bluuebunny/crossref_metadata_2025 huggingface dataset:
OVEREXPRESSION OF TEMPRANILLO-LIKE PROTEINS PROMOTES ENDORMANCY RELEASE IN POPLAR
Authors: Gómez-Soto D. , Triozzi P. M. , Conde D. , del Barrio C. , Allona I. , Perales M.
Published: January 2025

Trees in temperate and boreal latitudes synchronize their growth-dormancy cycles with seasonal environmental variations to ensure their survival over the years. Dormancy control is crucial during winter when plants cease growth and establish buds to protect their apical meristems from cold temperatures. To overcome endormancy, initiate bud break, and restore growth, plants must be exposed to a specific duration of chilling, referred to as the chilling requirement, which is species- and ecotype-dependent. In this work, we study the novel roles of twoTEMPRANILLO-like genes (TEML1andTEML2) in the annual cycle of poplar. We demonstrated thatPopulus TEMLgenes are regulated by photoperiod, cold temperatures and the circadian clock, and they play a role in the control of endodormancy. Notably, their function diverges from the role of its Arabidopsis orthologAtTEM, which regulatesFLOWERING LOCUS T (FT)transcription and the photoperiodic flowering transcription. Transcriptomic analysis of endodormant buds during winter revealed that the activation ofTEML1andTEML2promotes endodormancy release by modulating the expression of endodormancy regulators and growth-promoting genes.
Diacylglycerol kinase ζ dictates CD40-mediated immune synapse formation, mTORC1 signaling and plasma cell fate in B lymphocytes
Authors: Fernández-Barrecheguren Ana , Fernández-Rego Adrián , Fuentes-Cantos Lucía , Pons Tirso , Estrada Belén S. , Iborra-Pernichi Marta , Ebrahimi Tahmineh , Sagrega-Aparisi Ana , Cogliati Sara , Martínez-Martín Nuria , Jiménez-Saiz Rodrigo , Carrasco Yolanda R.
Published: January 2025

To mount a robust T-dependent immune response, antigen-specific B lymphocytes require CD40 stimulation through immune synapse formation with CD4+T follicular helper cells. CD40 triggers the activation of mammalian target of rapamycin complex-1 (mTORC1) and remodels the mitochondria to meet increased bioenergetic and anabolic demands. We show that diacylglycerol-kinase-ζ (DGKζ) has a crucial role in activating the mTORC1 pathway and remodeling mitochondria downstream of CD40 signaling in B cells. DGKζ governs organelle translocation to the CD40-mediated immune synapse and the recruitment of mTORC1 to lysosomes. DGKζ−/−B cells exhibited impaired mitochondria function, protein biosynthesis, metabolite transporter expression and cell cycle progression, accompanied by dysregulation of the transcriptional network governing B cell fate. These defects lead to a blockage in the progression of the germinal center response and plasma cell differentiationin vivo. Our findings establish DGKζ as a key mediator of CD40 functions in the B cell response.
Joint single-cell measurements of surface proteins, intracellular proteins and gene expression with icCITE-seq
Authors: Chen Kelvin Y. , Takeshima Yusuke , Zorzetto-Fernandes Andre L. , Makino Katsuhiro , Kibayashi Tatsuya , Hata Mayu , Ichiyama Kenji , Croteau Josh , Taylor Kevin , Sakaguchi Shimon
Published: January 2025

The development of single-cell RNA-sequencing assays has transformed our understanding of cellular and tissue heterogeneity, yielding significant insights into disease biology and its underlying mechanisms. In this work, we describe icCITE-seq (intracellular cellular indexing of transcriptomes and epitopes), a scalable method that simultaneously measures surface and intracellular protein levels alongside gene expression across thousands of cells. We validate the specificity of intracellular staining and demonstrate the utility of this multi-omic approach in interrogating phenotypic cellular states through targeted genetic perturbations in primary human T cells. icCITE-seq enables systematic profiling of gene expression, coupled with cytoplasmic, nuclear and PTM epitopes, providing an integrated approach towards understanding cellular identity, complexity and disease regulatory mechanisms.
CellSP: Module discovery and visualization for subcellular spatial transcriptomics data
Authors: Aggarwal Bhavay , Sinha Saurabh
Published: January 2025

Spatial transcriptomics has enabled the study of mRNA distributions within cells, a key aspect of cellular function. However, there is a dearth of tools that can identify and interpret functionally relevant spatial patterns of subcellular transcript distribution. To address this, we present CellSP, a computational framework for identifying, visualizing, and characterizing consistent subcellular spatial patterns of mRNA. CellSP introduces the concept of “gene-cell modules”, which are gene sets with non-random subcellular transcript distributions in many cells. It provides intuitive visualizations of the captured patterns and offers functional insights into each discovered module. We demonstrate that CellSP reliably identifies functionally significant modules across diverse tissues and technologies. We use the tool to discover subcellular spatial phenomena related to myelination, axonogenesis and synapse formation in the mouse brain. We find immune response-related modules that change between kidney cancer and healthy samples, and myelination-related modules specific to mouse models of Alzheimer’s Disease.
Differentiation of human T follicular helper cellsin vitrorequires co-operation between STAT3 and SMAD signaling cytokines that is restrained by IL-2
Authors: Foster William S. , Labeur-Iurman Lucía , Williams Geoffrey S. , Robinson Matthew J. , Carlesso Gianluca , Lloyd Clare M. , Harker James A.
Published: January 2025

