Search Results (8)

The murine Alpha Mouse Liver 12 (AML12) cell line, established over four decades ago, is one of the most commonly used non-transformed hepatocyte models in basic and pre-clinical liver research. Despite its widespread use, a comprehensive and current molecular characterization has been lacking. In this study, we combined cytogenetics with high-resolution genomic technologies to establish a detailed genetic reference profile of AML12. Inverted DAPI banding and multicolor fluorescence in situ hybridization (m-FISH) revealed a complex yet stable, near-tetraploid karyotype featuring double X-chromosome deletions [del(X)(A3)×2], a recurrent derivative chromosome der(3)t(2;3)(A2;H4), biallelic deletions of 17D1, two dicentric chromosomes dic(X;17), and multiple whole-chromosome gains (e.g., +1, +6, +15, +19×4) and losses (e.g., −4, −12, −16, −18). Multicolor banding (mcb) further pinpointed cryptic inversions on chromosomes 7 and 11. Copy number imbalances were visualized as in silico array comparative genomic hybridization (aCGH)-style profiles inferred from these metaphase-based assays, and no independent array- or sequencing-based copy number variation (CNV) experiment was performed in this study. Short tandem repeat (STR) profiling created a unique 16-locus authentication barcode that unambiguously distinguishes AML12 from other murine cell lines in public databases. Bulk RNA sequencing (RNA-seq) further demonstrated a transcriptional profile in AML12 cells that is indicative of hepatocyte origin while also revealing partial de-differentiation and reduced expression of selected urea cycle, gluconeogenic, and xenobiotic-metabolizing transcripts, consistent with limited mature hepatocyte functions. These functional inferences are likely based on gene expression patterns rather than on direct physiological assays. In summary, our study provides (i) the first integrated cytogenetic, STR, and next-generation sequencing dataset for AML12, (ii) a practical authentication panel for routine laboratory use, and (iii) reference information that will enhance the interpretation, reproducibility, and translational relevance of future studies using this versatile hepatocyte model. Full article

Genetically encoded fluorescent protein (FP)-based biosensors have revolutionized cell biology research by enabling real-time monitoring of molecular activities in live cells with exceptional spatial and temporal resolution. Multiplexed biosensing advances this capability by allowing the simultaneous tracking of multiple signaling pathways to uncover network interactions and dynamic coordination. However, challenges in spectral overlap limit broader implementation. Innovative strategies have been devised to address these challenges, including spectral separation through FP palette expansion and novel biosensor designs, temporal differentiation using photochromic or reversibly switching FPs, and spatial segregation of biosensors to specific subcellular regions or through cell barcoding techniques. Combining multiplexed biosensors with artificial intelligence-driven analysis holds great potential for uncovering cellular decision-making processes. Continued innovation in this field will deepen our understanding of molecular networks in cells, with implications for both fundamental biology and therapeutic development. Full article

Herein, we applied DNA barcoding for the genetic characterization of Sideritis syriaca subsp. syriaca (Lamiaceae; threatened local Cretan endemic plant) using seven molecular markers of cpDNA. Five fertilization schemes were evaluated comparatively in a pilot cultivation in Crete. Conventional inorganic fertilizers (ChFs), integrated nutrient management (INM) fertilizers, and two biostimulants were utilized (foliar and soil application). Plant growth, leaf chlorophyll fluorescence, and color were assessed and leaf content of chlorophyll, key antioxidants (carotenoids, flavonoids, phenols), and nutrients were evaluated. Fertilization schemes induced distinct differences in leaf shape, altering quality characteristics. INM-foliar and ChF-soil application promoted yield, without affecting tissue water content or biomass partitioning to inflorescences. ChF-foliar application was the most stimulatory treatment when the primary target was enhanced antioxidant contents while INM-biostimulant was the least effective one. However, when the primary target is yield, INM, especially by foliar application, and ChF, by soil application, ought to be employed. New DNA sequence datasets for the plastid regions of petB/petD, rpoC1, psbK-psbI, and atpF/atpH were deposited in the GenBank for S. syriaca subsp. syriaca while the molecular markers rbcL, trnL/trnF, and psbA/trnH were compared to those of another 15 Sideritis species retrieved from the GenBank, constructing a phylogenetic tree to show their genetic relatedness. Full article

Single-nucleus RNA sequencing (sNucRNA-seq) is an emerging technology that has been rapidly adopted and demonstrated to be a powerful tool for detailed characterization of each cell- and sub cell-types in complex tissues of higher eukaryotes. sNucRNA-seq has also been used to dissect cell-type-specific transcriptional responses to environmental or developmental signals. In plants, this technology is being utilized to identify cell-type-specific trajectories for the study of several tissue types and important traits, including the single-cell dissection of the genetic determinants regulating plant–microbe interactions. The isolation of high-quality nuclei is one of the prerequisite steps to obtain high-quality sNucRNA-seq results. Although nuclei isolation from several plant tissues is well established, this process is highly troublesome when plant tissues are associated with beneficial or pathogenic microbes. For example, root tissues colonized with rhizobium bacteria (nodules), leaf tissue infected with bacterial or fungal pathogens, or roots infected with nematodes pose critical challenges to the isolation of high-quality nuclei and use for downstream application. Therefore, isolation of microbe-free, high-quality nuclei from plant tissues are necessary to avoid clogging or interference with the microfluidic channel (e.g., 10× Genomics) or particle-templated emulsion that are used in sNucRNA-seq platforms. Here, we developed a simple, effective, and efficient method to isolate high-quality nuclei from soybean roots and root nodules, followed by washing out bacterial contamination. This protocol has been designed to be easily implemented into any lab environment, and it can also be scaled up for use with multiple samples and applicable to a variety of samples with the presence of microbes. We validated this protocol by successfully generating a barcoded library using the 10× Genomics microfluidic platform from tissue subjected to this procedure. This workflow was developed to provide an accessible alternative to instrument-based approaches (e.g., fluorescent cell sorting) and will expand the ability of researchers to perform experiments such as sNucRNA-seq and sNucATAC-seq on inherently heterogeneous plant tissue samples. Full article

