtl.DTLVardef@1bf5ebdorg.highwire.dtl.DTLVardef@e4e5dcorg.highwire.dtl.DTLVardef@1aee276org.highwire.dtl.DTLVardef@95f170_HPS_FORMAT_FIGEXP M_FIG C_FIG

Human centromeres are built over large stretches of repetitive, divergent -satellite DNA. Within these sequences lies a conserved, defined 17-bp sequence named the CENP-B box that is bound by the DNA-binding protein CENP-B. Recent studies have proposed that CENP-B box motifs along chromosome arms exist and may represent conserved, ectopic binding sites with functional relevance. Here, we evaluate the genomic distribution, conservation, and binding capacity of different CENP-B box motifs outside canonical centromeres. Analysis of thousands of complete human centromere assemblies reveals exceptional conservation of a unique and canonical CENP-B box motif within centromeres. This is in contrast with the high sequence variability and stochastic occurrence of more degenerate CENP-B box motifs along chromosome arms. Consistently, CENP-B binds only at canonical CENP-B box motifs embedded within -satellite sites, with no evidence of functional binding at any ectopic sites. Together, these results indicate an adaptive selection of canonical CENP-B boxes within centromeric regions, in contrast to random sequence occurrences for ectopic CENP-B box-like motifs.

Immunoglobulin genes are a central component of jawed-vertebrate adaptive immunity. A previous study showed that the blunt-snouted clingfish Gouania willdenowi lacks immunoglobulin genes and T-cell receptor gamma/delta loci, while retaining T-cell receptor alpha/beta genes, MHC genes, and RAG1 /RAG2. Here I extend that observation to the family Gobiesocidae using all seven chromosome-level Gobiesocidae genome assemblies currently available. Manual tblastn and synteny-guided searches found no convincing immunoglobulin heavy-chain or light-chain loci in G. willdenowi, Gouania pigra, Gobiesox punctulatus, Apletodon dentatus, Lepadogaster candolii, Lepadogaster purpurea, or Diplecogaster bimaculata. Thus, the absence of antibody genes is best interpreted as a root-level character of clingfishes. The latest seven-species screen of 40 additional immune-associated genes shifts the broader interpretation in the same direction: the B-cell/adaptive core genes CD79A, CD79B, CIITA, TNFRSF13B, and TNFSF13B lack strong tblastn support in all sampled Gobiesocidae, and 37 of the 40 tested targets show an all-zero binary pattern at the presence threshold. Only IL21R.1, TYROBP, and TNFRSF11A show strong hits in one or more species. I therefore interpret the principal immune-gene erosion as occurring at or near the Gobiesocidae root rather than as a recent Gouania-specific process, while keeping weak, paralog-sensitive, and patchy loci provisional. RAG2 comparisons show a shared Gobiesocidae PHD-domain C-to-S replacement in the zinc-binding motif, with apparently intact RAG2 coding sequence. A family-wide TRG/TRD screen did not recover TRGV V segments or accepted TRDC constant-region exons, but it did detect TRGC-like constant exons in several genomes. These TRGC-like sequences are probably not canonical TRG constant exons without further validation, so I treat the gamma/delta system as eroded or rearranged rather than as a complete root-level loss equivalent to the Ig loss. The RAG2 variant provides a plausible molecular context for antigen-receptor remodeling, but it is not evidence that RAG genes are pseudogenized, because TCR alpha/beta, MHC genes, and RAG1 /RAG2 are retained. Gobiesocidae are therefore best described as a vertebrate family with ancestral loss of canonical immunoglobulin genes and associated root-level erosion of B-cell and immune-related genes, not as a lineage lacking adaptive immunity in its entirety. HighlightsO_LISeven chromosome-level Gobiesocidae genomes lack convincing canonical IgH and IgL loci. C_LIO_LIThe strongest non-Ig losses map to the B-cell/adaptive core: CD79A, CD79B, CIITA, TNFRSF13B, and TNFSF13B. C_LIO_LITCR alpha/beta, MHC genes, and RAG1 /RAG2 are retained, so Gobiesocidae should not be described as lacking adaptive immunity in full. C_LIO_LIA shared Gobiesocidae RAG2 PHD-domain C-to-S variant provides candidate molecular context for antigen-receptor remodeling. C_LI

