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Gene expression studies are widely used in medical, biological, and pharmaceutical research. Obtaining high-quality RNA from tissues is a prerequisite for high-quality data that should accurately represent gene expression levels in-vivo. The main source of technical bias, which could affect the results from transcriptomic studies, is variation in RNA quality. In this regard, tissue preparation is critical: different disruption techniques can affect RNA quality, influencing further applications. Mechanical disaggregation is a common, inexpensive, and simple method to obtain a high cell yield, demonstrated to efficiently disrupt the extracellular matrix and release single cells. However, its efficacy is operator-dependent, leading to poorly reproducible results. A fast, reproducible, and standardized technique could undoubtedly overcome this problem, avoiding wasting time and resources. In this study, our goal was to evaluate the impact of two mechanical tissue disruption techniques on the purity and quality of RNA extracted from fresh lung biopsies. The samples were processed in parallel using manual mechanical disaggregation or an automated mechanical device. The results showed that samples processed with the automated device had a higher integrity compared to those processed manually with a median Fragmentation Index of 0.86 and 0.71 respectively. This difference is statistically significant (p = 0.0084). Overall, our results indicated that the use of automatic mechanical disaggregation could undoubtedly help to overcome the technical biases related to fresh tissues processing. [ABSTRACT FROM AUTHOR]

“Somehow scientists still pursue the same questions, if now on higher levels of theoretical abstraction rooted in deeper layers of empirical evidence… To paraphrase an old philosophy joke, science is more like it is today than it has ever been. In other words, science remains as challenging as ever to human inquiry. And the need to communicate its progress… remains as essential now as then.” — Tom Siegfried, Science News 2021 In fact, essential questions about pain have not changed since IASP’s creation in Issaquah: what causes it and how can we treat it? Are we any closer to answering these questions, or have we just widened the gap between bench and bedside? The technology used to answer questions about pain mechanisms has certainly changed, whether the focus is on sensory neurons, spinal cord circuitry, descending controls or cortical pain processing. In this paper, we will describe how transgenics, transcriptomics, optogenetics, calcium imaging, fMRI, neuroimmunology and in silico drug development have transformed the way we examine the complexity of pain processing. But does it all, as our founders hoped, help people with pain? Are voltage-gated Na channels the new holy grail for analgesic development, is there a pain biomarker, can we completely replace opioids, will proteomic analyses identify novel targets, is there a “pain matrix,” and can it be targeted? Do the answers lie in our tangible discoveries, or in the seemingly intangible? Our founders could barely imagine what we know now, yet their questions remain. [ABSTRACT FROM AUTHOR]

Traditional risk assessment methodologies in toxicology have relied upon animal testing, despite concerns regarding interspecies consistency, reproducibility, costs, and ethics. New Approach Methodologies (NAMs), including cell culture and multi-level omics analyses, hold promise by providing mechanistic information rather than assessing organ pathology. However, NAMs face limitations, like lacking a whole organism and restricted toxicokinetic interactions. This is an inherent challenge when it comes to the use of omics data from in vitro studies for the prediction of organ toxicity in vivo. One solution in this context are comparative in vitro–in vivo studies as they allow for a more detailed assessment of the transferability of the respective NAM data. Hence, hepatotoxic and nephrotoxic pesticide active substances were tested in human cell lines and the results subsequently related to the biology underlying established effects in vivo. To this end, substances were tested in HepaRG and RPTEC/tERT1 cells at non-cytotoxic concentrations and analyzed for effects on the transcriptome and parts of the proteome using quantitative real-time PCR arrays and multiplexed microsphere-based sandwich immunoassays, respectively. Transcriptomics data were analyzed using three bioinformatics tools. Where possible, in vitro endpoints were connected to in vivo observations. Targeted protein analysis revealed various affected pathways, with generally fewer effects present in RPTEC/tERT1. The strongest transcriptional impact was observed for Chlorotoluron in HepaRG cells (increased CYP1A1 and CYP1A2 expression). A comprehensive comparison of early cellular responses with data from in vivo studies revealed that transcriptomics outperformed targeted protein analysis, correctly predicting up to 50% of in vivo effects. [ABSTRACT FROM AUTHOR]

Cyclosporine A (CsA) has shown efficacy against immunity-related diseases despite its toxicity in various organs, including the liver, emphasizing the need to elucidate its underlying hepatotoxicity mechanism. This study aimed to capture the alterations in genome-wide expression over time and the subsequent perturbations of corresponding pathways across species. Six data from humans, mice, and rats, including animal liver tissue, human liver microtissues, and two liver cell lines exposed to CsA toxic dose, were used. The microtissue exposed to CsA for 10 d was analyzed to obtain dynamically differentially expressed genes (DEGs). Single-time points data at 1, 3, 5, 7, and 28 d of different species were used to provide additional evidence. Using liver microtissue-based longitudinal design, DEGs that were consistently up- or down-regulated over time were captured, and the well-known mechanism involved in CsA toxicity was elucidated. Thirty DEGs that consistently changed in longitudinal data were also altered in 28-d rat in-house data with concordant expression. Some genes (e.g. TUBB2A, PLIN2, APOB) showed good concordance with identified DEGs in 1-d and 7-d mouse data. Pathway analysis revealed up-regulations of protein processing, asparagine N-linked glycosylation, and cargo concentration in the endoplasmic reticulum. Furthermore, the down-regulations of pathways related to biological oxidations and metabolite and lipid metabolism were elucidated. These pathways were also enriched in single-time-point data and conserved across species, implying their biological significance and generalizability. Overall, the human organoids-based longitudinal design coupled with cross-species validation provides temporal molecular change tracking, aiding mechanistic elucidation and biologically relevant biomarker discovery. [ABSTRACT FROM AUTHOR]

Cooking oil fumes (COFs) are widely acknowledged as substantial contributors to indoor air pollution, having detrimental effects on human health. Despite the existence of commercialized in vitro aerosol exposure platforms, assessment risks of aerosol pollutants are primarily evaluated based on multiwell plate experiments by trapping and redissolving aerosols to conduct comprehensive in vitro immersion exposure manner. Therefore, an innovative real-time exposure system for COF aerosol was constructed, featuring a self-designed microfluidic chip as its focal component. The chip was used to assess toxicological effects of in vitro exposure to COF aerosol on cells cultured at the gas–liquid interface. Meanwhile, we used transcriptomics to analyze genes that exhibited differential expression in cells induced by COF aerosol. The findings indicated that the MAPK signaling pathway, known for its involvement in inflammatory response and oxidative stress, played a crucial role in the biological effects induced by COF aerosol. Biomarkers associated with inflammatory response and oxidative stress exhibited corresponding alterations. Furthermore, the concentration of COF aerosol exposure and post-exposure duration exert decisive effects on these biomarkers. Thus, the study suggests that COF can induce oxidative stress and inflammatory response in BEAS-2B cells, potentially exerting a discernible impact on human health. [ABSTRACT FROM AUTHOR]

Cordyceps chanhua , an important cordycipitoid medical mushroom with wide use in Asia, has gained attention for its bioactive component beauvericin (BEA), which is of medicinal value as a drug lead, but also of food safety risk. Recent observations by our group revealed a significant decrease of BEA content in C. chanhua when exposed to light, but the underlying regulatory mechanisms remain elusive. In this study, a comprehensive approach combining metabolomics and transcriptomics was employed to investigate the effects of white light on the secondary metabolism of C. chanhua for elucidation of the influence of light on BEA biosynthesis in this fungus. The result showed that the genes and metabolites involved in the synthesis of D-hydroxyisovaleric acid, a precursor of BEA synthesis, were down-regulated under light exposure, while those associated with the synthesis of phenylalanine, another precursor of BEA synthesis, were up-regulated leading to elevated phenylalanine levels. It suggested that the suppressive effect of light on BEA synthesis in C. chanhua occurred primarily through the inhibition of D-hydroxyisovaleric acid synthesis, while the enhanced phenylalanine biosynthesis likely directed towards other metabolic pathway such as pigment synthesis. These results contributed to a better understanding on how light modulates the secondary metabolism of C. chanhua and provided valuable guidance for optimizing BEA production in cultivation practices. [Display omitted] • Light inhibits the production of beauvericin (BEA) in Cordyceps chanhua. • Light reduces BEA by suppressing D-hydroxyisovaleric acid, a precursor. • Light boosts phenylalanine, another precursor, but changes its metabolic path. • Light exposure induces oxidative stress and stimulates the consumption of BEA. [ABSTRACT FROM AUTHOR]