Clonal heterogeneity in acute myeloid leukemia (AML) forms the basis for treatment failure and relapse. Attempts to decipher clonal evolution and clonal competition primarily depend on deep sequencing approaches. However, this prevents the experimental confirmation of the identified disease-relevant traits on the same cell material. Here, we describe the development and application of a complex fluorescent genetic barcoding (cFGB) lentiviral vector system for the labeling and subsequent multiplex tracking of up to 48 viable AML clones by flow cytometry. This approach allowed the visualization of longitudinal changes in the in vitro growth behavior of multiplexed color-coded AML clones for up to 137 days. Functional studies of flow cytometry-enriched clones documented their stably inherited increase in competitiveness, despite the absence of growth-promoting mutations in exome sequencing data. Transplantation of aliquots of a color-coded AML cell mix into mice revealed the initial engraftment of similar clones and their subsequent differential distribution in the animals over time. Targeted RNA-sequencing of paired pre-malignant and de novo expanded clones linked gene sets associated with Myc-targets, embryonic stem cells, and RAS signaling to the foundation of clonal expansion. These results demonstrate the potency of cFGB-mediated clonal tracking for the deconvolution of verifiable driver-mechanisms underlying clonal selection in leukemia. Full article

Acute Chagas disease (ACD) caused by Trypanosoma cruzi has emerged as a major food-borne disease in Brazilian Amazonia. For the first time, we characterized an outbreak of orally acquired ACD in Acre, in the forest community of Seringal Miraflores, affecting 13 individuals who shared the pulp of açai palm berries: 11 adults and two children (one newborn), all diagnosed by thick-drop blood smears. The fluorescent fragment length barcoding method, which simultaneously identifies species/genotypes of trypanosomes in blood samples, uncovered an unprecedented genetic diversity in patients from a single outbreak of ACD: T. cruzi TcI in all patients, mostly concomitantly with the non-pathogenic Trypanosoma rangeli of genotypes TrA or TrB, and TcI, TcIV, and TrB in the child. The patients presented persistent fever, asthenia, myalgia, edema of the face and lower limbs, hepatosplenomegaly and, rarely, cardiac arrhythmia. The clinical symptoms were not correlated to gender, age, or to trypanosome species and genotypes. The inferred SSU rRNA phylogenetic analyses of trypanosomes from humans, triatomines and sylvatic hosts included the first sequences of T. cruzi and T. rangeli from humans in southwestern (Acre and Rondônia) Amazonia, and the first TcI/TcIV sequences from Rhodnius spp. from Acre. The sylvatic transmission cycles of genetically different trypanosomes in landscapes changed by deforestation for human settlements and increasing açai production is a novel scenario favoring trypanosome transmission to humans in Acre. Full article

The barcode probe is a convenient and efficient tool for molecular cytogenetics. Tripidium arundinaceum, as a polyploid wild allied genus of Saccharum, is a useful genetic resource that confers biotic and abiotic stress resistance for sugarcane breeding. Unfortunately, the basic cytogenetic information is still unclear due to the complex genome. We constructed the Cot-20 library for screening moderately and highly repetitive sequences from T. arundinaceum, and the chromosomal distribution of these repetitive sequences was explored. We used the barcode of repetitive sequence probes to distinguish the ten chromosome types of T. arundinaceum by fluorescence in situ hybridization (FISH) with Ea-0907, Ea-0098, and 45S rDNA. Furthermore, the distinction among homology chromosomes based on repetitive sequences was constructed in T. arundinaceum by the repeated FISH using the barcode probes including Ea-0663, Ea-0267, EaCent, 5S rDNA, Ea-0265, Ea-0070, and 45S rDNA. We combined these probes to distinguish 37 different chromosome types, suggesting that the repetitive sequences may have different distributions on homologous chromosomes of T. arundinaceum. In summary, this method provide a basis for the development of similar applications for cytogenetic analysis in other species. Full article

HOXA9 and MEIS1 are frequently upregulated in acute myeloid leukemia (AML), including those with MLL-rearrangement. Because of their pivotal role in hemostasis, HOXA9 and MEIS1 appear non-druggable. We, thus, interrogated gene expression data of pre-leukemic (overexpressing Hoxa9) and leukemogenic (overexpressing Hoxa9 and Meis1; H9M) murine cell lines to identify cancer vulnerabilities. Through gene expression analysis and gene set enrichment analyses, we compiled a list of 15 candidates for functional validation. Using a novel lentiviral multiplexing approach, we selected and tested highly active sgRNAs to knockout candidate genes by CRISPR/Cas9, and subsequently identified a H9M cell growth dependency on the cytosolic phospholipase A2 (PLA2G4A). Similar results were obtained by shRNA-mediated suppression of Pla2g4a. Remarkably, pharmacologic inhibition of PLA2G4A with arachidonyl trifluoromethyl ketone (AACOCF3) accelerated the loss of H9M cells in bulk cultures. Additionally, AACOCF3 treatment of H9M cells reduced colony numbers and colony sizes in methylcellulose. Moreover, AACOCF3 was highly active in human AML with MLL rearrangement, in which PLA2G4A was significantly higher expressed than in AML patients without MLL rearrangement, and is sufficient as an independent prognostic marker. Our work, thus, identifies PLA2G4A as a prognostic marker and potential therapeutic target for H9M-dependent AML with MLL-rearrangement. Full article