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At least 19 recordsLinked to original sources
PubMed2025

Translation of the downstream ORF from bicistronic mRNAs by human cells: Impact of codon usage and splicing in the upstream ORF.

Biochemistry textbooks describe eukaryotic mRNAs as monocistronic. However, increasing evidence reveals the widespread presence and translation of upstream open reading frames preceding the "main" ORF. DNA and RNA viruses infecting eukaryotes often produce polycistronic mRNAs and viruses have evolved multiple ways of manipulating the host's translation machinery. Here, we introduce an experimental model to study gene expression regulation from virus-like bicistronic mRNAs in human cells. The model consists of a short upstream ORF and a reporter downstream ORF encoding a fluorescent protein. We have engineered synonymous variants of the upstream ORF to explore large parameter space, including codon usage preferences, mRNA folding features, and splicing propensity. We show that human translation machinery can translate the downstream ORF from bicistronic mRNAs, albeit reporter protein levels are thousand times lower than those from the upstream ORF. Furthermore, synonymous recoding of the upstream ORF exclusively during elongation significantly influences its own translation efficiency, reveals cryptic splice signals, and modulates the probability of downstream ORF translation. Our results are consistent with a leaky scanning mechanism facilitating downstream ORF translation from bicistronic mRNAs in human cells, offering new insights into the role of upstream ORFs in translation regulation.

Humans↗
PubMed2026

Daylight, Daily Rhythms, and Downstream Physiology: A Translational Study in Diurnal Nile Grass Rats (Arvicanthis niloticus).

The circadian system evolved under natural light-dark cycles, while modern humans spend much of their time indoors under electric lighting that differs substantially from daylight in intensity, spectral composition, and temporal structure. How such lighting environments influence the circadian system has not been systematically examined in a diurnal animal model under ecologically relevant conditions. In this study, we used the diurnal Nile grass rat (Arvicanthis niloticus) to assess daily locomotor rhythms across 4 lighting conditions designed to approximate common human exposure scenarios: rectangular daylight (D65‑R; ~5600 lux), semi‑sigmoidal daylight mimicking natural intensity dynamics (D65‑S; matched peak intensity with ~50% lower cumulative energy), fluorescent indoor light (F12; ~150 lux), and fluorescent light supplemented with a 1‑h midday daylight pulse (F12 + D65‑P). Using a within‑subject design (n = 8), male grass rats were housed under each condition for 2 weeks. D65‑R produced the highest daytime activity levels and the strongest day-night activity ratio, consistent with robust circadian entrainment. Despite matching peak intensity, D65‑S did not yield comparable circadian outcomes, indicating that cumulative photon exposure, rather than peak intensity alone, contributes to entrainment strength. Notably, the addition of a 1‑h midday daylight pulse (D65-P) partially increased circadian amplitude under otherwise fluorescent conditions, with higher periodogram amplitude relative to F12 alone. A separate cohort of males was exposed to D65-R or F12 for 6 weeks (n = 10/condition) to assess physiological outcomes, including metabolic and reproductive measures. Compared with the D65-R group, the F12 group showed a higher diabetic rate (10% vs. 40%) and reduced sperm motility (45 ± 6 vs. 19 ± 1%), consistent with potential downstream correlates of circadian rhythm disruption. Together, these findings demonstrate that lighting conditions characteristic of indoor environments produce weaker circadian organization than daylight-equivalent lighting in a diurnal rodent, which underscore the importance of light quality in shaping circadian rhythms and downstream physiological processes.

daylight↗
PubMed2024

Heat Inactivation of Nipah Virus for Downstream Single-Cell RNA Sequencing Does Not Interfere with Sample Quality.

Single-cell RNA sequencing (scRNA-seq) technologies are instrumental to improving our understanding of virus-host interactions in cell culture infection studies and complex biological systems because they allow separating the transcriptional signatures of infected versus non-infected bystander cells. A drawback of using biosafety level (BSL) 4 pathogens is that protocols are typically developed without consideration of virus inactivation during the procedure. To ensure complete inactivation of virus-containing samples for downstream analyses, an adaptation of the workflow is needed. Focusing on a commercially available microfluidic partitioning scRNA-seq platform to prepare samples for scRNA-seq, we tested various chemical and physical components of the platform for their ability to inactivate Nipah virus (NiV), a BSL-4 pathogen that belongs to the group of nonsegmented negative-sense RNA viruses. The only step of the standard protocol that led to NiV inactivation was a 5 min incubation at 85 °C. To comply with the more stringent biosafety requirements for BSL-4-derived samples, we included an additional heat step after cDNA synthesis. This step alone was sufficient to inactivate NiV-containing samples, adding to the necessary inactivation redundancy. Importantly, the additional heat step did not affect sample quality or downstream scRNA-seq results.