Summary: Effector secretion is crucial for root endophytes to establish and protect their ecological niche.We used time‐resolved transcriptomics to monitor effector gene expression dynamics in two closely related Sebacinales, Serendipita indica and Serendipita vermifera, during symbiosis with three plant species, competition with the phytopathogenic fungus Bipolaris sorokiniana, and cooperation with root‐associated bacteria.We observed increased effector gene expression in response to biotic interactions, particularly with plants, indicating their importance in host colonization. Some effectors responded to both plants and microbes, suggesting dual roles in intermicrobial competition and plant–microbe interactions. A subset of putative antimicrobial effectors, including a GH18‐CBM5 chitinase, was induced exclusively by microbes. Functional analyses of this chitinase revealed its antimicrobial and plant‐protective properties.We conclude that dynamic effector gene expression underpins the ability of Sebacinales to thrive in diverse ecological niches with a single fungal chitinase contributing substantially to niche defense. [ABSTRACT FROM AUTHOR]

To understand whether domestication had an impact on susceptibility and responsiveness to arbuscular mycorrhizal fungi (AMF) in tomato (Solanum lycopersicum), we investigated two tomato cultivars ("M82" and "Moneymaker") and a panel of wild relatives including S. neorickii, S. habrochaites and S. pennellii encompassing the whole Lycopersicon clade. Most genotypes revealed good AM colonisation levels when inoculated with the AMF Funneliformis mosseae. By contrast, both S. pennellii accessions analysed showed a very low colonisation, but with normal arbuscule morphology, and a negative response in terms of root and shoot biomass. This behaviour was independent of fungal identity and environmental conditions. Genomic and transcriptomic analyses revealed in S. pennellii the lack of genes identified within QTLs for AM colonisation, a limited transcriptional reprogramming upon mycorrhization and a differential regulation of strigolactones and AM‐related genes compared to tomato. Donor plants experiments indicated that the AMF could represent a cost for S. pennellii: F. mosseae could extensively colonise the root only when it was part of a mycorrhizal network, but a higher mycorrhization led to a higher inhibition of plant growth. These results suggest that genetics and functional traits of S. pennellii are responsible for the limited extent of AMF colonisation. Summary Statement: Contrary to our hypothesis (domesticated plants are less dependent on mycorrhization than their wild relatives), we found a very low AM colonisation and a negative growth response in Solanum pennelli. Genetic and functional traits were identified as responsible for the limited colonisation success. [ABSTRACT FROM AUTHOR]

The proper function of the placenta is essential for the health and growth of the fetus and the mother. The placenta relies on dynamic gene expression for its correct and timely development and function. Although numerous studies have identified genes vital for placental functions, equine placental molecular research has primarily focused on single placental locations, in sharp contrast with the broader approach in human studies. Here, we hypothesized that the molecular differences across different regions of the equine placenta are negligible because of its diffuse placental type with a macroscopic homogenous distribution of villi across the placental surface. We compared the transcriptome and stereological findings of the body, pregnant horn, and non-pregnant horn within the equine chorioallantois. Our transcriptomic analysis indicates that the variation between regions of the placenta within individuals is less than the variation observed between individuals. A low number of differentially expressed genes (DEGs) (n = 8) was identified when comparing pregnant and non-pregnant horns within the same placenta, suggesting a remarkable molecular uniformity. A higher number of DEGs was identified when comparing each horn to the body (193 DEGs comparing pregnant horn with body and 207 DEGs comparing non-pregnant horn with body). Genes with a higher expression in the body were associated with processes such as extracellular matrix synthesis and remodeling, which is relevant for placental maturation and placenta-endometrial separation at term and implies asynchrony of these processes across locations. The stereological analysis showed no differences in microcotyledonary density, and width between the locations. However, we observed a greater chorioallantoic thickness in the body and pregnant horn compared to the non-pregnant horn. Overall, our findings reveal a uniform transcriptomic profile across the placental horns, alongside a more distinct gene expression pattern between the uterine body and horns. These regional differences in gene expression suggest a different pace in the placental maturation and detachment among the placental locations. [Display omitted] • Gene expression variation observed across different placental locations. • Genes related to ECM degradation vary between the body and horns. • Microscopic features are uniform across the entire placenta. [ABSTRACT FROM AUTHOR]

Congenital lung malformations are fatal at birth in their severe forms. Prevention and early intervention of these birth defects require a comprehensive understanding of the molecular mechanisms of lung development. We find that the loss of inturned (Intu), a cilia and planar polarity effector gene, severely disrupts growth and branching morphogenesis of the mouse embryonic lungs. Consistent with our previous results indicating an important role for Intu in ciliogenesis and hedgehog (Hh) signaling, we find greatly reduced number of primary cilia in both the epithelial and mesenchymal tissues of the lungs. We also find significantly reduced expression of Gli1 and Ptch1 , direct targets of Hh signaling, suggesting disruption of cilia-dependent Hh signaling in Intu mutant lungs. An agonist of the Hh pathway activator, smoothened, increases Hh target gene expression and tubulogenesis in explanted wild type, but not Intu mutant, lungs, suggesting impaired Hh signaling response underlying lung morphogenetic defects in Intu mutants. Furthermore, removing both Gli2 and Intu completely abolishes branching morphogenesis of the lung, strongly supporting a mechanism by which Intu regulates lung growth and patterning through cilia-dependent Hh signaling. Moreover, a transcriptomics analysis identifies around 200 differentially expressed genes (DEGs) in Intu mutant lungs, including known Hh target genes Gli1, Ptch1/2 and Hhip. Genes involved in muscle differentiation and function are highly enriched among the DEGs, consistent with an important role of Hh signaling in airway smooth muscle differentiation. In addition, we find that the difference in gene expression between the left and right lungs diminishes in Intu mutants, suggesting an important role of Intu in asymmetrical growth and patterning of the mouse lungs. [Display omitted] • Loss of Intu leads to reduced size and abnormal branching of mouse embryonic lungs. • Cilia are reduced in Intu mutant lungs. • Hh signaling is reduced in Intu mutant lungs. • Tubulogenesis and branching morphogenesis ae disrupted in Intu mutant lung explants even with a Smoothened agonist. • Hh target genes and smooth muscle-related genes are differentially expressed in Intu mutant lungs. [ABSTRACT FROM AUTHOR]

Background: Monocyte Chemotactic Protein 1-Induced Protein 1 (MCPIP1, also called Regnase-1) is a negative modulator of inflammation with tumor-suppressive properties. Mice with keratinocyte-specific deletion of the Zc3h12a gene, encoding MCPIP1, (Mcpip1eKO mice) are more susceptible to the development of epidermal papillomas initiated by 7,12-dimethylbenz[a]-anthracene (DMBA) and promoted by 2-O-tetradecanoylphorbol-13-acetate (TPA). Methods: The aim of this study was to investigate the MCPIP1 RNase-dependent microRNA (miRNA)‒mRNA regulatory network in chemically induced squamous cell carcinoma (SCC)-like skin papillomas. Next-generation sequencing (NGS) coupled with bioinformatic analysis was used to shortlist the MCPIP1-dependent changes in protein-coding genes and miRNAs. The expression levels of the selected miRNAs were analyzed by quantitative PCR in human keratinocytes with MCPIP1 silencing. Functional studies were performed in human keratinocytes transfected with appropriate miRNA mimics. The DIANA-microT-CDS algorithm and DIANA-TarBase v7 database were used to predict potential target genes and identify the experimentally validated targets of differentially expressed (DE) miRNAs. Results: RNA sequencing (RNA-Seq) analysis of control and Mcpip1eKO DMBA/TPA-induced papillomas revealed transcriptome changes, with 2400 DE protein-coding genes and 33 DE miRNAs. The expression of miR-223-3p, miR-376c-3p, and miR-139-5p was confirmed to be dependent on MCPIP1 activity in both murine and human models. We showed that MCPIP1 directly regulates the expression of miR-376c-3p via direct cleavage of the corresponding precursor miRNA. The pro-proliferative activity of miR-223-3p, miR-376c-3p, and miR-139-5p was experimentally confirmed in SCC-like keratinocytes. Bioinformatic prediction of the mRNA targets of the DE-miRNAs revealed 416 genes as putative targets of the 18 upregulated miRNAs and 425 genes as putative targets of the 15 downregulated miRNAs. Further analyses revealed the murine interactions that are conserved in humans. Functional analysis indicated that during the development of cutaneous SCC, the most important pathways/processes mediated by the miRNA‒mRNA MCPIP1-dependent network are the regulation of inflammatory processes, epithelial cell proliferation, Wnt signaling, and miRNA transcription. Conclusions: Loss of MCPIP1 modulates the expression profiles of 33 miRNAs in chemically induced Mcpip1eKO papillomas, and these changes directly affect the miRNA‒mRNA network and the modulation of pathways and processes related to carcinogenesis. [ABSTRACT FROM AUTHOR]