T follicular helper cells (TFH) are a CD4 T cell subset required for effective humoral immunity; promoting antibody responses to vaccines and natural infections. The mechanisms by which human TFH differentiate are not fully understood, and given the anatomical localization to the secondary lymphoid tissues, and asynchronous nature ofin vivoTFH populations, studying TFH differentiationin vitrorepresents an attractive alternative, through which the mechanisms which govern TFH differentiation can be explored. Here we demonstrate reliable differentiation ofin vitroTFH from naïve human CD4 T cells in a STAT3 dependent fashion. Cooperation between STAT3 and SMAD signaling cytokines led to expression of the lineage defining transcriptional regulator BCL6, multiple downstream proteins required for TFH function and localization (including PD1 and CXCR5), and potent IL-21 production capacity by thesein vitroTFH. Critically we found that autocrine production of IL-2 acted as an intrinsic brake on TFH differentiationin vitro; promoting proliferation but limiting the acquisition of a TFH phenotype resembling that found in germinal centersin vivo; neutralization of IL-2 resulting in the emergence of this phenotype. Overall, we show that human TFH expansion, differentiation and function relies on careful regulation of pro- and anti-TFH cytokine signaling.
Structural Basis for the Assembly of Amyloid Fibrils by the Master Cell-Signaling Regulator Human Receptor-Interacting Protein Kinase 1
Authors: Polonio Paula , López-Alonso Jorge Pedro , Jiang Hanxing , Escobedo Fátima , Titaux Gustavo , Ubarretxena-Belandia Iban , Mompeán Miguel
Published: January 2025

Amyloid fibrils, typically associated with neurodegenerative diseases, also play critical roles as functional assemblies in biological processes. The RIP homotypic interaction motifs (RHIMs) in receptor-interacting protein kinases 1 and 3 (RIPK1 and RIPK3) are essential for necroptosis, orchestrating the formation of amyloid-like fibrils that assemble into necrosomes. These supramolecular complexes propagate cell death signals and activate effectors like MLKL. While the structures of human RIPK3 (hRIPK3) homomeric fibrils and RIPK1-RIPK3 heteromeric fibrils have been resolved, the atomic structure of human RIPK1 (hRIPK1) homomeric fibrils has remained elusive.

Here, we present a high-resolution structure of hRIPK1 RHIM-mediated amyloid fibrils, determined using an integrative approach combining cryoprobe-detected solid-state nuclear magnetic resonance spectroscopy and cryo-electron microscopy. The fibrils adopt an N-shaped amyloid fold consisting of three β-sheets stabilized by the conserved IQIG RHIM motif through hydrophobic interactions and hydrogen bonding. A key hydrogen bond between N545 and G542 closes the β2-β3 loop, resulting in denser side-chain packing compared to hRIPK3 homomeric fibrils. This structural feature likely contributes to the compact architecture of hRIPK1 fibrils, in contrast to the more relaxed S-shaped fold observed in hRIPK3.

These findings provide structural insights into how hRIPK1 homomeric fibrils nucleate hRIPK3 recruitment and fibrillization during necroptosis, offering broader perspectives on the molecular principles governing RHIM-mediated amyloid assembly and functional amyloids.
Novel Insights into T-Cell Exhaustion and Cancer Biomarkers in PDAC Using ScRNA-Seq
Authors: Saleem Muhammad Usman , Sajid Hammad Ali , Shabbir Muhammad Imran
Published: January 2025

One of the aggressive and lethal cancers, pancreatic ductal adenocarcinoma (PDAC) is characterised by poor prognosis and resistance to conventional treatments. Moreover, tumor immune microenvironment (TIME) plays a crucial role in the progression and therapeutic resistance of PDAC. It is associated with T-cells exhaustion, leading to the progressive loss of T-cell functions with impaired ability to kill tumor cells. Therefore, this study employed single cell RNA sequencing (scRNA-seq) analysis and identified upregulated genes of cancer cells in two groups (“cancer cells_vs_all-PDAC” and “cancer-PDAC_vs_all-normal”), while of T-cells, including CD8+ NKT-like cells, memory CD4+ T-cells, and naive CD4+ T-cells were identified between conditions (PDAC_vs_Normal) with their respective cell types. Subsequently, common and unique markers of cancer cells from both groups were identified, resulting in three sub-groups (common in both groups, unique to “cancer cells_vs_all-PDAC” and unique to “cancer-PDAC_vs_all-normal” group). Moreover, the top-10 enriched Reactome pathways of cancer cells (common and unique) and T-cells were identified; and the genes implicated in those pathways were selected to perform PPI analysis, eventually revealing the top-10 hub-genes of each group (common and unique) of cancer cells (GAPDH, AKT1, EGFR, CS, RHOA, TPI1, SDHA, TFRC, FASN, HIF1A, H4C6, MYC, H3C12, DDX21, USP7, RFC4, APEX1, CDK9, H2BC9, NOP2, FN1, COL1A1, COL1A2, COL3A1, COL5A2, COL6A1, COL5A1, BGN, COL6A2,andFBN1) and T-cells (ACTB, AKT1, CD4, CTNNB1, FN1, H3-3B, HIF1A, HSP90AA1, HSP90AB1, HSPA8, IFNG, ITGB1, JUN, MAPK3, MMP9, NFKB1, TP53, UBB,andUBC). Furthermore, the gene expression validation of cancer cell and T-cells hub-genes was performed using GEPIA2 and TISCH2, while overall survival analysis of cancer cell hub-genes was performed using GEPIA2. Conclusively, this study unravelled a total of 16 novel markers (CS, H4C6, H3C12, H2BC9, TP53, FN1, MMP9, ITGB1, HSP90AB1, AKT1, ACTB, MAPK3, UBC, UBB, CTNNB1, and H3-3B) of cancer and T-cells, indicating that these might be implicated in the pathways leading to T-cell exhaustion, rendering growth, proliferation, and survival of cancer cells. This study provides the groundwork for future research into the immune landscape of PDAC, particularly T-cell exhaustion. However, further clinical studies are needed to validate these novel markers as potential therapeutic targets in PDAC patients.
Where were the Caribbean mangroves during the Last Glacial Maximum? A microtopographical approach
Authors: Rull Valentí
Published: January 2025