Nipah Virus↗
PubMed2025

KIF18A promotes chromosome congression in cooperation with CENP-E downstream of CENP-C.

Chromosome congression is a key process that acts to align chromosomes at the spindle equator via kinetochore-microtubule interactions, with defects in chromosome alignment leading to chromosomal instability. However, defining the mechanisms that underlie chromosome congression is limited due to the multiple factors that act in parallel to regulate chromosome movement. Here, we conducted a genome-wide Cas9-based functional genetics screen using a hypomorphic CENP-C mutant that affects its kinetochore interactions. Our analysis identified KIF18A, whose knockout resulted in synthetic lethality with the CENP-C mutant. Further analysis revealed that the synthetic defect was due to a reduction in CENP-E function in the CENP-C mutant. Our work suggests that KIF18A promotes chromosome alignment in cooperation with CENP-E downstream of CENP-C during early prometaphase. Thus, our analysis enables us to dissect parallel molecular mechanisms for chromosome congression and identify sensitivities and biomarkers that might guide anti-KIF18A chemotherapeutics.

Kinesins↗
PubMed2026

Identification of CD55 as a downstream factor of EP4 receptor signaling in colorectal cancer cells.

Prostaglandin E2 (PGE2) signaling through the E-type prostanoid 4 (EP4) receptor has been implicated in the pathophysiology of colorectal cancer (CRC). We herein identified decay-accelerating factor, also known as CD55, as a novel CRC-associated downstream factor of the EP4 receptor. The integration of transcriptomic profiling of PGE2-stimulated HCA-7 human colon cancer cells with analyses of cancer genomic databases predicted CD55 as a potential EP4 receptor-regulated target. Inhibitor-based experiments showed the induction of CD55 after a PGE2 stimulation required the EP4 receptor and Gi protein in HCA-7 cells, whereas protein kinase A signaling was dispensable. In combination with a toxicogenomic database analysis, p38 mitogen-activated protein kinase (MAPK) was identified as the predominant effector connecting the EP4 receptor to CD55 upregulation. A single-cell RNA-seq re-analysis of human CRC tissues revealed CD55 upregulation and p38 MAPK-related gene set enrichment in epithelial cells expressing the EP4 receptor, suggesting that this induction mechanism may operate in a subset of epithelial cells in clinical specimens. Collectively, these results delineate a PGE2/EP4 receptor/Gi protein/p38 MAPK signaling axis that induces CD55 expression in HCA-7 cells and epithelial tumor cells, provide new mechanistic clues for understanding the regulation of complement regulatory molecule CD55 expression by prostaglandin signaling.

Humans↗
PubMed2026

Global downstream BMP15 pathway analysis in human ovarian granulosa cells reveals novel genetic variations associated with primary ovarian insufficiency.

OBJECTIVES: Primary ovarian insufficiency (POI) is a fertility disorder with a well-established genetic component, but many cases still remain idiopathic. Approximately 1.5-12% of patients with POI can carry a variant in the BMP15 gene, depending on the population and the diagnostic criteria. We hypothesize that genetic variations within pathways downstream of BMP15 activity in ovarian granulosa cells (GCs) may contribute to unexplained cases of POI. The main goal of this study is to identify novel variants associated with POI in genes induced by BMP15 in GCs. STUDY DESIGN: Primary cultures of human GCs were stimulated with recombinant human BMP15. Microarray analysis profiled the BMP15-induced transcriptome in GCs. Validation was achieved by qPCR and immunoblot. Further, target exome sequencing of the differentially expressed genes was performed on 64 women with early POI onset in search of novel variants. MAIN OUTCOME MEASURES: Transcriptome profiling of human GCs stimulated with BMP15 and target exome sequencing in women with early onset of POI. RESULTS: Transcriptome analysis revealed significant upregulation of 19 genes (p&#xa0;<&#xa0;0.05). Ontology analysis of these genes converged towards two main pathways: TGF-beta signaling and regulation of stem cell pluripotency. Target exome sequencing identified six novel rare variants in five BMP15-induced genes (SAMD11, SMAD6, ID1, USP35, GPCR137C) in 9 of the 64 women with early POI (14%). CONCLUSIONS: BMP15 action in human ovarian GCs defines TGF-beta signaling and pluripotency fate in ovarian follicles. In addition, this study uncovers new potential candidate genes for the pathogenesis of POI.