This study into the response of two Ipomoea batatas (L.) Lam cultivars, Melinda and Tio Joe, to coinfection with sweet potato chlorotic stunt virus (SPCSV) and sweet potato pakakuy virus (SPPV), employed a comprehensive approach encompassing symptomatology, real‐time quantitative polymerase chain reaction, metagenomics, and transcriptomics. SPCSV is a quarantine virus with synergistic effects, which decimate yields. SPPV is the most prevalent DNA virus in sweet potato germplasm, with a tendency to persist in meristems, posing a significant risk for germplasm transfer between territories. Graft inoculation experiments revealed that while Tio Joe remained asymptomatic for 12 weeks and suppressed virus replication, Melinda displayed symptoms early on and exhibited high virus titers. Metagenomic analyses corroborated these observations and confirmed that SPCSV and SPPV were responsible. Transcriptomic analysis unveiled disparities in gene expression between Melinda and Tio Joe. Differential gene expression was heightened and altered in Melinda as the viruses disrupted its gene expression. Its defense strategies, such as inducing abscisic acid signaling, were insufficient to overcome disruptive viral effects like oxidative stress, rendering it susceptible. In contrast, Tio Joe had relatively stable differential gene expression, indicating resistance to SPPV–SPCSV coinfection. Overexpressed genes such as sirtuin, rapid alkalization factor, and nuclear pore anchor triggered quantitative resistance, supported with enriched organelles such as mitochondrion and pathways such as proteasome and cutin, suberine, and wax biosynthesis. Tio Joe maintained its genome integrity and inhibited viral replication by tightly controlling gene expression and preventing reactive oxygen species accumulation. [ABSTRACT FROM AUTHOR]

Clear-cell renal cell carcinoma (ccRCC) is the most common origin of pancreatic metastases (PM). Distinct genomic aberrations, favorable prognosis, and clinical observations on high angiogenesis, and succeeding tyrosine kinase inhibitor (TKI) sensitivity have been reported in PM-ccRCC. However, no functional or single-cell studies have been conducted thus far. We recruited five PM-ccRCC patients and investigated the genomic, single-cell transcriptomic, and drug sensitivity profiles of their patient-derived cells (PDCs). The PM depicted both expected and novel genomic alterations. Further, the transcriptomics differed from both primary and metastatic ccRCC, with upregulations of the PI3K/mTOR and – supporting the clinical observations – angiogenesis pathways. Data integration at pathway level showed that transcriptomics explained drug sensitivities the best. Accordingly, PM-ccRCC PDCs shared sensitivity to many PI3K/mTOR inhibitors. Altogether, we show distinct genomic and transcriptomic signatures in PM-ccRCC, highlight the superiority of transcriptomics in interpreting drug sensitivities, and encourage the use of TKIs and PI3K/mTOR inhibitors in PM-ccRCC. Functional precision medicine approach reveals genomic and transcriptomic aberrations that distinguish pancreatic metastases from other types of ccRCC metastases and suggest potential therapeutic targets at the individual level. [ABSTRACT FROM AUTHOR]

SUMMARY C4 species have evolved more than 60 times independently from C3 ancestors. This multiple and parallel evolution of the complex C4 trait suggests common underlying evolutionary mechanisms, which could be identified by comparative analysis of closely related C3 and C4 species. Efficient C4 function depends on a distinctive leaf anatomy that is characterised by enlarged, chloroplast‐rich bundle sheath cells and narrow vein spacing. To elucidate the molecular mechanisms that generate the Kranz anatomy, we analysed a developmental series of leaves from the C4 plant Flaveria bidentis and the closely related C3 species Flaveria robusta by comparing anatomies and transcriptomes. Vascular density measurements of all nine leaf developmental stages identified three leaf anatomical zones whose proportions vary with respect to the developmental stage. We then deconvoluted the transcriptome datasets using non‐negative matrix factorisation, which identified four distinct transcriptome patterns in the growing leaves of both species. By integrating the leaf anatomy and transcriptome data, we were able to correlate the different transcriptional profiles with different developmental zones in the leaves. These comparisons revealed an important role for auxin metabolism, in particular auxin homeostasis (conjugation and deconjugation), in establishing the high vein density typical of C4 species. [ABSTRACT FROM AUTHOR]

Human blood vessel walls show concentric layers, with the outermost tunica adventitia harboring mesenchymal progenitor cells. These progenitor cells maintain vessel homeostasis and provide a robust cell source for cell-based therapies. However, human adventitial stem cell niche has not been studied in detail. Here, using spatial and single-cell transcriptomics, we characterized the phenotype, potential, and microanatomic distribution of human perivascular progenitors. Initially, spatial transcriptomics identified heterogeneity between perivascular layers of arteries and veins and delineated the tunica adventitia into inner and outer layers. From this spatial atlas, we inferred a hierarchy of mesenchymal progenitors dictated by a more primitive cell with a high surface expression of CD201 (PROCR). When isolated from humans and mice, CD201Low expression typified a mesodermal committed subset with higher osteogenesis and less proliferation than CD201High cells, with a downstream effect on canonical Wnt signaling through DACT2. CD201Low cells also displayed high translational potential for bone tissue generation. [Display omitted] • Integrated transcriptomics delineates the topography of human blood vessels • CD201-expressing progenitors are spatially restricted to the outer adventitia • Human and mouse CD201High progenitors are primitive with higher stem-like activity • Wnt signaling inhibitor DACT2 controls the functional role of CD201 progenitors Wang et al. investigated the perivascular microenvironmental niche within human blood vessels using an integrated transcriptomic approach, including spatial sequencing. The study identified perivascular progenitor cells expressing CD201 within the outer layers of the tunica adventitia with distinct stemness and osteogenic potential. [ABSTRACT FROM AUTHOR]

Breast cancer is one of the most prevalent cancers in women globally. Its treatment and prognosis are significantly influenced by the tumor microenvironment and tumor heterogeneity. Precision therapy enhances treatment efficacy, reduces unwanted side effects, and maximizes patients' survival duration while improving their quality of life. Spatial transcriptomics is of significant importance for the precise treatment of breast cancer, playing a critical role in revealing the internal structural differences of tumors and the composition of the tumor microenvironment. It offers a novel perspective in studying the spatial structure and cell interactions within tumors, facilitating more effective personalized treatments for breast cancer. This article will summarize the latest findings in the diagnosis and treatment of breast cancer from the perspective of spatial transcriptomics, focusing on the revelation of the tumor microenvironment, identification of new therapeutic targets, enhancement of disease diagnostic accuracy, comprehension of tumor progression and metastasis, assessment of drug responses, creation of highresolution maps of tumor cells, representation of tumor heterogeneity, and support for clinical decision-making, particularly in elucidating the tumor microenvironment, tumor heterogeneity, immunotherapy and their correlation with clinical outcomes. [ABSTRACT FROM AUTHOR]