During the Last Glacial Maximum (LGM), when global sea levels dropped by ∼132 m, the Caribbean continental shelf was fully exposed, which drastically reduced the flat topographical habitat necessary for mangrove growth. It has been proposed that mangroves survived in flat microsites beyond the shelf break and later expanded from these microrefugia to their current distribution after the LGM. However, this hypothesis remains untested. This study aims to identify potential refugia by locating flat areas around the −132 m isobath using Global Multiresolution Topography (GMRT) images. A significant ∼200-km-long potential refugium was identified on the northern Trinidad (NT) shelf, along with several scattered kilometer-scale microrefugia near the Cariaco Basin (CB) in northeastern Venezuela. Additionally, two isolated prospective microrefugia were detected in northern Colombia (NC) and western Hispaniola (WH). The remaining LGM Caribbean coasts were considered unsuitable for mangrove growth. The NT refugium, along with the CB microrefugia, may have served as the primary sources for subsequent mangrove expansion. This expansion was likely facilitated by postglacial sea-level rise and the SE-NE Caribbean Current (CC), which would have acted as a major agent for propagule dispersal. This microtopographical survey not only supports the microrefugial hypothesis but also narrows the focus to the most promising areas, significantly reducing the time, effort and resources required for future seismic and coring campaigns.
Asparagine endopeptidase promotes radioresistance in breast cancer through ATR pathway modulation
Authors: Morillo-Huesca Macarena , Tomé Mercedes , Watts Colin , Huertas Pablo , Moreno-Bueno Gema , Durán Raúl V. , Martinez-Fabregas Jonathan
Published: January 2025

Background

Tumor resistance represents a major challenge in the current oncology landscape. Asparagine endopeptidase (AEP) overexpression correlates with worse prognosis and reduced overall survival in most human solid tumors. However, the underlying mechanisms of the connection between AEP and reduced overall survival in cancer patients remain unclear.

Methods

High-throughput proteomics, cellular and molecular biology approaches and clinical data from breast cancer (BC) patients were used to identify novel, biologically relevant AEP targets. Immunoblotting and qPCR analyses were used to quantify mRNA and protein levels. Flow cytometry, confocal microscopy, chemical inhibitors, siRNA- and shRNA-silencing and DNA repair assays were used as functional assays. In-silico analyses using the TCGA BC dataset and immunofluorescence assays in an independent cohort of invasive ductal (ID) BC patients were used to validate the clinical relevance of our findings.

Results

Here we showed a dual role for AEP in genomic stability and radiotherapy resistance in BC patients by suppressing ATR and PPP1R10 levels. Reduced ATR and PPP1R10 levels were found in BC patients expressing high AEP levels and correlated with worst prognosis. Mechanistically, AEP suppresses ATR levels, reducing DNA damage-induced cell death, and PPP1R10 levels, promoting Chek1/P53 cell cycle checkpoint activation, allowing BC cells to efficiently repair DNA. Functional studies revealed AEP-deficiency results in genomic instability, increased DNA damage signaling, reduced Chek1/P53 activation, impaired DNA repair and cell death, with phosphatase inhibitors restoring the DNA damage response in AEP-deficient BC cells. Furthermore, AEP inhibition sensitized BC cells to the chemotherapeutic reagents cisplatin and etoposide. Immunofluorescence assays in an independent cohort of IDBC patients showed increased AEP levels in ductal cells. These analyses showed that higher AEP levels in radioresistant IDBC patients result in ATR nuclear eviction, revealing AEPhigh/ATRlowprotein levels as an efficient predictive biomarker for the stratification of radioresistant patients.

Conclusion

The newly identified AEP/ATR/PPP1R10 axis plays a dual role in genomic stability and radiotherapy resistance in BC. Our work provides new clues to the underlying mechanisms of tumor resistance and strong evidence validating the AEP/ATR axis as a novel predictive biomarker and therapeutic target for the stratification and treatment of radioresistant BC patients.
EWS-FLI1 Expression in Human Embryonic MSCs Leads to Transcriptional Reprograming, Defective DNA Damage Repair and Ewing Sarcoma
Authors: Hernández-Muñoz Inmaculada , Cuervas Irene , Prada Estela , Pulecio Julian , Gimeno Ramón , Andrades Evelyn , Gómez-González Soledad , Berenguer-Molins Pau , Acedo-Terrades Ariadna , Perera-Bel Júlia , Bódalo Marta , Nonell Lara , Pérez Elena , Grases Daniela , Mata Caterina , Yélamos José , Richaud Yvonne , Vidal Enrique , Cuartero Yasmina , Le Dily François , Suñol Mariona , Manzanares Alejandro , Raya Angel , Mora Jaume
Published: January 2025