Synthetic Genetic Array (SGA) analysis comprises the high-throughput crossing of a query deletion strain against a genome-wide deletion library to score fitness interactions in thousands of double mutants. SGAs have produced comprehensive genetic interaction maps in yeasts and have emerged as a leading platform for pharmacogenomics: mapping genetic modifiers of drug response, identifying synthetic lethal targets and illuminating mechanisms of drug action and resistance. We have previously demonstrated that in fission yeast, the ade6 mutant is functionally neutral relative to the parental library and can serve as a standard negative control for SGA screens. Here, while we confirm our previous observation, we show that this neutrality fails under pharmacological stress. Using Torin1, an ATP-competitive TOR kinase inhibitor, we demonstrate that the ade6 SGA fitness profile diverges from that of the parental library in a dose-dependent and genomically widespread manner. At 2 M Torin1 only 12.2% of scored genes exceed a 1.5-fold fitness difference between backgrounds; at 3 M this proportion rises to 43.2% -a 3.5-fold increase driven by qualitative reorganisation of the genetic interaction landscape rather than simple scaling of pre-existing differences. Gene ontology analysis of divergent genes implicates autophagy, iron starvation responses, central carbon metabolism, and vesicle trafficking, consistent with TOR-regulated nutrient adaptation being differentially affected by the ade6-M210/M216 point mutations in the library versus the ade6 null in the SGA control. Our results have implications in fission yeast and beyond and we propose solutions towards reliable retrieval of genetic interactions.

Collective cell migration is coordinated by adherens junctions (AJs) which serve both as structural links between the actin cytoskeleton of adjacent cells, as well as mechano-transductory structures allowing cells to transmit mechanical signals. Vinculin contributes to AJ maturation in multiple ways, binding to actin and the cadherin-catenin complex, bundling actin filaments, antagonising branched actin polymerisation and recruiting late AJ proteins. Here we have analysed the effect of vinculin on junctional actin organisation and its role in collective migration during unjamming in the human epithelial cell line MCF10A. At the apical surface of MCF10A monolayers, we found transcellular actin fibres (TAFs) that are directionally coordinated across long ranges, up to 10 cells. These TAFs are contractile and "cross" cell-cell contacts at AJs. Analysis of a vinculin knockout cell line revealed that this protein is essential for the coordination of TAFs across multiple cells. Arp2/3 activity must be tightly regulated to establish a long-range network of TAFs, since its downregulation by CK666 treatment, as well as its upregulation by the expression of an activated Rac1 mutant or a mutation that prevents the vinculin-Arp2/3 interaction, all impair TAF formation. During hypotonic unjamming of monolayers, we found that MCF10A cells with long-range TAFs migrate more collectively than vinculin KO cells in which TAFs only connect adjacent cells. Similarly, space-induced unjamming of MCF10A and vinculin knockout monolayers showed that cells connected by the long-range TAFs can collectively coordinate the direction in which they extend their lamellipodia. Thus, we show that vinculin plays a novel role in organising long-range actin networks across multiple cells and coordinating collective migration within cell monolayers.

During meiosis, homologous chromosomes pair to form synaptonemal complexes (SCs) and exchange genetic material through a process known as meiotic recombination. First, programmed DNA double-strand breaks form, followed by the assembly of recombination foci on SCs. These foci mark the sites of recombination intermediates and future crossovers. Distributions of recombination foci along SCs have been studied in many eukaryotes, revealing the interplay between recombination patterns and genome evolution. However, in fish, data on recombination patterns are scarce, and, for the majority of groups, completely absent. Here, we measure the positions of MLH1 foci in 3,504 SCs from 219 male meiotic cells of an African annual killifish Nothobranchius virgatus, a representative of a genus with remarkable karyotype and genome diversity, and present a detailed statistical analysis of its recombination patterns. We found that, in contrast to the several other fish species characterised to date, recombination in N. virgatus occurs across almost entire chromosome arms, excluding (peri)centromeres and telomeres. In the longest SCs, we observed a proximal and a distal peak of the recombination focus frequency and explained the peaks by chromosome pairing dynamics. We also revealed the typical positions of focus pairs, demonstrated interference between foci, with the minimal interfocus distance of 4 m, and described regions of the total recombination suppression near centromeres and telomeres. In sum, our study provides a detailed analysis of recombination patterns in a killifish with a fully acrocentric karyotype and contributes to cytogenomic and statistical methodology for future exploration of meiotic recombination patterns.

