Ether lipids influence cancer cell fate by modulating iron uptake.

Cancer cell fate has been widely ascribed to mutational changes within protein-coding genes associated with tumor suppressors and oncogenes. In contrast, the mechanisms through which the biophysical properties of membrane lipids influence cancer cell survival, dedifferentiation and metastasis have received little scrutiny. Here, we report that cancer cells endowed with a high metastatic ability and cancer stem cell-like traits employ ether lipids to maintain low membrane tension and high membrane fluidity. Using genetic approaches and lipid reconstitution assays, we show that these ether lipid-regulated biophysical properties permit non-clathrin-mediated iron endocytosis via CD44, leading directly to significant increases in intracellular redox-active iron and enhanced ferroptosis susceptibility. Using a combination of in vitro three-dimensional microvascular network systems and in vivo animal models, we show that loss of ether lipids also strongly attenuates extravasation, metastatic burden and cancer stemness. These findings illuminate a mechanism whereby ether lipids in carcinoma cells serve as key regulators of malignant progression while conferring a unique vulnerability that can be exploited for therapeutic intervention.

Effects of glucose and trehalose on tris-citric acid-egg yolk-fructose diluents for semen cryopreservation in goat.

Objectives: This study aimed to examine the impacts of the wide range of concentrations of glucose and trehalose on the tris-citric acid-egg yolk-fructose (TCEF) extenders for cryopreservation of goat semen. Materials and Methods: The sperm sample was pooled, washed, and diluted in control (TCEF without glucose and trehalose), TCEF + glucose (75, 150, 450, and 900 mm), and TCEF + trehalose (75, 150, 450, and 900 mm). After equilibrations, the semen straws were frozen under LN2 in the LN2 tank. After LN2 storage, the straws were thawed at 37°C for 30 seconds. The sperm parameters of all study groups were checked after equilibration and freezing. Results: After equilibration, the progressive motility (PM), total motility (TM), and viability of sperm in G-75, G-150, G-450, T-75, T-150, and T-450 were not significantly different (p < 0.05) from those in control. After cryopreservation and thawing, the PM, TM, and plasma membrane integrity (PMI) of T-150 were significantly higher (p < 0.05) than in control, G-75, G-900, T-75, and T-900. The viability of sperm in T-150 was substantially higher (p < 0.05) than in the control, whereas there was no significant difference among the control, G-75, G-900, T-75, and T-900. However, the acrosome integrity (AI) of sperm in G-900 was significantly decreased (p < 0.05) compared to the control, G-75, G-150, G-450, T-75, T-150, and T-450. Conclusion: According to the findings, the supplementation of 150 mm trehalose in the TCEF diluent was more efficient for sperm cryopreservation in the buck as reflected by PM, TM, viability, PMI, and AI.

Poultry farmers' knowledge, attitude, and practices toward poultry waste management in Bangladesh.

Background and Aim: The improper handling of poultry litter and waste poses risks to humans and environment by introducing certain compounds, elements, and pathogenic microorganisms into the surrounding environment and food chain. However, understanding the farmers' knowledge, attitude, and practices (KAP) could provide insights into the constraints that hinder the appropriate adoption of waste management. Therefore, this study aimed to assess poultry farmers' KAP regarding waste management issues. Materials and Methods: A cross-sectional KAP study was conducted with native poultry keepers and small-scale commercial poultry farmers in seven districts of Bangladesh. In the survey, 385 poultry producers were interviewed using validated structured questionnaires through face-to-face interviews to collect the quantitative data in their domiciles. Results: The overall KAP of farmers regarding poultry waste management issues demonstrated a low level of KAP (p = 0.001). The analysis shows that roughly 5% of farmers have a high level of knowledge of poultry waste management issues, followed by around one-third of respondents having a moderate level of knowledge. Considering the attitude domain, more than one-fifth of native poultry keepers and nearly two-thirds of commercial producers demonstrated a low level of attitude toward poultry waste management. Considering the overall analysis, roughly half of the respondents found a high level of attitude, and over half of the farmers showed a moderate level of attitude toward poultry waste management issues. The analysis showed that the level of good practices for native and commercial poultry production systems is estimated at 77.3% versus 45.9%, respectively, despite the farmers' lesser knowledge and attitudes toward poultry waste management systems. Overall, analysis showed that nearly 60% and 40% of poultry producers had high and moderate levels, respectively, of good practices in poultry waste management issues. Conclusion: Analysis of the KAP data shows that farmers had a low level of KAP toward poultry waste management. The result of this study will assist in formulating appropriate strategies and to adopt poultry waste management solutions by poultry farmers to reduce environmental degradation.