Ewing sarcoma (ES) is an aggressive bone and soft tissue neoplasm characterized byEWSR1/ETSrearrangements and whose cellular origin remains unclear. EWS-FLI1 expression in human pediatric mesenchymal stem cells (hpMSCs) induces a quantitatively and qualitatively different transcriptional response than its expression in human adult MSCs (haMSCs), but fails to form tumorsin vivo. ES cells have early developmental lineage signatures distinct from postnatal MSCs. Here, we have generated MSCs from experimental teratomas out of human embryonic stem cells (heSCs). Transduction of these human embryonic mesenchymal stem cells (heMSCs) with EWS-FLI1 results in the acquisition of an ES transcriptome, although the oncogene does not preferentially bind to promoters, but to intronic and intergenic microsatellites with >10 CA dinucleotides and GGAA repeats, respectively. In heMSCs, EWS-FLI1 directly regulates BRCA1 expression, although EWS-FLI1-expressing cells show defects in DNA damage repair. Xenografting of EWS-FLI1-transduced heMSCs resulted in the formation of tumors expressing characteristic ES markers. In summary, EWS-FLI1 enforces an aberrant transcriptome and endowsin vivotransforming capacity when expressed in an undifferentiated early heMSC. Our approach represents an innovative experimental method for understanding critical aspects of the biology of developmental tumors, from leukemia to sarcomas, in which few (even single) genetic alterations are able to transform a fetal stem cell.
Reactivation of developmentally silenced globin genes through genomic deletions reveals that enhancer distance matters
Authors: Felder Anna-Karina , Tjalsma Sjoerd J.D. , Verhagen Han J.M.P. , Majied Rezin , Verstegen Marjon J.A.M. , Verheul Thijs C.J. , Mohnani Rebecca , Gremmen Richard , Krijger Peter H.L. , Philipsen Sjaak , van den Akker Emile , de Laat Wouter
Published: January 2025

The human genome contains regulatory DNA elements, enhancers, that can activate gene transcription over long chromosomal distances. Here, we show that enhancer distance can be critical for gene silencing. We demonstrate that linear recruitment of the normally distalHBBsuper-enhancer to developmentally silencedHBGpromoters, through deletion or inversion of intervening DNA sequences, results in strongly reactivatedHBGexpression in adult erythroid cells andex vivodifferentiated hematopoietic stem and progenitor cells. A similar observation is made in theHBAlocus, where deletion-to-recruit of the distal enhancer strongly reactivates embryonicHBZexpression. Overall, our work assigns function to seemingly non-regulatory genomic segments: by providing linear separation they may support genes to autonomously control their transcriptional response to distal enhancers.
Cortical Response to Acute Implantation of the Utah Optrode Array in Macaque Cortex
Authors: Villamarin-Ortiz Adrián , Reiche Christopher F. , Federer Frederick , Clark Andrew M. , Rolston John D. , Soto-Sánchez Cristina , Fernandez Eduardo , Blair Steve , Angelucci Alessandra
Published: January 2025

Optogenetics has transformed the study of neural circuit function, but limitations in its application to species with large brains, such as non-human primates (NHPs), remain. A major challenge in NHP optogenetics is delivering light to sufficiently large volumes of deep neural tissue with high spatiotemporal precision, without simultaneously affecting superficial tissue. To overcome these limitations, we recently developed and testedin vivoin NHP cortex, the Utah Optrode Array (UOA). This is a 10×10 array of penetrating glass shanks, tiling a 4×4mm2area, bonded to interleaved needle-aligned and interstitial µLED arrays, which allows for independent photostimulation of deep and superficial brain tissue. Here, we investigate the acute biological response to UOA implantation in NHP cortex, with the goal of optimizing device design for reduced insertion trauma and subsequent chronic response. To this goal, we systematically vary UOA shank diameter, surface texture, tip geometry, and insertion pressure, and assess their effects on astrocytes, microglia, and neuronal viability, following acute implantation. We find that UOAs with shanks of smaller diameter, smooth surface texture and round tips cause the least damage. Higher insertion pressures have limited effects on the inflammatory response, but lead to greater tissue compression. Our results highlight the importance of balancing shank diameter, tip geometry, and insertion pressure in UOA design for preserving tissue integrity and improving long-term UOA performance and biocompatibility.
Conservation of symmetry breaking at the level of chromatin accessibility between fly species with unrelated anterior determinants
Authors: Amiri Ezra E. , Tenger-Trolander Ayse , Li Muzi , Julian Alexander Thomas , Kasan Koray , Sanders Sheri A. , Blythe Shelby , Schmidt-Ott Urs
Published: January 2025

Establishing the anterior-posterior body axis is a fundamental process during embryogenesis, and the fruit fly,Drosophila melanogaster, provides one of the best-known case studies of this process. In Drosophila, localized mRNA ofbicoidserves as anterior determinant (AD). Bicoid engages in a concentration-dependent competition with nucleosomes and initiates symmetry-breaking along the AP axis by promoting chromatin accessibility at the loci of transcription factor (TF) genes that are expressed in the anterior of the embryo. However, ADs of other fly species are unrelated and structurally distinct, and little is known about how they function. We addressed this question in the moth fly,Clogmia albipunctata, in which a maternally expressed transcript isoform of the pair-rule segmentation geneodd-pairedis localized in the anterior egg and has been co-opted as AD. We provide ade novoassembly and annotation of the Clogmia genome and describe how knockdown ofzeldaand maternalodd-pairedtranscript affect chromatin accessibility and expression of TF-encoding loci. The results of these experiments suggest direct roles of Cal-Zld in opening and closing chromatin during nuclear cleavage cycles and show that Clogmia’s maternalodd-pairedactivity promotes chromatin accessibility and anterior expression during the early phase of zygotic genome activation at Clogmia’shomeobrainandsloppy-pairedloci. We conclude that unrelated dipteran ADs initiate anterior-posterior axis-specification at the level of enhancer accessibility and thathomeobrainandsloppy-pairedhomologs may serve a more widely conserved role in the initiation of anterior pattern formation given their early anterior expression and function in head development in other insects.
ECM Signatures Reveal Quiescent Stem Cell Diversity in the Colonic Niche
Authors: Hickey Séamus E. , Andreatta Massimo , Enright Christina , Boucrot Emmanuel , Kiely Patrick , Cashman Siobhán B. , Carmona Saintiago J. , McGourty Kieran
Published: January 2025