Humans↗
PubMed2026

Gene regulatory mechanisms downstream of DNA methylation.

Cytosine DNA methylation is a conserved epigenetic modification that regulates gene expression, represses transposable elements and maintains genome stability across diverse eukaryotes. Although major advances have uncovered the pathways involved in the establishment, maintenance and removal of DNA methylation, the downstream mechanisms by which this mark influences transcriptional programmes and shapes chromatin structure are less well understood. Here, we review how specialized reader proteins and transcription factors interpret DNA methylation to preserve methylation patterns, recruit effector complexes, regulate chromatin accessibility and interact with parallel epigenetic systems to mediate transcriptional silencing and activation across mammals and plants. We highlight that robust transcriptional and epigenetic states emerge from overlapping, layered and partially redundant DNA methylation-dependent mechanisms. Together, these insights provide a framework for understanding how DNA methylation shapes the epigenome to regulate development, differentiation and disease progression.

Journal Article↗
PubMed2025

Nuclear basket proteins Nup2 and Mlp1 drive heat shock-induced 3D genome restructuring downstream of transcriptional activation.

The nuclear pore complex (NPC), a multisubunit complex located within the nuclear envelope, regulates RNA export and the import and export of proteins. Here we address the role of the NPC in driving thermal stress-induced 3D genome repositioning of Heat Shock Responsive (HSR) genes in budding yeast. We found that two nuclear basket proteins, Nup2 and Mlp1, although dispensable for NPC integrity, are required for driving HSR genes into coalesced chromatin clusters, consistent with their strong, heat shock-dependent recruitment to HSR gene regulatory and coding regions. HSR gene clustering occurs predominantly within the nucleoplasm and is independent of the essential scaffold-associated proteins Nup1 and Nup145. Notably, acute double depletion of Nup2 and Mlp1 has little effect on the formation of Heat Shock Factor 1 (Hsf1)-containing transcriptional condensates, Hsf1 and Pol II recruitment to HSR genes, or HSR mRNA abundance. Our results define a 3D genome restructuring role for nuclear basket proteins extrinsic to the NPC and downstream of HSR gene activation.

3D genome architecture↗
PubMed2026

Comparative Transcriptomics Reveals Shared Downstream Pathways in Craniofacial Pathology.

Treacher Collins syndrome and Nager syndrome are craniofacial developmental disorders caused by defects in ribosome biogenesis and RNA splicing, respectively, yet they exhibit overlapping abnormalities affecting neural crest cell-derived craniofacial structures. To investigate shared downstream pathogenic mechanisms, we performed a comparative transcriptomic analysis of zebrafish polr1c and sf3b4 mutant models from our previous studies. Comparative analysis identified 17 shared differentially expressed genes (DEGs) between polr1c and sf3b4 mutants, with the majority of shared genes dysregulated in the same direction, indicating a coordinated rather than random transcriptional response. Gene ontology analysis identified ATP-dependent protein folding chaperone activity as the only shared molecular function, driven in part by upregulation of hsp90aa1.2, indicating a common proteostasis response. Because chaperone activity is linked to extracellular matrix (ECM) protein processing, we cross-referenced DEGs from both mutants against the curated zebrafish matrisome. Three of the 17 shared DEGs (serpinh1b, il11a, and lepa) were matrisome-associated and upregulated in both mutants. Serpinh1b, a collagen-specific chaperone, was strongly expressed in craniofacial cartilage and mesenchymal populations during pharyngeal arch development and exhibited nearly identical fold changes in both mutants. Il11a is of particular interest because its receptor, IL11RA, is known to be associated with human craniosynostosis, suggesting potential relevance to craniofacial development. Together, it is possible to hypothesize that shared chaperone-associated transcriptional changes, together with altered ECM-related gene expression, may contribute to polr1c- and sf3b4-associated craniofacial disorders, warranting further functional validation.

Extracellular Matrix↗
PubMed1979

Hemodynamic responses of dog lung lobe to lobar venous occlusion.