Centromeres are composed of DNA repeats within chromosomes primary constriction. CENP-B is the only centromeric protein known to bind a specific motif, the CENP-B box, promoting kinetochore stability. We recently uncovered degenerate CENP-B binding motifs outside centromeres, whose position and orientation defines chromosome specific banding patterns. Here, we leveraged telomere-to-telomere assemblies to map conservation of these ectocentromeric sequences (ECS) across hundreds of haplotypes. We found strong negative selection acting on their occurrence along chromosome arms, implying functional constraints incompatible with stochastic drift. We classified four categories: (i) ECSs that lack CENP-B binding ([~]84%); (ii) ECSs bound by CENP-B ([~]10%); (iii) ECSs near CENP-B-enriched accessible chromatin ([~]6%); (iv) we further identified [~]700 CENP-B binding sites outside centromeres without CENP-B boxes. Integrating chromatin conformation capture (HiC), neocentromeres and meiotic recombination mapping with CENP-B CUT&RUN, methylation and ATAC-seq data, we found heterogenous functionalities driven by distance-dependent enrichment and local contacts of boxes in inverted orientation on the same strand, analogous to ALU repeats affecting topological folding. CENP-B knockdown significantly reduced neighboring gene expression, revealing a moonlighting regulatory role outside centromeres. Our findings characterizes human ectocentromeric sites as evolutionarily constrained and functionally heterogeneous elements along chromosome arms with context-dependent roles in chromatin state. Graphical AbstractEctocentromeric sites exhibit heterogeneous CENP-B occupancy and context-dependent chromatin functions. Ectocentromeric sequences (ECSs) along chromosome arms fall into four categories: (i) CENP-B box motifs alone, lacking protein binding, embedded within repressed or boundary chromatin and contributing to TAD organization. Motifs with opposite orientation (forward and reverse complement) paired on the same strand may further promote self-complementary chromatin contacts analogous to Alu inverted repeats, reshaping topology with long-range looping contacts; (ii/iii) ECSs bound by CENP-B protein, associated with specific open chromatin state downstream of H3K27me3-marked compacted chromatin that modulate local accessibility and gene expression; and (iv) CENP-B binding peaks lacking a canonical box motif, located proximal to transcription start sites and linked to active gene expression. Together, ectocentromeric sites represent functionally heterogeneous elements with context-dependent roles spanning chromosome architecture, chromatin state, and transcription regulation. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=112 SRC="FIGDIR/small/728588v1_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@1683fb7org.highwire.dtl.DTLVardef@12f1c9dorg.highwire.dtl.DTLVardef@1ffc699org.highwire.dtl.DTLVardef@14790dd_HPS_FORMAT_FIGEXP M_FIG C_FIG