Colonic stem cells have a key role in the continuous regeneration of a healthy intestinal epithelium. Despite considerable advances in single-cell omics technologies, the transcriptional heterogeneity of rare cell types such as colonic stem cells, as well as their functional states and niche-specific behaviors, remain poorly characterised. In this study, we leverage a meta-analysis of scRNA-seq and spatial transcriptomic datasets to comprehensively map the heterogeneity of colonic stem cells. We identify multiple, previously underappreciated stem cell states, including distinct quiescent subtypes marked byCDKN1A(P21),CDKN1B(P27), andCDKN1C(P57), proliferative populations defined byMKI67(Ki67) andLRIG1, and a lineage-committed intermediate subpopulation expressingMUC2. Strikingly, we find that these states can be robustly identified solely by their extracellular matrix (ECM) gene expression signatures, revealing ECM composition as a critical determinant of stem cell identity. Notably, LAMA1 expression is highly specific to the P57+ quiescent population, linking laminin-mediated microenvironments to the active maintenance of deep quiescence, consistent with our recent findings associating LAMA1 with quiescent cell survival. By applying these ECM gene signatures, we delineate discrete “micro-niches” of quiescent stem cells in healthy tissue and provide evidence that analogous states persist in the colorectal cancer (CRC) environment. Extending our approach to an unrelated tissue, the pancreas, we detect parallel quiescent cell subtypes, illustrating the broader applicability of ECM-based signatures. Taken together, our findings redefine the concept of stem cell heterogeneity in the colon, establish ECM-driven gene signatures as a powerful tool for characterizing stem cell states, and offer new perspectives on the niche-dependent regulation of both healthy and cancerous stem cell populations.
EPSD 2.0: An Updated Database of Protein Phosphorylation Sites across Eukaryotic Species
Authors: Chen Miaomiao , Gou Yujie , Lei Ming , Xiao Leming , Zhao Miaoying , Huang Xinhe , Liu Dan , Feng Zihao , Peng Di , Xue Yu
Published: January 2025

As one of the most crucial post-translational modifications (PTMs), protein phosphorylation regulates a broad range of biological processes in eukaryotes. Biocuration, integration and annotation of reported phosphorylation events will deliver a valuable resource for the community. Here, we present an updated database, the eukaryotic phosphorylation site database 2.0 (EPSD 2.0), which includes 2,769,163 experimentally identified phosphorylation sites (p-sites) in 362,707 phosphoproteins from 223 eukaryotes. From the literature, 873,718 new p-sites identified through high-throughput phosphoproteomic research were first collected, and 1,078,888 original phosphopeptides together with primary references were reserved. Then, this dataset was merged into EPSD 1.0, comprising 1,616,804 p-sites within 209,326 proteins across 68 eukaryotic organisms [1]. We also integrated 362,190 additional known p-sites from 10 public databases. After redundancy clearance, we manually re-checked each p-site and annotated 88,074 functional events for 32,762 p-sites, covering 58 types of downstream effects on phosphoproteins, and regulatory impacts on 107 biological processes. In addition, phosphoproteins and p-sites in 8 model organisms were meticulously annotated utilizing information supplied by 100 external platforms encompassing 15 areas. These areas included kinase/phosphatase, transcription regulators, three-dimensional structures, physicochemical characteristics, genomic variations, functional descriptions, protein domains, molecular interactions, drug-target associations, disease-related data, orthologs, transcript expression levels, proteomics, subcellular localization, and regulatory pathways. We expect that EPSD 2.0 will become a useful database supporting comprehensive studies on phosphorylation in eukaryotes. The EPSD 2.0 database is freely accessible online athttps://epsd.biocuckoo.cn/.
Challenges and facilitators in pathways to cancer diagnosis in Southern Africa: A qualitative study
Authors: Day S. , Arendse KD. , Scott SE. , Moyo M. , Mzeche S. , Guzha BT. , Tegama N. , Sills VA. , Ras T. , Walter FM. , Moodley J.
Published: January 2025

Objectives

To explore healthcare workers (HCW) experiences, barriers and facilitators in managing patients with symptoms of possible breast, cervical or colorectal cancer.

Design

A qualitative in-depth interview study with HCWs managing patients with breast, cervical and colorectal cancer symptoms. We also conducted workshops with a group of HCWs to check the credibility of the interview findings.

Setting

The study was conducted with staff working in primary, secondary and tertiary health facilities in the Eastern and Western Cape in South Africa (SA) and Harare and Bulawayo and their referral provinces in Zimbabwe.

Participants

HCWs with experience in managing patients with symptoms of possible breast, cervical or colorectal cancer were recruited for the study. Participants were purposively sampled, according to region, healthcare level, and job role. A total of 56 participants (26 in SA and 30 in Zimbabwe) participated in the in-depth interviews. Twenty-six (12 in SA and 14 in Zimbabwe) participated in four clinical advisory group workshops across both countries.