We perfused the left lower lobe of the dog lung with constant flow. When the lobar venous outflow was occluded, the lobar venous pressure rose suddenly to a level somewhere below the arterial pressure, and then the arterial and venous pressures began to rise more slowly. A possible explanation for this response is that, when the outflow was occluded, flow through some downstream segment of the bed ceased. Because flow into the lung continued, the arteriovenous pressure difference after occlusion represents the pressure drop across some upstream segment through which the flow continued. We designated the arteriovenous pressure difference just after outflow occlusion as the upstream pressure drop. The arteriovenous pressure difference before occlusion minus the upstream pressure drop was designated the downstream pressure drop. In an attempt to better understand the meaning of the upstream and downstream pressure drops, we examined the influence of pulmonary vasoconstriction and flow direction on the size of the upstream and downstream pressure drops. We also compared these pressure drops with the pressure drops occurring upstream and downstream from the midpoint of the lobar vascular volume, using the low-viscosity bolus technique. The results indicate that changes in the upstream and downstream pressure drops, as evaluated by outflow occlusion, reflect changes in the lobar arterial and venous resistances.

Animals↗
PubMed2026

scGPA: an LLM-assisted workflow for directional virtual gene perturbation analysis from single-cell transcriptomes.

BACKGROUND: Existing virtual perturbation methods can often infer directional changes by comparing predicted post-perturbation expression profiles with control cells. However, workflows that directly return direction-specific downstream candidate genes together with confidence scores, evidence support and interpretable summaries remain limited. We developed scGPA, an LLM-assisted workflow system for directional single-cell virtual gene perturbation analysis. METHODS: scGPA starts from raw single-cell RNA sequencing data and performs quality control, normalization, dimensionality reduction, clustering and cell-group selection. It then constructs cell-group-specific wild-type regulatory networks using repeated subsampling, principal component regression (PCR)/Ridge-based network inference and CP tensor denoising. Based on these networks, scGPA simulates dose-aware virtual knockdown of the target gene and applies signed perturbation propagation to estimate the magnitude and direction of downstream transcriptional responses. LLM assistance is used for marker-based cell-type annotation, evidence-guided candidate prioritization and user-facing biological summarization. RESULTS: We benchmarked scGPA across five public Perturb-seq datasets and compared its performance with GEARS, scGPT and a random baseline. The overall correct prediction rate of scGPA was 23.0%, exceeding those of GEARS (20.7%), scGPT (15.1%) and the random baseline (13.6%). These results indicate that scGPA achieved a higher correct prediction rate than the two comparator models and the random baseline. We subsequently evaluated scGPA using a public osteosarcoma single-cell dataset and performed qRT-PCR validation in 143B osteosarcoma cells. Among genes with significant experimental changes, scGPA achieved a directional concordance of 76.9%. When all tested downstream genes were counted, 37.0% were directionally correct, 51.9% showed no significant change and 11.1% changed in the opposite direction. CONCLUSIONS: scGPA provides a practical workflow system for predicting and prioritizing direction-specific downstream transcriptional responses after target-gene perturbation. By integrating single-cell regulatory network inference, signed virtual perturbation and LLM-assisted interpretation, scGPA supports target-gene function inference and downstream mechanistic investigation from single-cell transcriptomic data.

Single-Cell Gene Expression Analysis↗
PubMed2026

Small Copy Number Neutral Intrachromosomal Translocation of PAX6 and Aniridia.

IMPORTANCE: Approximately 5% to 10% of individuals with classic aniridia do not receive a molecular diagnosis after clinical testing for variants in PAX6 and its downstream regulatory region. OBJECTIVE: To apply optical genome mapping (OGM) and long-read whole-genome sequencing (lrWGS) to diagnose an individual with unexplained classic aniridia. DESIGN, SETTING, AND PARTICIPANTS: High-quality DNA was extracted from the blood of a 16-year-old male patient with classic aniridia and prior negative clinical test results that included sequencing and copy number analysis of PAX6 exons and downstream regulatory region as well as genomic analysis via short-read whole-genome sequencing (srWGS) and analyzed using OGM and lrWGS. All analyses were performed in a research laboratory in Wisconsin from January 2019 to September 2025. INTERVENTIONS: OGM and lrWGS. MAIN OUTCOMES AND MEASURES: Identification of a structural variant disrupting PAX6 expression in an individual with classic aniridia, following negative prior testing including srWGS. RESULTS: OGM identified a 55-kb deletion on 11p13 encompassing all PAX6 exons and exon 12 of ELP4, with insertion of this segment into 11q21. lrWGS delineated the exact breakpoints, confirming that the downstream regulatory region, required for normal PAX6 expression, remained at the 11p13 locus. Consequently, the translocated copy of PAX6 at 11q21 is expected to lack expression due to the loss of its essential regulatory elements. CONCLUSIONS AND RELEVANCE: These findings in an individual with classic aniridia harboring an intrachromosomal rearrangement at the PAX6 locus identified by OGM and lrWGS may represent the smallest reported structural variant to separate the PAX6 coding sequence from its downstream regulatory region. This structural variant may have fallen below the detection threshold of srWGS due to its balanced nature and small size, suggesting OGM and lrWGS would be needed for definitive identification.