The Xishuangbanna game fowl, a local Chinese chicken breed renowned for its athleticism, is facing extinction. Despite its significance, research on its genome assembly and resequencing remains sparse, limiting our understanding of its genetic structure and function. In this study, we assembled a chromosome-level genome using second-generation sequencing and high-throughput chromosome conformation capture (Hi-C), along with transcriptome sequencing. We also resequenced 62 individuals. The resulting assembly yielded a 1.09 gigabase (Gb) high-quality genome, 994.34 Gb of resequencing data, and 141.38 Gb of transcriptome data from 15 tissues. This comprehensive dataset provides valuable insights into the artificial selection processes shaping the breed and offers a solid foundation for further genetic and population studies. [ABSTRACT FROM AUTHOR]

Introduction: The majority of patients with mycosis fungoides (MF) have an indolent disease course, but a substantial fraction (20-30%) of patients progress to advanced stages - usually with a grave prognosis. Early differentiation between indolent and aggressive types of MF is important for the choice of treatment regimen and monitoring of the individual patient. Good biomarkers are therefore desired. Methods: Here, we used spatial transcriptomics on skin samples at time-of-diagnosis to enable prediction of patients who later progressed to advanced stages of MF. Formalin-fixed, paraffin-embedded skin biopsies at time of diagnosis from six patients with MF who progressed to advanced stages of disease within 4 months to 12 years after diagnosis, and nine patients who remained in early-stage disease over 9 to 27 years were analyzed using the GeoMx Digital Spatial Profiler to capture spatially resolved high-plex RNA gene expression data. Five different regions of interest (the epidermis, the basal layer of epidermis, CD4+ T-cells and neighboring cells, and Pautrier's microabscesses) were profiled for further assessment. Results and discussion: Interestingly, RUNX2, SHMT2, and MCM7 were upregulated in the enriched population of malignant T-cells in Pautrier's microabscesses in patients who later developed advanced stages of disease. Expression of RUNX2, SHMT2 and MCM7 in malignant T-cells was confirmed in a subset of patients in MF skin using scRNA-seq datasets across multiple studies and correlating with stage of disease. Taken together, we provide first evidence that RUNX2 has potential as a biomarker to identify MF patients progressing to advanced stage disease. As RUNX2 has not previously been linked to MF, our data also shows the analytical strength of combining spatial transcriptomics with scRNA-seq analysis. [ABSTRACT FROM AUTHOR]

Semen Ziziphi Spinosae (SZS) is a traditional Chinese herbal medicine widely used to treat insomnia and anxiety in clinical practice. Currently, the demand for SZS is increasing every year, but the production of wild SZS is unstable due to environmental factors. Grafting sour jujube scions onto sour jujube or jujube tree stocks can achieve a high production rate within a short period of time. However, the effects of grafting on the quality of SZS have not been reported. This study investigated the differences between wild-type and grafted SZS from three aspects: phenotype, chemical composition, and molecular mechanism. The findings revealed that the grafted specimens were generally larger in morphology and lighter in color than the wild-type samples. The dimensions of both the grafted specimens were generally larger than those of the wild specimens. The HPLC-ELSD results revealed that the three main chemical components in the grafted SZS, namely, spinosin, jujuboside A, and jujuboside B, had higher contents than their wild-type counterparts. Comprehensive transcriptome sequencing analysis and KEGG annotation revealed that DEG enrichment between grafted and wild-type SZS occurred mainly during stress resistance and rootstock scion healing. There were 23 DEGs that may encode enzymes involved in the biosynthetic pathway of flavonoids and 21 genes encoding terpenoid saponins. Further investigation revealed that the expression of the genes C4H, CHS, CHI, and F3'5'H in the flavonoid biosynthesis pat.hway and HMGR, MVK, MVD, and FPPS in the saponin biosynthesis pathway accounted for the difference in quality between grafted and wild SZS. Furthermore, WGCNA identified 15 core genes related to medicinal ingredients between grafted and wild SZS. These results provide support for further research on the differences in the quality of medicinal ingredients between grafted and wild SZS. [ABSTRACT FROM AUTHOR]

Background: Neuroblastoma (NB) is one of the most devastating malignancies in children, accounting for a high mortality rate due to limited treatment options. This study is aimed at elucidating the role of the ferroptosis‐related EIF2S1 gene in NB pathogenesis and exploring its potential as a therapeutic target. Methods: We conducted comprehensive bioinformatics analyses utilizing the FerrDb database and NB‐related transcriptomics data to investigate the role of EIF2S1 in NB. Changes in EIF2S1 expression were subsequently validated in NB tissues and cell lines. Loss‐of‐function experiments were performed in SK‐N‐SH and IMR‐32 cell lines through shRNA‐mediated EIF2S1 knockdown. The impact of EIF2S1 knockdown on the tumorigenesis of SK‐N‐SH cells was assessed in nude mice. Results: Bioinformatics analyses revealed a significant association between elevated EIF2S1 expression and poor prognosis in NB patients. The increased levels of EIF2S1 expression were confirmed in NB tissues and cancerous cell lines. Furthermore, EIF2S1 overexpression was linked to translational regulation and immune cell infiltration modulation. Silencing of EIF2S1 resulted in the suppression of cell proliferation, migration, and tumorigenicity in NB cells. Additionally, EIF2S1 knockdown led to an accumulation of iron and oxidative stress, as well as a reduction in GPX4 and SLC7A11 expression. Conclusion: Our findings indicate that EIF2S1 appears to facilitate the progression of NB by protecting tumor cells from ferroptosis through modulating GPX4 and SLC7A11 expression. Consequently, EIF2S1 may serve as a potential therapeutic target for the management of NB. [ABSTRACT FROM AUTHOR]

Torpor encompasses diverse adaptations to extreme environmental stressors such as hibernation, aestivation, brumation, and daily torpor. Here we introduce StrokeofGenus, an analytic pipeline that identifies distinct transcriptomic states and shared gene expression patterns across studies, tissues, and species. We use StrokeofGenus to study multiple and diverse forms of torpor from publicly-available RNA-seq datasets that span eight species and two classes. We identify three transcriptionally distinct states during the cycle of heterothermia: euthermia, torpor, and interbout arousal. We also identify torpor-specific gene expression patterns that are shared both across tissues and between species with over three hundred million years of evolutionary divergence. We further demonstrate the general sharing of gene expression patterns in multiple forms of torpor, implying a common evolutionary origin for this process. Although here we apply StrokeofGenus to analysis of torpor, it can be used to interrogate any other complex physiological processes defined by transient transcriptomic states. [ABSTRACT FROM AUTHOR]

Background: Variation in snake venoms is well documented, both between and within species, with intraspecific venom variation often correlated with geographically distinct populations. The puff adder, Bitis arietans, is widely distributed across sub-Saharan Africa and into the Arabian Peninsula where it is considered a leading cause of the ~310,000 annual snakebites across the region, with its venom capable of causing substantial morbidity and mortality. Despite its medical importance and wide geographic distribution, there is little known about venom variation between different B. arietans populations and the potential implications of this variation on antivenom efficacy. Methodology: We applied a range of analyses, including venom gland transcriptomics, in vitro enzymatic assays and reverse phase chromatography to comparatively analyse B. arietans venoms originating from Nigeria, Tanzania, and South Africa. Immunological assays and in vitro enzymatic neutralisation assays were then applied to investigate the impact of venom variation on the potential efficacy of three antivenom products; SAIMR Polyvalent, EchiTAb-Plus and Fav-Afrique. Findings: Through the first comparison of venom gland transcriptomes of B. arietans from three geographically distinct regions (Nigeria, Tanzania, and South Africa), we identified substantial variation in toxin expression. Findings of venom variation were further supported by chromatographic venom profiling, and the application of enzymatic assays to quantify the activity of three pathologically relevant toxin families. However, the use of western blotting, ELISA, and in vitro enzymatic inhibition assays revealed that variation within B. arietans venom does not appear to substantially impact upon the efficacy of three African polyvalent antivenoms. Conclusions: The large distribution and medical importance of B. arietans makes this species ideal for understanding venom variation and the impact this has on therapeutic efficacy. The findings in this study highlight the likelihood for considerable venom toxin variation across the range of B. arietans, but that this may not dramatically impact upon the utility of treatment available in the region. Author summary: The puff adder (Bitis arietans) is found across sub-Saharan Africa and the Arabian Peninsula and is capable of causing life threatening pathology due to its potent venom. The extensive range of B. arietans exposes populations to different ecological pressures which may impact upon the composition of venom toxins. In this study, we examined the venom composition of B. arietans from three countries separated by large geographic distance: Nigeria, Tanzania and South Africa. By integrating venom gland transcriptomes, venom chromatography, and in vitro functional assays to profile B. arietans venom composition, we uncovered extensive variation between the three locales. Given that venom variation can have a significant impact on the efficacy of antivenom treatment, we also investigated the ability of three African antivenoms to recognise and inhibit in vitro venom activity. Through these analyses, we were able to determine that venom variation did not have a substantial impact on the neutralising effect of selected antivenoms. This study has highlighted the potentially extensive venom variation found across the range of B. arietans and initiated valuable investigations into the efficacy of African antivenoms to protect human populations vulnerable to snakebite envenoming. [ABSTRACT FROM AUTHOR]