Results

Drawing on the Model of Pathways to Treatment, HCW perceptions of patient-level factors influencing the diagnostic interval included financial limitations, and patients’ absence and delays in attendance. Healthcare provider and system factors included: challenges with referral and feedback systems; training needs; low awareness of protocols and guidelines; inappropriate and suboptimal clinical assessments; and broader socio-economic factors and resource limitations.

Conclusion

Improving the timely diagnosis of breast, cervical, and colorectal cancer in Southern Africa necessitates targeted strategies that address both patient-related, provider and health-system delays.

Strengths and Limitations

To our knowledge, this is the first study in SA and Zimbabwe that explores healthcare workers across all levels of care perceptions of the challenges and facilitators in the pathways to breast, cervical and colorectal cancer diagnosis.

The qualitative nature of the inquiry and strong theoretical underpinning with the Model of Pathways to Treatment enabled in-depth exploration of the research question.

The study setting included both urban and rural settings that represent differences in access to cancer care and health systems.

We note that the discussion of patient level facilitators and barriers to early diagnosis are the perceptions of HCWs and not the views of the patients themselves.

By examining cancer care in two distinct countries, the study provides valuable insights into how different levels of human development index impact healthcare systems and identify unique challenges and best practices. A comparative analysis reveals important differences in health outcomes and resource allocation, which can inform targeted interventions.
The landscape of plasma proteomic links to human organ imaging
Authors: Fan Zirui , Chirinos Julio , Yang Xiaochen , Shu Juan , Li Yujue , O'Brien Joan M. , Witschey Walter , Rader Daniel J. , Gur Ruben , Zhao Bingxin
Published: January 2025

Plasma protein levels provide important insights into human disease, yet a comprehensive assessment of plasma proteomics across organs is lacking. Using large-scale multimodal data from the UK Biobank, we integrated plasma proteomics with organ imaging to map their phenotypic and genetic links, analyzing 2,923 proteins and 1,051 imaging traits across multiple organs. We uncovered 5,067 phenotypic protein-imaging associations, identifying both organ-specific and organ-shared proteomic relations, along with their enriched protein-protein interaction networks and biological pathways. By integrating external gene expression data, we observed that plasma proteins associated with the brain, liver, lung, pancreas, and spleen tended to be primarily produced in the corresponding organs, while proteins associated with the heart, body fat, and skeletal muscle were predominantly expressed in the liver. We also mapped key protein predictors of organ structures and showed the effective stratification capability of plasma protein-based prediction models. Furthermore, we identified 8,116 genetic-root putative causal links between proteins and imaging traits across multiple organs. Our study presents the most comprehensive pan-organ imaging proteomics map, bridging molecular and structural biology and offering a valuable resource to contextualize the complex roles of molecular pathways underlying plasma proteomics in organ systems.
Cell heterogeneity and fate bistability drive tissue patterning during intestinal regeneration
Authors: Schwayer C. , Barbiero S. , Brückner D. B. , Baader C. , Repina N. A. , Diaz O. E. , Meylan L. Challet , Kalck V. , Suppinger S. , Yang Q. , Schnabl J. , Kilik U. , Camp J. G. , Stockinger B. , Bühler M. , Stadler M. B. , Hannezo E. , Liberali P.
Published: January 2025

Tissue regeneration relies on the ability of cells to undergode novopatterning. While tissue patterning has been viewed as the transition from initially identical un-patterned cells to an arrangement of different cell types, recent evidence suggests that initial heterogeneities between cells modulate tissue-scale pattern formation. Yet, how such heterogeneities arise and, thereafter, regulate cell type emergence in a population of cells is poorly understood. Usingin vivoandin vitromouse regenerative systems, we identify a critical tissue density that is required to induce heterogeneous inactivation of the mechanosensor YAP1. Experimental and biophysical approaches demonstrate that YAP1 cell-to-cell heterogeneity pre-patterns the first cell fate decision, via both chromatin remodelling and a supracellular feedback between FOXA1 and Delta-Notch signalling. This feedback motif induces cell fate bistability endowing memory to the system and the maintenance of patterns during homeostasis. These findings reveal a generalisable framework in which transient cell-to-cell heterogeneity, regulated by tissue-scale properties, serves as a critical control parameter for the emergence of cell fate and stable patterning during regeneration.
High resolution multi-scale profiling of embryonic germ cell-like cells derivation reveals pluripotent state transitions in humans
Authors: Stucchi Sarah , Sepulveda-Rincon Lessly P. , Dion Camille , Matassa Gaja , Valenti Alessia , Cheroni Cristina , Vitriolo Alessandro , Prazzoli Filippo , Young George , Rigoli Marco Tullio , Ciprietti Martina , Muda Benedetta , Heckhausen Zoe , Hajkova Petra , Caporale Nicolò , Testa Giuseppe , Leitch Harry G.
Published: January 2025

Primordial germ cells (PGCs) are the embryonic precursors of the gametes. In mice and rats, PGCs can readily acquire pluripotency in vitro by forming embryonic germ cells (EGCs). To date, a comparable in vitro system has not been established in humans, despite the fact that human PGCs (hPGCs) readily undergo pluripotent conversion in the context of germ cell tumorigenesis. Here we report that hPGC-like cells (hPGCLCs) undergo conversion to human embryonic germ-like cells (hEGCLCs) upon exposure to the same inductive signals previously used to derive mouse EGCs. This defined, feeder-free culture system allows efficient derivation of human EGCLCs which can be expanded and maintained in standard human pluripotent stem cell medium. hEGCLCs are transcriptionally similar to human pluripotent stem cells (hPSCs) and can differentiate into all three germ layers, as well as giving rise to PGCLCs once more - demonstrating the interconvertibility of pluripotent states. This is also evident at the epigenetic level, as the initial DNA demethylation that occurs in hPGCLCs is largely reversed in hEGCLCs, restoring DNA methylation to the level observed in hPSCs. This new in vitro model captures the transition from the pluripotent stem cell state to a germ cell identity and back again, and therefore represents a highly tractable system to study pluripotent and epigenetic transitions, including those which occur during human germ cell tumorigenesis.