Aniridia↗
PubMed2026

Pesticide occurrence, transformation, and transport from wastewater treatment plants into stream networks with diverse land uses.

Neonicotinoid insecticides and strobilurin fungicides are detected in many environmental compartments and have been associated with negative environmental and human health implications. Wastewater treatment plants (WWTPs) are often hotspots for introducing such contaminants into the environment. Therefore, the occurrence of strobilurin fungicides, neonicotinoids, and their metabolites at two WWTPs with varying land uses and population sizes was investigated. Polar organic chemical integrative samplers were deployed in WWTP influent and effluent and placed upstream and downstream of the effluent mixing zone for 2 weeks in April and July 2022. Biosolids were also collected at each time point. Neonicotinoids were detected with the highest frequency (68%), followed by strobilurin fungicides (49%) and neonicotinoid metabolites (31%). Time-weighted average concentrations for influent/effluent ranged from 85.2&#xa0;&#xb1;&#xa0;87.8 to 409.4&#xa0;&#xb1;&#xa0;74.5&#xa0;ng/L. Pesticide concentrations, specifically the metabolites, typically increased from influent to effluent, resulting in effluent having higher pesticide loads than influent. Pesticide concentrations varied between the upstream and downstream monitoring locations by analyte, with WWTP samples in the highly developed region having significantly higher concentrations of pesticides and less variation by monitoring period. Chronic ecotoxicity benchmarks for freshwater invertebrates for imidacloprid were surpassed in treated effluent at both WWTPs in July and in the downstream monitoring location in the heavily developed area. Findings support the need for further exploration of pesticide contributions from WWTPs to river systems, specifically related to metabolite contributions to downstream streams and their effects on aquatic environments.

Water Pollutants, Chemical↗
PubMed2022

WNK1-OSR1 Signaling Regulates Angiogenesis-Mediated Metastasis towards Developing a Combinatorial Anti-Cancer Strategy.

Lysine-deficient protein kinase-1 (WNK1) is critical for both embryonic angiogenesis and tumor-induced angiogenesis. However, the downstream effectors of WNK1 during these processes remain ambiguous. In this study, we identified that oxidative stress responsive 1b (osr1b) is upregulated in endothelial cells in both embryonic and tumor-induced angiogenesis in zebrafish, accompanied by downregulation of protein phosphatase 2A (pp2a) subunit ppp2r1bb. In addition, wnk1a and osr1b are upregulated in two liver cancer transgenic fish models: [tert x p53-/-] and [HBx,src,p53-/-,RPIA], while ppp2r1bb is downregulated in [tert x p53-/-]. Furthermore, using HUVEC endothelial cells co-cultured with HepG2 hepatoma cells, we confirmed that WNK1 plays a critical role in the induction of hepatoma cell migration in both endothelial cells and hepatoma cells. Moreover, overexpression of OSR1 can rescue the reduced cell migration caused by shWNK1 knockdown in HUVEC cells, indicating OSR1 is downstream of WNK1 in endothelial cells promoting hepatoma cell migration. Overexpression of PPP2R1A can rescue the increased cell migration caused by WNK1 overexpression in HepG2, indicating that PPP2R1A is a downstream effector in hepatoma. The combinatorial treatment with WNK1 inhibitor (WNK463) and OSR1 inhibitor (Rafoxanide) plus oligo-fucoidan via oral gavage to feed [HBx,src,p53-/-,RPIA] transgenic fish exhibits much more significant anticancer efficacy than Regorafenib for advanced HCC. Importantly, oligo-fucoidan can reduce the cell senescence marker-IL-1&#x3b2; expression. Furthermore, oligo-fucoidan reduces the increased cell senescence-associated &#x3b2;-galactosidase activity in tert transgenic fish treated with WNK1-OSR1 inhibitors. Our results reveal the WNK1-OSR1-PPP2R1A axis plays a critical role in both endothelial and hepatoma cells during tumor-induced angiogenesis promoting cancer cell migration. By in vitro and in vivo experiments, we further uncover the molecular mechanisms of WNK1 and its downstream effectors during tumor-induced angiogenesis. Targeting WNK1-OSR1-mediated anti-angiogenesis and anti-cancer activity, the undesired inflammation response caused by inhibiting WNK1-OSR1 can be attenuated by the combination therapy with oligo-fucoidan and may improve the efficacy.

Animals↗