Background. Type 2 diabetes mellitus (T2D) is defined by progressive pancreatic {beta}-cell dysfunction whose molecular underpinnings remain incompletely understood. Single-cohort transcriptomic analyses of donor islets have yielded heterogeneous gene lists of limited cross-study reproducibility, constraining both mechanistic interpretation and biomarker development. Methods. We combined two complementary analytical strategies applied to four public human islet transcriptomic cohorts (GSE25724, GSE20966, GSE38642, and GSE164416; n = 7-57 donors per contrast). For the integrative arm, three microarray datasets and one bulk RNA-seq dataset were processed independently and unified through gene-level random-effects meta-analysis, hallmark pathway scoring (GSVA/MSigDB), and iterative module refinement, yielding a two-axis disease framework. For the diagnostic arm, a consensus multi-method machine learning pipeline, combining LASSO penalized logistic regression, Support Vector Machine Recursive Feature Elimination (SVM-RFE), and Random Forest importance scoring, was applied to 184 differentially expressed genes from the RNA-seq cohort, with all normalization steps performed within leave-one-out cross-validation (LOOCV) folds to prevent data leakage. Machine learning classification of the RNA-seq cohort was additionally subjected to external transportability testing in the independent bulk human islet RNA-seq cohort GSE50244 using an overlap-restricted reduced score and a threshold fixed in the discovery cohort. Results. Meta-analysis across all four cohorts identified 337 high-confidence T2D-associated genes (96.1% directional concordance in beta-cell-enriched tissue). These were distilled into two refined 14-gene modules: ImmuneStress (MICB, HLA-DRA, HLA-DPA1, IL1R2, and others) and BetaCellIdentitySecretion (RASGRP1, PPP1R1A, SLC2A2, and others), whose composite IsletDysfunctionScore provided the most stable cross-platform separation of non-diabetic from T2D islets (Hedges' g = 1.80, p = 9.83 x $10^-17$, $\text{I}^2$= 0%). Consistent with progressive disease, IsletDysfunctionScore increased monotonically from non-diabetic to impaired glucose tolerance to T2D. Separately, the machine learning pipeline derived a 10-gene diagnostic panel: GABRA2, SLC2A2, ARG2, DKK3, PRIMA1, TAFA4, HHATL, PARVG, RNU1-70P, and the novel lncRNA ENSG00000284653, that achieved perfect discrimination in LOOCV (AUC = 1.000, sensitivity = 1.000, specificity = 1.000, zero misclassifications across all 57 donors). A leakage-verification experiment confirmed that this performance reflected genuine biological signal: global quantile normalization prior to cross-validation collapsed AUC to 0.380. External testing showed that 8 of the 10 panel genes were measurable in GSE50244. The frozen 8-gene reduced score retained strong discrimination (external AUC = 0.907), with 6 of 8 genes preserving directional concordance, but the discovery-derived threshold did not transfer because the external score distribution was shifted upward and compressed, yielding complete sensitivity but zero specificity at the frozen cutoff Conclusions. Integrating pathway-level meta-analysis with machine learning classification, we present a coherent two-axis model: immune/stress activation and loss of beta-cell identity/secretory competence, together with a compact, biologically interpretable 10-gene diagnostic signature. Panel genes converge on GABA signaling, glucose transport, arginine metabolism, WNT pathway inhibition, and a novel lncRNA, providing both mechanistic hypotheses and high-priority targets for external validation. These findings offer a reproducible transcriptomic scaffold for future mechanistic, biomarker, and clinical translation studies of human islet dysfunction. They also support external transportability of the core biological signal, while indicating that absolute operating thresholds are cohort-dependent and would require recalibration before deployment in independent datasets.

Ribosome biogenesis is a highly coordinated pathway that involves the assembly of ribosomal RNAs (rRNAs) with ribosomal proteins (r-proteins) to generate functional ribosomal subunits (r-subunits). The Saccharomyces cerevisiae (yeast) large 60S r-subunit consists of three rRNA molecules and 46 r-proteins. The contributions of nearly all r-proteins of the yeast large r-subunit have been characterized; however, a few non-essential proteins remain poorly understood. Although non-essential, human eL22 has been identified as a key player in p53 regulation during ribosomal stress and as a highly mutated target in cancers. Despite this function, the role of eL22 in ribosome maturation is still ill-defined. In this study, we characterized yeast eL22 r-protein. Our results show that eL22 assembles into intermediate nucleolar pre-60S ribosomal particles. Loss of eL22 impairs cell growth and reduces 60S r-subunit accumulation, phenotypes that are exacerbated at low temperatures. Analysis of pre-rRNA processing by pulse-chase labeling, northern blot hybridization, and primer extension reveals a defect in 27S pre-rRNA maturation, specifically at the level of 27SB pre-rRNA processing. Consequently, nuclear export of eL22-deficient pre-60S particles is mildly impaired. Furthermore, we identify genetic interactions between eL22 and neighboring r-proteins, eL38 and eL31. We conclude that eL22 assembly is required for optimal pre-60S maturation during middle nucleolar stages, particularly at low temperatures, a function likely supported by the cooperative action of other r-proteins associated with common elements of 25S rRNA. HighlightsO_LIWe have studied the role of r-protein eL22 in yeast ribosome assembly. C_LIO_LIeL22 is required for 60S ribosomal subunit production. C_LIO_LIThe absence of eL22 is critical at low temperatures. C_LIO_LIeL22 is important for 27SB pre-rRNA processing and nuclear export of pre-ribosomes. C_LIO_LIeL22 functionally interacts with r-proteins eL38 and eL31 in domain III of 25S rRNA. C_LI