Graphical abstract

In brief

We report the first fully defined system to efficiently convert hPGCLCs to a pluripotent stem cell (PSC) state. We tracked pluripotent state transitions by multi-omic analysis and provided a high-resolution map of the transcriptional and epigenomic transitions upon entry to and exit from the human germline.

Highlights

Efficient derivation of hEGCLC in fully defined feeder-free conditions

Single-cell transcriptomic profiling of transitions from the hPSC state to hPGCLCs and back.

Longitudinal DNA methylation profiling highlights the overall reversibility of epigenetic states

Multi-omic gene regulatory network analysis identifies key regulators of pluripotent transitions
A highly stable engineered disulfide bond in the dimer interface ofE. coliorotate phosphoribosyl transferase
Authors: Kurauskas Vilius , Johansson Kristoffer E. , Willemoës Martin , Winther Jakob R.
Published: January 2025

Structural disulfides are very rarely found in cytoplasmic proteins of mesophilic organisms. Nevertheless, disulfides can form in the cytosol if these are stabilized sufficiently by the supporting protein structure. To investigate the redox properties of structural disulfide bonds, we introduced disulfide bonds into the cytosolic enzyme fromE. coli, orotate phosphoribosyl transferase (OPRT). Because OPRT is a homodimer the introduction of opposing cysteine residues (R44C and D92C) into separate monomers of the enzyme meant that disulfide bond formation could easily be followed by non-reducing SDS-PAGE. Redox titration experiments in DTT buffers revealed that the designed disulfide bond was exceedingly stable with a redox potential of −314 mV, close to that of dithiothreitol (DTT). This interchain disulfide bond improved global thermostability by 5.9°C relative to wild type protein and 21.4°C above its reduced form. The catalytic activity of the enzyme in either its thiol or disulfide state was not substantially affected, indicating structural integrity. Combining an inactive subunit with the asymmetric nature of disulfide bond allowed determination of rates of subunit rearrangement and disulfide formation. The R44C/D92C double mutant resulted in the formation of a symmetric homodimer with two interchain disulfides. The redox potential of this dimer variant was further decreased to −321 mV. However, the global stability of the two-disulfide dimer was not substantially increased relative to the dimer with the single disulfide bond, suggesting that the stabilizing effect was gained by preventing dimer dissociation. Finally, disulfide bond exchange is possible by thioredoxin. With a redox potential similar to that of the eukaryotic cytosol, these mutant variants may provide a new tool for cytosolic redox sensingin vivo, possibly targeting the thioredoxin system.
Perdurant TTC21B protein in the early mouse embryo is required for proper forebrain neural progenitor proliferation
Authors: Niewoehner Rebekah , Paulding David , Leal Jesus , Stottmann Rolf W.
Published: January 2025

Primary cilia play a pivotal role in cellular signaling and development and disruptions in ciliary form and/or function leads to human ciliopathies. Here, we examine the role ofTtc21b, a key component of the intraflagellar transport-A complex, in mouse forebrain development using aTtc21baliennull allele. Our findings reveal significant microcephaly in homozygous mutants is caused by disrupted neural progenitor proliferation and differentiation. Histological and immunohistochemical analyses show an enlarged ventricular zone and reduced cortical plate thickness, accompanied by altered mitotic spindle angles, suggesting defects in symmetric versus asymmetric cell divisions. Despite lowTtc21bexpression in the forebrain epithelium, early embryonic expression patterns imply that perdurant TTC21B protein may underlie these phenotypes. Progenitor proliferation kinetics were disrupted, with fewer cells re-entering the cell cycle, correlating with reduced TBR2-positive intermediate progenitors and altered neurogenesis dynamics. Neuronal processes in the cortical plate were significantly shortened, suggesting cytoskeletal defects specific to terminal differentiation stages. Our findings support a model where earlyTtc21bexpression in precursors destined for the forebrain is critical for sustaining later neural progenitor proliferation and differentiation. These results advance our understanding of primary cilia in cortical development and provide a framework for exploring cytoskeletal contributions to ciliopathies.
Massively parallel metabarcoding of droplet co-cultures
Authors: Tan James Yi , Li Jessica Du , Bahr Auden , Lin Xiaoxia Nina
Published: January 2025