ΔNp63/p73 drive metastatic colonization by controlling a regenerative epithelial stem cell program in quasi-mesenchymal cancer stem cells.

Cancer stem cells (CSCs) may serve as the cellular seeds of tumor recurrence and metastasis, and they can be generated via epithelial-mesenchymal transitions (EMTs). Isolating pure populations of CSCs is difficult because EMT programs generate multiple alternative cell states, and phenotypic plasticity permits frequent interconversions between these states. Here, we used cell-surface expression of integrin ß4 (ITGB4) to isolate highly enriched populations of human breast CSCs, and we identified the gene regulatory network operating in ITGB4+ CSCs. Specifically, we identified ΔNp63 and p73, the latter of which transactivates ΔNp63, as centrally important transcriptional regulators of quasi-mesenchymal CSCs that reside in an intermediate EMT state. We found that the transcriptional program controlled by ΔNp63 in CSCs is largely distinct from the one that it orchestrates in normal basal mammary stem cells and, instead, it more closely resembles a regenerative epithelial stem cell response to wounding. Moreover, quasi-mesenchymal CSCs repurpose this program to drive metastatic colonization via autocrine EGFR signaling.

Genome-wide CRISPR screen identifies PRC2 and KMT2D-COMPASS as regulators of distinct EMT trajectories that contribute differentially to metastasis.

Epithelial-mesenchymal transition (EMT) programs operate within carcinoma cells, where they generate phenotypes associated with malignant progression. In their various manifestations, EMT programs enable epithelial cells to enter into a series of intermediate states arrayed along the E-M phenotypic spectrum. At present, we lack a coherent understanding of how carcinoma cells control their entrance into and continued residence in these various states, and which of these states favour the process of metastasis. Here we characterize a layer of EMT-regulating machinery that governs E-M plasticity (EMP). This machinery consists of two chromatin-modifying complexes, PRC2 and KMT2D-COMPASS, which operate as critical regulators to maintain a stable epithelial state. Interestingly, loss of these two complexes unlocks two distinct EMT trajectories. Dysfunction of PRC2, but not KMT2D-COMPASS, yields a quasi-mesenchymal state that is associated with highly metastatic capabilities and poor survival of patients with breast cancer, suggesting that great caution should be applied when PRC2 inhibitors are evaluated clinically in certain patient cohorts. These observations identify epigenetic factors that regulate EMP, determine specific intermediate EMT states and, as a direct consequence, govern the metastatic ability of carcinoma cells.

CRISPR mediated targeting of DUX4 distal regulatory element represses DUX4 target genes dysregulated in Facioscapulohumeral muscular dystrophy.

Facioscapulohumeral muscular dystrophy (FSHD) is a debilitating muscle disease that currently does not have an effective cure or therapy. The abnormal reactivation of DUX4, an embryonic gene that is epigenetically silenced in somatic tissues, is causal to FSHD. Disease-specific reactivation of DUX4 has two common characteristics, the presence of a non-canonical polyadenylation sequence within exon 3 of DUX4 that stabilizes pathogenic transcripts, and the loss of repressive chromatin modifications at D4Z4, the macrosatellite repeat which encodes DUX4. We used CRISPR/Cas9 to silence DUX4 using two independent approaches. We deleted the DUX4 pathogenic polyadenylation signal, which resulted in downregulation of pathogenic DUX4-fl transcripts. In another approach, we transcriptionally repressed DUX4 by seeding heterochromatin using the dCas9-KRAB platform within exon 3. These feasibility of targeting DUX4 experiments were initially tested in a non-myogenic carcinoma cell line that we have previously characterized. Subsequently, in an immortalized patient myoblast cell line, we demonstrated that targeting DUX4 by either approach led to substantial downregulation of not only pathogenic DUX4 transcripts, but also a subset of its target genes that are known biomarkers of FSHD. These findings offer proof-of-concept of the effect of silencing the polyadenylation sequence on pathogenic DUX4 expression.

Stability of patient-specific features of altered DNA replication timing in xenografts of primary human acute lymphoblastic leukemia.