Spatial transcriptomics is an emerging technology that enables the profiling of gene expression in tissues while preserving spatial location information. This innovative approach is anticipated to provide a comprehensive understanding of the spatial distribution of different cells within tissues and facilitate in-depth analysis of tissue structure. To accurately recognize spatial domains from spatial transcriptomics, we have introduced a generalized deep learning method called GAAEST (Graph Attention-based Autoencoder for Spatial Transcriptomics). Our proposed approach effectively integrates both spatial location information and gene expression data from spatial transcriptomics. Specifically, it leverages spatial location details to construct a neighborhood graph and employs a graph attention network-based encoder to embed gene expression information into a spatially informed space. At the same time, to further optimize the learned potential embedding, self-supervised contrastive learning is introduced to capture spatial information at three levels: local, global and contextual feature of spots. Finally, the decoder reconstructs gene expressions, which are then clustered to identify spatial domains with similar expression patterns and spatial proximity. Based on our experiments conducted on multiple datasets, GAAEST consistently outperforms existing state-of-the-art methods. The proposed GAAEST demonstrates excellent capabilities in spatial domain recognition, positioning it as an ideal tool for advancing spatial transcriptomics research. The graph attention based automatic encoder utilizing contrastive learning effectively combines spatial location information and gene expression data from spatial transcriptomics for enhanced domain recognition capacity. [ABSTRACT FROM AUTHOR]

Fluid management remains a critical challenge in the treatment of septic shock, with individualized approaches lacking. This study aims to develop a statistical model based on transcriptomics to identify subgroups of septic shock patients with varied responses to fluid strategy. The study encompasses 494 septic shock patients. A benefit score is derived from the transcriptome space, with higher values indicating greater benefits from restrictive fluid strategy. Adherence to the recommended strategy is associated with a hazard ratio of 0.82 (95% confidence interval: 0.64–0.92). When applied to the baseline hospital mortality rate of 16%, adherence to the recommended fluid strategy could potentially lower this rate to 13%. A proteomic signature comprising six proteins is developed to predict the benefit score, yielding an area under the curve of 0.802 (95% confidence interval: 0.752–0.846) in classifying patients who may benefit from a restrictive strategy. In this work, we develop a proteomic signature with potential utility in guiding fluid strategy for septic shock patients. Fluid management in septic shock lacks personalized approaches, which are critical for improving patient outcomes. Here, the authors show that a proteomic signature can help identify patients who may benefit from a restrictive fluid strategy, potentially reducing hospital mortality rates. [ABSTRACT FROM AUTHOR]

H3K27M diffuse midline gliomas (DMG), including diffuse intrinsic pontine gliomas (DIPG), exhibit cellular heterogeneity comprising less-differentiated oligodendrocyte precursors (OPC)-like stem cells and more differentiated astrocyte (AC)-like cells. Here, we establish in vitro models that recapitulate DMG-OPC-like and AC-like phenotypes and perform transcriptomics, metabolomics, and bioenergetic profiling to identify metabolic programs in the different cellular states. We then define strategies to target metabolic vulnerabilities within specific tumor populations. We show that AC-like cells exhibit a mesenchymal phenotype and are sensitized to ferroptotic cell death. In contrast, OPC-like cells upregulate cholesterol biosynthesis, have diminished mitochondrial oxidative phosphorylation (OXPHOS), and are accordingly more sensitive to statins and OXPHOS inhibitors. Additionally, statins and OXPHOS inhibitors show efficacy and extend survival in preclinical orthotopic models established with stem-like H3K27M DMG cells. Together, this study demonstrates that cellular subtypes within DMGs harbor distinct metabolic vulnerabilities that can be uniquely and selectively targeted for therapeutic gain. Pediatric brain cancers are lethal malignancies driven by less-differentiated stem-like cells. Here the authors show that these cells exhibit distinct mitochondrial metabolism programs with targetable vulnerabilities. [ABSTRACT FROM AUTHOR]

Alternative splicing is an important process that contributes to highly diverse transcripts and protein products, which can affect the development of disease in various organisms. Cardiovascular disease (CVD) represents one of the greatest global threats to humans, particularly acute myocardial infarction (MI) and subsequent ischemic reperfusion (IR) injury, which involve complex transcriptomic changes in heart tissues associated with metabolic reshaping and immunological response. In this study, we used a newly developed ONT full-length transcriptomic approach and performed transcript-resolved differential expression profiling in murine models of MI and IR. We built an analytical pipeline to reliably identify and quantify alternative splicing products (isoforms), expanding on the currently available catalog of isoforms described in mice. The updated alternative splicing landscape included transcripts, genes, and pathways that were differentially regulated during IR and MI. Our study establishes a pipeline to profile highly diverse isoforms using state-of-the-art long-read sequencing, builds a landscape of alternative splicing in the mouse heart during MI and IR. [ABSTRACT FROM AUTHOR]

The emergence of vaccinomics and system vaccinology represents a transformative shift in immunization strategies, advocating for personalized vaccines tailored to individual genetic and immunological profiles. Integrating insights from genomics, transcriptomics, proteomics, and immunology, personalized vaccines offer the promise of enhanced efficacy and safety, revolutionizing the field of vaccinology. However, the development of personalized vaccines presents multifaceted challenges, including technical, ethical, economic, and regulatory considerations. Addressing these challenges is essential to ensure equitable access and safety of personalized vaccination strategies. Despite these hurdles, the potential of personalized vaccines to optimize responses and mitigate disease burden underscores the significance of ongoing research and collaboration in advancing precision medicine in immunization. [ABSTRACT FROM AUTHOR]

Introduction: Ewing Sarcoma (EWS) has been reported in seven children with Down syndrome (DS). To date, a detailed assessment of this solid tumour in DS patients is yet to be made. Methods: Here,we characterise a chemo-resistantmediastinal EWS in a 2-year-old DS child, the youngest ever reported case, by exploiting sequencing approaches. Results: The tumour showed a neuroectodermal development driven by the EWSR1-FLI1 fusion. The inherited myeloperoxidase deficiency of the patient caused failure of neutrophil-mediated cell death and promoted genomic instability. Discussion: In this context, the tumour underwent genome-wide near haploidisation resulting in a massive overexpression of pro-inflammatory cytokines. Recruitment of defective neutrophils fostered rapid evolution of this EWS. [ABSTRACT FROM AUTHOR]

Formalin-fixed paraffin-embedded (FFPE) samples are valuable but underutilized in single-cell omics research due to their low RNA quality. In this study, leveraging a recent advance in single-cell genomic technology, we introduce snPATHO-seq, a versatile method to derive high-quality single-nucleus transcriptomic data from FFPE samples. We benchmarked the performance of the snPATHO-seq workflow against existing 10x 3' and Flex assays designed for frozen or fresh samples and highlighted the consistency in snRNA-seq data produced by all workflows. The snPATHO-seq workflow also demonstrated high robustness when tested across a wide range of healthy and diseased FFPE tissue samples. When combined with FFPE spatial transcriptomic technologies such as FFPE Visium, the snPATHO-seq provides a multi-modal sampling approach for FFPE samples, allowing more comprehensive transcriptomic characterization. A combination of an FFPE nuclei preparation protocol and a probe-based transcriptomic profiling technique enables snRNA-seq characterization of archival human FFPE tissues, holding promise for retrospective studies involving aged clinical cohorts. [ABSTRACT FROM AUTHOR]