A predictive understanding of microbiomes is necessary to engineer them for human and environmental health. While omics data provide insight into patterns of microbial diversity and function, we lack understanding on how microbial interactions contribute to community function and emergent ecological properties. Synthetic ecology approaches seek to address this by constructing and interrogating defined co-cultures of cultured representatives. This provides phenotypic observations on how interactions change in different contexts and contribute to overall community function. However, this cannot be applied to systems where relevant isolates are not available. Here we developed Cocoa-seq (combinatorial co-cultivation and amplicon sequencing), a microfluidic workflow utilizing the high throughput nature of nanoliter-scale, water-in-oil droplets to generate and profile co-cultures generated by the stochastic co-encapsulation of cells from samples, circumventing laborious axenic isolation. The workflow multiplexes over a thousand 16S amplicon libraries from droplet co-cultures into one, with potential for even higher scalability, to enable metabarcoding of each droplet community. To validate Cocoa-seq, we compared community distributions generated from Cocoa-seq with two benchmark communities, a model synthetic bi-culture and synthetic mock communities of various compositions, to those from other measurements or expectations. Benchmarking showed qualitative agreement with fluorescence microscopy and quantitatively similar compositions and beta-diversity patterns compared to expectations. We also introduced discrete spike-in molecular standards to estimate absolute abundance, but uneven amplification due to stochastic bias prevented accurate quantification. We recommend how Cocoa-seq can be employed to identify interactions or functionally critical consortia as well as critical limitations to consider.
Phylogenetic and taxonomic insights intoBetula: low-coverage whole genome sequencing and plastome analysis with focus on the rare Ukrainian endemic speciesBetula klokoviiZaverucha
Authors: Tarieiev Andrii , Karbstein Kevin , Gailing Oliver
Published: January 2025

Betula klokoviiZaverucha is a rare endemic species of Ukraine that is still not well taxonomically studied. In the current pilot study, we performed low-coverage whole genome sequencing forB. klokovii, related species (B. pendulaRoth andB. pubescensEhrh.) and the assumed hybridB. klokovii × B. pendula,assessed the genomic structure of taxa with different mapping settings using UMAP non-linear dimension reduction algorithm, extracted and assembled whole plastomes.

Single Nucleotide Polymorphism (SNP) analysis based on low-coverage whole genome sequencing (LC-WGS) followed by UMAP visualization reveals the separation ofB. klokoviifrom the other analysed taxa. The best taxonomic resolution was achieved with reads filtered for contamination. In contrast, the best result in obtaining complete plastome assemblies was achieved via NOVOPlasty pipeline and raw reads.

The size for the eight newly assembled plastid genomes ranges between 160,535 and 160,625bp, GC content is 36,1%. We annotated 130 genes (113 unique) for all eight assemblies. In addition, we investigatedB. klokovii’srelationships with 20 other birch species and two intraspecific taxa by reconstructing plastome-based Bayesian inference and maximum likelihood phylogenies. Overall, plastome phylogeny provides a better resolution in comparison to phylogenies based on a few plastid or nuclear molecular markers. However, it could be affected by chloroplast capture, and some other factors like quality of sequencing and assembly, and is not suitable to detect hybrids when used alone.

In particular, we found thatB. klokoviiis likely a separate taxon that is closely related toB. pubescensbut morphologically and genetically distinct. The study shows that genome-wide SNP data and plastome phylogenies have a certain potential for addressing issues with specific taxa within the genusBetulaL. However, to fully leverage the potential of this approach, we suggest collecting a much larger number of birch plastome sequences to be sequenced and assembled. For a better understanding of birch phylogeny there is a need for reference-grade chromosome scale genome assemblies for polyploid species.
Mixed gain detector configurations for time-resolved X-ray solution scattering
Authors: Haubro Morten Lunn , Pon Joseph , Hart Philip Adam , Haldrup Kristoffer , van Driel Tim Brandt
Published: January 2025

X-ray detection at X-ray free-electron lasers is challenging in part due to the XFEL's extremely short and intense X-ray pulses. Experimental measurements are further complicated by the large fluctuations inherent to the self-amplified spontaneous emission process producing the X-rays. At the Linac Coherent Light Source the ePix10ka2M detector offers multiple gain modes, and auto-ranging between these, to increase the dynamic range while retaining low noise. For diffuse scattering techniques, such as time-resolved X-ray solution scattering, where the shape of the scattering pattern largely does not change between exposures, a fixed mix of different gain modes offers many of the same advantages as auto-ranging. We find that configuring individual ASICs in separate gain modes does not impact the intensity linearity of the gain response and has a limited effect on the effective dynamic range in regions with different gain mode settings while avoiding the complexities of auto-ranging. Small (<5%) non-linear gain contributions arise when pixels on the same ASIC are configured in different gain modes. We present a configuration scheme that is designed to select the optimal mixed gain configuration to minimize effects of saturation in the high-/medium-gain region, while maximizing the number of pixels with higher gain to improve the signal-to-noise ratio.
Damping autoregressive grey model and its application to the prediction of losses caused by meteorological disasters
Authors: Yan Shuli , Gong Xiaoyu , Zeng Xiangyan
Published: January 2025

Purpose

Meteorological disasters pose a significant risk to people’s lives and safety, and accurate prediction of weather-related disaster losses is crucial for bolstering disaster prevention and mitigation capabilities and for addressing the challenges posed by climate change. Based on the uncertainty of meteorological disaster sequences, the damping accumulated autoregressive GM(1,1) model (DAARGM(1,1)) is proposed.

Design/methodology/approach

Firstly, the autoregressive terms of system characteristics are added to the damping-accumulated GM(1,1) model, and the partial autocorrelation function (PACF) is used to determine the order of the autoregressive terms. In addition, the optimal damping parameters are determined by the optimization algorithm.

Findings

The properties of the model were analyzed in terms of the stability of the model solution and the error of the restored value. By fitting and predicting the losses affected by meteorological disasters and comparing them with the results of four other grey models, the validity of the new model in fitting and prediction was verified.

Originality/value

The dynamic damping trend factor is introduced into the grey generation operator so that the grey model can flexibly adjust the accumulative order of the sequence. On the basis of the damping accumulated grey model, the autoregressive term of the system characteristics is introduced to take into account the influence of the previous data, which is more descriptive of the development trend of the time series itself and increases the effectiveness of the model.