Genome-wide DNA replication timing (RT) profiles reflect the global three-dimensional chromosome architecture of cells. They also provide a comprehensive and unique megabase-scale picture of cellular epigenetic state. Thus, normal differentiation involves reproducible changes in RT, and transformation generally perturbs these, although the potential effects of altered RT on the properties of transformed cells remain largely unknown. A major challenge to interrogating these issues in human acute lymphoid leukemia (ALL) is the low proliferative activity of most of the cells, which may be further reduced in cryopreserved samples and difficult to overcome in vitro. In contrast, the ability of many human ALL cell populations to expand when transplanted into highly immunodeficient mice is well documented. To examine the stability of DNA RT profiles of serially passaged xenografts of primary human B- and T-ALL cells, we first devised a method that circumvents the need for bromodeoxyuridine incorporation to distinguish early versus late S-phase cells. Using this and more standard protocols, we found consistently strong retention in xenografts of the original patient-specific RT features. Moreover, in a case in which genomic analyses indicated changing subclonal dynamics in serial passages, the RT profiles tracked concordantly. These results indicate that DNA RT is a relatively stable feature of human ALLs propagated in immunodeficient mice. In addition, they suggest the power of this approach for future interrogation of the origin and consequences of altered DNA RT in ALL.

Influence of Repressive Histone and DNA Methylation upon D4Z4 Transcription in Non-Myogenic Cells.

We looked at a disease-associated macrosatellite array D4Z4 and focused on epigenetic factors influencing its chromatin state outside of the disease-context. We used the HCT116 cell line that contains the non-canonical polyadenylation (poly-A) signal required to stabilize somatic transcripts of the human double homeobox gene DUX4, encoded from D4Z4. In HCT116, D4Z4 is packaged into constitutive heterochromatin, characterized by DNA methylation and histone H3 tri-methylation at lysine 9 (H3K9me3), resulting in low basal levels of D4Z4-derived transcripts. However, a double knockout (DKO) of DNA methyltransferase genes, DNMT1 and DNMT3B, but not either alone, results in significant loss of DNA and H3K9 methylation. This is coupled with upregulation of transcript levels from the array, including DUX4 isoforms (DUX4-fl) that are abnormally expressed in somatic muscle in the disease Facioscapulohumeral muscular dystrophy (FSHD) along with DUX4 protein, as indicated indirectly by upregulation of bondafide targets of DUX4 in DKO but not HCT116 cells. Results from treatment with a chemical inhibitor of histone methylation in HCT116 suggest that in the absence of DNA hypomethylation, H3K9me3 loss alone is sufficient to facilitate DUX4-fl transcription. Additionally, characterization of a cell line from a patient with Immunodeficiency, Centromeric instability and Facial anomalies syndrome 1 (ICF1) possessing a non-canonical poly-A signal and DNA hypomethylation at D4Z4 showed DUX4 target gene upregulation in the patient when compared to controls in spite of retention of H3K9me3. Taken together, these data suggest that both DNA methylation and H3K9me3 are determinants of D4Z4 silencing. Moreover, we show that in addition to testis, there is appreciable expression of spliced and polyadenylated D4Z4 derived transcripts that contain the complete DUX4 open reading frame (ORF) along with DUX4 target gene expression in the thymus, suggesting that DUX4 may provide normal function in this somatic tissue.

A region of euchromatin coincides with an extensive tandem repeat on the mouse (Mus musculus) inactive X chromosome.

Euchromatic features are largely absent from the human inactive X chromosome (Xi), with the exception of several large tandem repeats that can be detected as euchromatin bands at metaphase. Despite residing megabases apart, these tandem repeats make frequent inactive X-specific interactions. The mouse homologue has been reported for at least one of the tandem repeats, but whether the mouse Xi is also characterized by distinct bands of euchromatin remains unknown. We examined the mouse Xi for the presence of euchromatin bands by examining the pattern of histone H3 dimethylated at lysine 4 and detected two major signals. The first band resides in the subtelomeric region of band XF5 and may correspond to the pseudoautosomal region. The second band localizes to XE3 and coincides with an extensive complex repeat composed of a large tandem and inverted repeat segment as well as several large short interspersed nuclear element (SINE)-rich tandem repeats. Fluorescence in situ hybridization reveals that sequences with homology to the repeat region are scattered along the length of the Y chromosome. Immunofluorescence analysis of histone H3 trimethylated at lysine 9 on metaphase chromosomes indicates that the repeat region corresponds to a band of constitutive heterochromatin on the male X and female active X chromosomes, whereas the euchromatin signal appears to be female specific. These data suggest that the band of euchromatin observed at XE3 is unique to the mouse Xi, comparable to the chromatin arrangement of several large tandem repeats located on the human X chromosome.