Venoms are complex mixtures produced by animals and consist of hundreds of components including small molecules, peptides, and enzymes selected for effectiveness and efficacy over millions of years of evolution. With the development of venomics, which combines genomics, transcriptomics, and proteomics to study animal venoms and their effects deeply, researchers have identified molecules that selectively and effectively act against membrane targets, such as ion channels and G protein-coupled receptors. Due to their remarkable physico-chemical properties, these molecules represent a credible source of new lead compounds. Today, not less than 11 approved venom-derived drugs are on the market. In this review, we aimed to highlight the advances in the use of venom peptides in the treatment of diseases such as neurological disorders, cardiovascular diseases, or cancer. We report on the origin and activity of the peptides already approved and provide a comprehensive overview of those still in development. [ABSTRACT FROM AUTHOR]

Tissue regeneration is orchestrated by macrophages that clear damaged cells and promote regenerative inflammation. How macrophages spatially adapt and diversify their functions to support the architectural requirements of actively regenerating tissue remains unknown. In this study, we reconstructed the dynamic trajectories of myeloid cells isolated from acutely injured and early stage dystrophic muscles. We identified divergent subsets of monocytes/macrophages and DCs and validated markers (e.g., glycoprotein NMB [GPNMB]) and transcriptional regulators associated with defined functional states. In dystrophic muscle, specialized repair-associated subsets exhibited distinct macrophage diversity and reduced DC heterogeneity. Integrating spatial transcriptomics analyses with immunofluorescence uncovered the ordered distribution of subpopulations and multilayered regenerative inflammation zones (RIZs) where distinct macrophage subsets are organized in functional zones around damaged myofibers supporting all phases of regeneration. Importantly, intermittent glucocorticoid treatment disrupted the RIZs. Our findings suggest that macrophage subtypes mediated the development of the highly ordered architecture of regenerative tissues, unveiling the principles of the structured yet dynamic nature of regenerative inflammation supporting effective tissue repair. [ABSTRACT FROM AUTHOR]

Salinity adaptation is an important issue in aquaculture. Understanding the immediate-early response to salinity stress helps in comprehending this process. In vitro experiments using cell lines can explain cell-independent reactions without the involvement of hormones in vivo. In this study, salinity stress experiments were conducted using cell line derived from the gills of Gymnocypris przewalskii (GPG cell line) to isolate immediate-early response-related genes and miRNAs using transcriptomics, followed by bioinformatics analysis. The results showed that intracellular free Ca2+ appeared to be a key factor in cell sensing and initiating downstream cell signaling in response to external salinity. Additionally, cell apoptosis was the most common feature of salinity stress, with multiple signaling pathways involved in salinity-induced cell apoptosis. Furthermore, MiRNAs played a crucial role in the rapid response to salinity stress by selectively inhibiting the expression of specific genes. Additionally, for the first time in the G. przewalskii genome, Tf2 and TY3 families of transposons were found to have responsive roles to the external salinity stress. This study contributes to a better understanding of osmotic sensing in G. przewalskii and provides theoretical assistance for improving salinity adaptation in aquaculture fish species. [ABSTRACT FROM AUTHOR]

Nicotine exhibits obvious heterosis, which can be used to create Nicotiana tabacum L. (tobacco) varieties with varying nicotine content. However, the reasons for the formation of nicotine heterosis and its relationship to nicotine transport and accumulation remain unknown. This study conducted a comprehensive analysis of six tobacco hybrids with varying heterosis levels and their parent materials from various aspects, such as phenotype, physiology, and transcriptomics. The results showed that the direct path coefficient of transport heterosis to nicotine heterosis was highest in hybrids, at 0.98, and a highly significant positive correlation between the two. The plant height, thick stalk circumference, large flow of tissue fluid in the stalk, and high nicotine concentration of tobacco were the underlying factors that led to the strong nicotine transport capacity of hybrids. The formation of nicotine transport heterosis in hybrids was mainly influenced by non-additive gene effects (accounting for 89.93%), with over-dominant effects playing a dominant role (accounting for 58.79%). Among non-additive expression DEGs, nicotine transporter related multi antimicrobial extrusion protein, drug/metabolite transporter, ABC family transporter, and glutathione S-transferase were significantly upregulated in hybrid strains. The RT-qPCR results indicated that these genes related nicotine transport also exhibited heterosis at the expression level. Our results revealed that the formation of nicotine heterosis is mainly achieved by enhancing the nicotine transport capacity in hybrids. The results are not only beneficial for promoting the theoretical study of nicotine heterosis in tobacco and the breeding and utilization of hybrids, but are also of great significance for guiding nicotine production and promoting its multipurpose utilization. [ABSTRACT FROM AUTHOR]

Temperature is an important limiting factor in the counter-seasonal cultivation of pepper. Currently, there are no studies on transcriptomic analysis of 'cold stress memory' in pepper. In this study, in order to understand the mechanism of 'cold stress memory' in pepper (Capsicum annuum L.), seedlings were subjected to the following treatments: normal temperature treatment (P0), the first cold treatment for 3 days (P3), the recovery temperature treatment for 3 days (R3), and another cold treatment for 3 days (RP3). The results showed that P3 plants wilted the most, RP3 the second and R3 the least. Leaf reactive oxygen species (ROS) and electrolyte leakage were the most in P3, the second in RP3 and the least in R3. In addition, RP3 had the highest accumulation of zeaxanthin, violaxanthin and β-cryptoxanthin, followed by P3, and R3 had the least. These results suggest that pepper seedlings are characterized by 'cold stress memory'. Transcriptomics was used to analyze the key genes and transcription factors involved in the biosynthesis of zeaxanthin, violaxanthin and β-cryptoxanthin during the formation of 'cold stress memory'. This study provides candidate genes and transcription factors for an in-depth study of the cold tolerance mechanism in pepper. [ABSTRACT FROM AUTHOR]

Erianin is one of the most representative bibenzyls with significant inhibitory activity against a wide range of tumor cells. However, the low erianin level in natural materials has severely inhibited its further development in health care. Our aim was to uncover the erianin biosynthetic pathway to lay the foundation for promoting its production. Firstly, we screened and obtained two Dendrobium species (Dendrobium thyrsiflorum stems with lower erianin content and D. chrysotoxum stems with higher erianin content), belonging to the same Dendrobium section (Chrysotoxae). A systematic analysis of bibenzyl structure and content in two stems revealed that gigantol might be an erianin biosynthetic intermediate, which was verified by introducing deuterium-labeled gigantol. Chemical structure analyses indicated that gigantol was modified by two kinds of enzymes (hydroxylases and O-methyltransferases), leading to erianin synthesis. Up-regulated hydroxylases and O-methyltransferases (OMTs) were screened out and were performed by molecular docking simulation experiments. We propose a rational biosynthetic pathway from gigantol to erianin, as well as relevant enzymes involved in the process. Our findings should significantly contribute to comprehensive resolution of the erianin biosynthetic pathway, promote its large-scale industrial production as well as contribute to biosynthesis studies of other bibenzyls. [ABSTRACT FROM AUTHOR]

Lung cancer is one of the most common malignant tumours worldwide and its high mortality rate makes it a leading cause of cancer-related deaths. To address this daunting challenge, we need a comprehensive understanding of the pathogenesis and progression of lung cancer in order to adopt more effective therapeutic strategies. In this regard, integrating multi-omics data of the lung provides a highly promising avenue. Multi-omics approaches such as genomics, transcriptomics, proteomics, and metabolomics have become key tools in the study of lung cancer. The application of these methods not only helps to resolve the immunotherapeutic mechanisms of lung cancer, but also provides a theoretical basis for the development of personalised treatment plans. By integrating multiomics, we have gained amore comprehensive understanding of the process of lung cancer development and progression, and discovered potential immunotherapy targets. This review summarises the studies on multi-omics and immunology in lung cancer, and explores the application of these studies in early diagnosis, treatment selection and prognostic assessment of lung cancer, with the aim of providing more personalised and effective treatment options for lung cancer patients. [ABSTRACT FROM AUTHOR]