Efficient transport of membrane proteins through the endosomal network is essential for brain development and function, with perturbation implicated in disease. Deficiencies in Retromer, a key regulator of endosomal transport, have been linked to aging-related neurodegenerative disorders including Alzheimers and Parkinsons disease. To better define the neuroprotective role of Retromer, we have applied cell surface restricted proteomics to identify those integral membrane proteins whose recycling to the plasma membrane is mediated by Retromer and associated cargo adaptors, sorting nexin 3 (SNX3), its paralogue sorting nexin 12 (SNX12), and sorting nexin 27 (SNX27) (data available via ProteomeXchange: PXD078277). By comparing primary rat cortical neurons and astrocytes we have identified several cargoes that require either SNX3/SNX12- or SNX27-Retromer complexes for endosomal recycling, including proteins involved in synapse organisation, synaptic signalling and Alzheimers disease pathology. We highlight that perturbed Retromer function leads to endosomal enlargement, and we establish a key role of SNX27-Retromer in modulating transport of glutamate across both neuronal and astrocytic membranes via recycling of glutamate transporters EAAT3 (SLC1A1) and EAAT1 (SLC1A3) respectively. Our study provides further mechanistic insight into the consequences of Retromer deficiency for neuronal and astrocytic function, offering new avenues of research in the treatment of neurodegenerative disease. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=194 SRC="FIGDIR/small/724903v1_ufig1.gif" ALT="Figure 1"> View larger version (59K): org.highwire.dtl.DTLVardef@98277forg.highwire.dtl.DTLVardef@1490534org.highwire.dtl.DTLVardef@f4a9feorg.highwire.dtl.DTLVardef@c48402_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical AbstractC_FLOATNO Suppression of Retromer and the sorting nexins (SNX27, SNX3/SNX12) leads to a significant change in the surface proteome of rat cortical neurons and astrocytes. Focusing on the glutamate transporters, SLC1A1 and SLC1A3, we have validated that SNX27-Retromer is required for their trafficking, with SNX27-Retromer suppression in astrocytes leading to a loss of glutamate uptake. C_FIG

In AL amyloidosis, monoclonal immunoglobulin light chains (LCs) aggregate as amyloid fibrils in tissues. In synergy with the intrinsic aggregation propensity of specific LC sequences, microenvironment factors may be involved in tuning the disease pathophysiology, in particular proteolytic LC remodelling and heterotypic interactions in the extracellular milieu. Accounting for extrinsic modulators is critical for understanding the phenotypic variability of AL, usually imputed mainly to the LC diversity. We investigated the effects of apolipoprotein E (allele 3, apoE3) and clusterin (CLU), two amyloid-signature proteins involved in extracellular proteostasis, on the fibrillogenesis kinetics of amyloidogenic LC fragments from patient-derived sequences, as well as on aggregate composition, fibril morphology and thermodynamic stability. We show that apoE3 and CLU act as heterotypic interactors of prefibrillar and fibrillar LCs, significantly modulating LC amyloidogenesis, with complex and non-monotypic effects that range from anti- to pro-amyloidogenic depending on their concentration and on the LCs intrinsic amyloidogenicity. ApoE3 and CLU also influence fibril morphology, possibly by modifying protofilament association, and alter their thermodynamic properties. LC interactors may play a significant and insofar underappreciated role in the AL pathophysiology in vivo, likely contributing to phenotypic variability and structural polymorphisms and, possibly, to fibril resilience to amyloid reabsorption strategies.

Existing databases of interphase chromosome conformations typically store three-dimensional coordinates of genomic segments. However, since interphase chromatin is highly dynamic, such databases are dominated by transient configurations and unstructured regions, whose positions vary continuously between cells and over time, unlike folded proteins such as globin, which adopt similar structures in every cell. These drawbacks motivated the inception of a database based on strion (a portmanteau of a string capturing structure and function). A strion concisely describes the structure and activity of all transcription units in one cell, by retaining only functionally relevant positional information. Sets of strions describing structures in different cells sampled at different times are compiled into a super-strion. Then, 46 super-strions summarise the range of structure and activity of a human cell type, including information on all transcription units, how often each co-fires and co-clusters with others in transcription factories/hubs, enhancer interactomes and small-world expression networks. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/724942v1_ufig1.gif" ALT="Figure 1"> View larger version (38K): org.highwire.dtl.DTLVardef@13a1263org.highwire.dtl.DTLVardef@18d2c78org.highwire.dtl.DTLVardef@162865corg.highwire.dtl.DTLVardef@1631d65_HPS_FORMAT_FIGEXP M_FIG C_FIG