Background: Psoriasis is an inflammatory skin condition where immune cells play a significant role. The importance of the cross-talk between keratinocytes and immune cells in the pathogenesis of psoriasis has recently been reaffirmed. Recent studies have found that several S1PR functional antagonists, other than S1PR2, are effective in improving psoriasis. This study aims to investigate the role of S1PR2 in psoriasis, that has not been investigated before. Methods: Spatial transcriptomics, RT-qPCR, and flow cytometry were used to map the immune cell landscape and its association with metabolic pathways in an imiquimod (IMQ)-induced psoriasis-like inflammation in S1pr2fl/fl K14-Cre mice that could not sense sphingosine-1-phosphate (S1P) in the epidermis through the S1PR2 receptor. Results: Our analysis suggests that S1PR2 in keratinocytes plays a major role in psoriasis-like inflammation compared to other S1PRs. It acts as a downregulator, inhibiting the recruitment of Th17 cells into the skin. In IMQ-induced psoriasis skin, both S1pr2-/- and S1pr2fl/fl K14-Cre mice showed higher expressions of proinflammatory cytokines such as TNF-a, IL-17A, and IL-1b together with higher expressions of MyD88/NF-kB pathway compared to the wild-type mice. Remarkably, in IMQ-treated mice, the deletion of S1pr2 in keratinocytes only resulted in a larger population of Th17 cells in skin-draining lymph nodes. Other S1PR modulators did not improve the worsening of psoriasis-like inflammation caused by S1PR2 deficiency in keratinocytes. Conclusion: This study reaches two main conclusions: signals from keratinocytes play a central role in creating an immune environment that promotes the development of psoriasis, and stimulating S1PR2, instead of suppressing it, represents a potential therapeutic approach for psoriasis. [ABSTRACT FROM AUTHOR]

The diagnosis of pediatric tuberculosis (TB) poses a challenge for clinical teams worldwide. TB-mediated changes in the expression of host genes in the peripheral blood can serve as diagnostic biomarkers and can provide better insights into the host immune mechanisms of childhood TB. Peripheral blood mononuclear cells (PBMCs) from children (n=102) with microbiologically confirmed TB disease, TB infection (TBI), pneumonia, and healthy controls (HC) were stimulated with either the Purified Protein Derivative (PPD) or the Early Secretory Antigen 6kDa-Culture Filtrate Protein 10 (ESAT6-CFP10) complex of Mycobacterium tuberculosis (Mtb). RNA was extracted and quantified using gene expression microarrays. Differential expression analysis was performed comparing microbiologically confirmed TB to the other diagnostic groups for the stimulated and unstimulated samples. Using variable selection, we identified sparse diagnostic gene signatures; one gene (PID1) was able to distinguish TB from pneumonia after ESAT6-CFP10 stimulation with an AUC of 100% in the test set, while a combination of two genes (STAT1 and IFI44) achieved an AUC of 91.7% (CI95% 75.0%-100%) in the test set after PPD stimulation. The number of significantly differentially expressed (SDE) genes was higher when contrasting TB to pneumonia or HC in stimulated samples, compared to unstimulated ones, leading to a larger pool of candidate diagnostic biomarkers. Our approach provides enlightened aspects of peripheral TB-specific responses and can form the basis for a point of care test meeting the World Health Organization (WHO) Target Product Profile (TPP) for pediatric TB. [ABSTRACT FROM AUTHOR]

Listeria monocytogenes is a ubiquitous and psychrotrophic foodborne pathogen commonly found in raw materials, ready-to-eat products, and food environments. We previously demonstrated that L. monocytogenes can grow faster at low temperature when unsaturated fatty acids (UFA) are present in its environment. This could question the maintenance of food safety for refrigerated foods, especially those reformulated with a higher ratio of UFA versus saturated fatty acids (SFA) to fit with nutritional recommendations. In this study, we used transcriptomics to understand the impact of UFA on the behavior of L. monocytogenes at low temperature. We first demonstrated that fabK, a key gene in SFA synthesis, is up-regulated in the presence of UFA but not SFA at low temperature. L. monocytogenes can thus regulate the synthesis of SFA in its membrane according to the type of FA available in its environment. Interestingly, we also observed up-regulation of genes involved in chemotaxis and flagellar assembly (especially cheY and flaA) in the presence of UFA but not SFA at low temperature. TEM observations confirmed that L. monocytogenes acquired a remarkable phenotype with numerous and long-looped flagella only in the presence of UFA at 5°C but not at 37°C. As flagella are well known to be involved in biofilm formation, this new finding raises questions about the structure and persistence of biofilms settled in refrigerated environments using unsaturated lipid-rich products. [ABSTRACT FROM AUTHOR]

The incidence of central nervous system (CNS) disease has persistently increased over the last several years. There is an urgent need for effective methods to improve the cure rates of CNS disease. However, the precise molecular basis underlying the development and progression of major CNS diseases remains elusive. A complete molecular map will contribute to research on CNS disease treatment strategies. Emerging technologies such as single-cell RNA sequencing (scRNA-seq) and Spatial Transcriptomics (ST) are potent tools for exploring the molecular complexity, cell heterogeneity, and functional specificity of CNS disease. scRNA-seq and ST can provide insights into the disease at cellular and spatial transcription levels. This review presents a survey of scRNA-seq and ST studies on CNS diseases, such as chronic neurodegenerative diseases, acute CNS injuries, and others. These studies offer novel perspectives in treating and diagnosing CNS diseases by discovering new cell types or subtypes associated with the disease, proposing new pathophysiological mechanisms, uncovering novel therapeutic targets, and identifying putative biomarkers. Implications of scRNA-seq and ST for CNS diseases. A comprehensive understanding of cellular information and spatial context using scRNA-seq and ST, helping to elucidate the biological functions, the intricate connection between crucial regulatory pathways, and cell communication after CNS diseases. [ABSTRACT FROM AUTHOR]

Pedicel length is a crucial agronomic trait of cucumbers. Fruit deformation can occur When the pedicel is too long or too short. Moreover, an appropriate pedicel length is advantageous for mechanized harvesting. Therefore, it is essential to investigate the molecular regulatory mechanisms underlying cucumber pedicel length. In the current study, we obtained a short pedicel mutant through EMS mutagenesis and discovered that the reduced cell number was the primary cause of the shortened pedicel. Upon analyzing the hormone content, we found that the level of trans zeatin in the long-pedicel material was significantly higher than that in the short-pedicel material. Further transcriptome sequencing analysis revealed that differentially expressed genes were enriched in cytokinin synthesis-related pathways. Based on these results, the present study concluded that cucumber pedicel length is regulated by genes related to the cytokinin synthesis pathway and that differences in length result from differences in zeatin content and cell number. [ABSTRACT FROM AUTHOR]

Usage of the phrase “biological age” has picked up considerably since the advent of aging clocks and it has become commonplace to describe an aging clock's output as biological age. In contrast to this labeling, biological age is also often depicted as a more abstract concept that helps explain how individuals are aging internally, externally, and functionally. Given that the bulk of molecular aging is tissue‐specific and aging itself is a remarkably complex, multifarious process, it is unsurprising that most surveyed scientists agree that aging cannot be quantified via a single metric. We share this sentiment and argue that, just like it would not be reasonable to assume that an individual with an ideal grip strength, VO2 max, or any other aging biomarker is biologically young, we should be careful not to conflate an aging clock with whole‐body biological aging. To address this, we recommend that researchers describe the output of an aging clock based on the type of input data used or the name of the clock itself. Epigenetic aging clocks produce epigenetic age, transcriptomic aging clocks produce transcriptomic age, and so forth. If a clock has a unique name, such as our recently developed epigenetic aging clock CheekAge, the name of the clock can double as the output. As a compromise solution, aging biomarkers can be described as indicators of biological age. We feel that these recommendations will help scientists and the public differentiate between aging biomarkers and the much more elusive concept of biological age. [ABSTRACT FROM AUTHOR]

Metabolic RNA labeling‐based time‐resolved single‐cell RNA sequencing (scRNA‐seq) has provided unprecedented tools to dissect the temporal dynamics and the complex gene regulatory networks of gene expression. However, this technology fails to reveal the spatial organization of cells in tissues, which also regulates the gene expression by intercellular communication. Herein, it is demonstrated that integrating time‐resolved scRNA‐seq with spatial transcriptomics is a new paradigm for spatiotemporal analysis. Metabolic RNA labeling‐based time‐resolved Well‐TEMP‐seq is first applied to profile the transcriptional dynamics of glioblastoma (GBM) cells and discover two potential pathways of EZH2‐mediated mesenchymal transition in GBM. With spatial transcriptomics, it is further revealed that the crosstalk between CCL2+ malignant cells and IL10+ tumor‐associated macrophages in the tumor microenvironment through an EZH2‐FOSL2‐CCL2 axis contributes to the mesenchymal transition in GBM. These discoveries show the power of integrative spatiotemporal scRNA‐seq to elucidate the complex gene regulatory mechanism and advance the understanding of cellular processes in disease. [ABSTRACT FROM AUTHOR]

T cells are dynamically regulated immune cells that are implicated in a variety of diseases ranging from infection, cancer and autoimmunity. Recent advancements in sequencing methods have provided valuable insights in the transcriptional and epigenetic regulation of T cells in various disease settings. In this review, we identify the key sequencing-based methods that have been applied to understand the transcriptomic and epigenomic regulation of T cells in diseases. [ABSTRACT FROM AUTHOR]

ABSTRACT Hemoglobin H (HbH) disease, a form of alpha‐thalassemia, poses significant clinical challenges due to its complex molecular underpinnings. It is characterized by reduced synthesis of the alpha‐globin chain. The integration of multi‐omics and precision medicine holds immense potential to comprehensively understand and capture interactions at the molecular and genetic levels. This review integrates current multi‐omics approaches and advanced technologies in HbH research. Furthermore, it delves into detailed pathophysiology and possible therapeutics in the upcoming future. We explore the role of genomics, transcriptomics, proteomics, and metabolomics studies, alongside bioinformatics tools and gene‐editing technologies like CRISPR/Cas9, to identify genetic modifiers, decipher molecular pathways, and discover therapeutic targets. Recent advancements are unveiling novel genetic and epigenetic modifiers impacting HbH disease severity, paving the way for personalized precision medicine interventions. The significance of multi‐omics research in unraveling the complexities of rare diseases like HbH is underscored, highlighting its potential to revolutionize clinical practice through precision medicine approaches. This paradigm shift can pave the way for a deeper understanding of HbH complexities and improved disease management. [ABSTRACT FROM AUTHOR]

Transposable elements (TEs) are crucial for genetic diversity and gene regulation. Current single-cell quantification methods often align multi-mapping reads to either 'best-mapped' or 'random-mapped' locations and categorize them at the subfamily levels, overlooking the biological necessity for accurate, locus-specific TE quantification. Moreover, these existing methods are primarily designed for and focused on transcriptomics data, which restricts their adaptability to single-cell data of other modalities. To address these challenges, here we introduce MATES, a deep-learning approach that accurately allocates multi-mapping reads to specific loci of TEs, utilizing context from adjacent read alignments flanking the TE locus. When applied to diverse single-cell omics datasets, MATES shows improved performance over existing methods, enhancing the accuracy of TE quantification and aiding in the identification of marker TEs for identified cell populations. This development facilitates the exploration of single-cell heterogeneity and gene regulation through the lens of TEs, offering an effective transposon quantification tool for the single-cell genomics community. Transposable elements (TEs) pose challenges for quantification due to multi-mapping reads. Here, authors present MATES, a deep learning method that accurately assigns reads to specific TE loci, enhancing TE quantification in single-cell omics datasets and identifying marker TEs in cell populations. [ABSTRACT FROM AUTHOR]

Splice site recognition is essential for defining the transcriptome. Drugs like risdiplam and branaplam change how human U1 snRNP recognizes particular 5′ splice sites (5′SS) and promote U1 snRNP binding and splicing at these locations. Despite the therapeutic potential of 5′SS modulators, the complexity of their interactions and snRNP substrates have precluded defining a mechanism for 5′SS modulation. We have determined a sequential binding mechanism for modulation of −1A bulged 5′SS by branaplam using a combination of ensemble kinetic measurements and colocalization single molecule spectroscopy (CoSMoS). Our mechanism establishes that U1-C protein binds reversibly to U1 snRNP, and branaplam binds to the U1 snRNP/U1-C complex only after it has engaged with a −1A bulged 5′SS. Obligate orders of binding and unbinding explain how reversible branaplam interactions cause formation of long-lived U1 snRNP/5′SS complexes. Branaplam targets a ribonucleoprotein, not only an RNA duplex, and its action depends on fundamental properties of 5′SS recognition. Splice sites must be correctly identified for introns to be removed from pre-mRNAs and for expression of the correct mRNA isoforms. Here, the authors elucidate how the human U1 snRNP splicing factor recognizes 5′ splice sites and how a splicing modulating drug can enhance splice site recognition. [ABSTRACT FROM AUTHOR]

Colorectal cancer (CRC) exhibits significant genetic and epigenetic diversity, evolving into sub-clonal populations with varied metastatic potentials and treatment responses. Predicting metastatic disease in CRC patients remains challenging, underscoring the need for reliable biomarkers. While most research on therapeutic targets and biomarkers has focused on proteins, non-coding RNAs such as long non-coding RNAs (lncRNAs) comprise most of the transcriptome and demonstrate superior tissue- and cancer-specific expression. We utilised spatial transcriptomics to investigate lncRNAs in CRC tumours, offering more precise cell-type-specific expression data compared to bulk RNA sequencing. Our analysis identified 301 lncRNAs linked to malignant CRC regions, which we validated with public data. Further validation using RNA–FISH revealed three lncRNAs (LINC01978, PLAC4, and LINC01303) that are detectable in stage II tumours but not in normal epithelium and are upregulated in metastatic tissues. These lncRNAs hold potential as biomarkers for early risk assessment of metastatic disease. [ABSTRACT FROM AUTHOR]

The polarization of cells often involves the transport of specific mRNAs and their localized translation in distal projections. Neurons and glia are both known to contain long cytoplasmic processes, while localized transcripts have only been studied extensively in neurons, not glia, especially in intact nervous systems. Here, we predict 1,740 localized Drosophila glial transcripts by extrapolating from our meta-analysis of seven existing studies characterizing the localized transcriptomes and translatomes of synaptically associated mammalian glia. We demonstrate that the localization of mRNAs in mammalian glial projections strongly predicts the localization of their high-confidence Drosophila homologs in larval motor neuron–associated glial projections and are highly statistically enriched for genes associated with neurological diseases. We further show that some of these localized glial transcripts are specifically required in glia for structural plasticity at the nearby neuromuscular junction synapses.We conclude that peripheral glial mRNA localization is a common and conserved phenomenon and propose that it is likely to be functionally important in disease. [ABSTRACT FROM AUTHOR]

Single‐cell RNA sequencing and spatial transcriptomics enable unprecedented insight into cellular and molecular pathways implicated in human skin aging and regeneration. Senescent cells are individual cells that are irreversibly cell cycle arrested and can accumulate across the human lifespan due to cell‐intrinsic and ‐extrinsic stressors. With an atlas of single‐cell RNA‐sequencing and spatial transcriptomics, epidermal and dermal senescence and its effects were investigated, with a focus on melanocytes and fibroblasts. Photoaging due to ultraviolet light exposure was associated with higher burdens of senescent cells, a sign of biological aging, compared to chronological aging. A skin‐specific cellular senescence gene set, termed SenSkin™, was curated and confirmed to be elevated in the context of photoaging, chronological aging, and non‐replicating CDKN1A+ (p21) cells. In the epidermis, senescent melanocytes were associated with elevated melanin synthesis, suggesting haphazard pigmentation, while in the dermis, senescent reticular dermal fibroblasts were associated with decreased collagen and elastic fiber synthesis. Spatial analysis revealed the tendency for senescent cells to cluster, particularly in photoaged skin. This work proposes a strategy for characterizing age‐related skin dysfunction through the lens of cellular senescence and suggests a role for senescent epidermal cells (i.e., melanocytes) and senescent dermal cells (i.e., reticular dermal fibroblasts) in age‐related skin sequelae. [ABSTRACT FROM AUTHOR]