Transcripts of repetitive DNA elements signal to block phagocytosis of hematopoietic stem cells.

Macrophages maintain hematopoietic stem cell (HSC) quality by assessing cell surface Calreticulin (Calr), an "eat-me" signal induced by reactive oxygen species (ROS). Using zebrafish genetics, we identified Beta-2-microglobulin (B2m) as a crucial "don't eat-me" signal on blood stem cells. A chemical screen revealed inducers of surface Calr that promoted HSC proliferation without triggering ROS or macrophage clearance. Whole-genome CRISPR-Cas9 screening showed that Toll-like receptor 3 (Tlr3) signaling regulated b2m expression. Targeting b2m or tlr3 reduced the HSC clonality. Elevated B2m levels correlated with high expression of repetitive element (RE) transcripts. Overall, our data suggest that RE-associated double-stranded RNA could interact with TLR3 to stimulate surface expression of B2m on hematopoietic stem and progenitor cells. These findings suggest that the balance of Calr and B2m regulates macrophage-HSC interactions and defines hematopoietic clonality.

Leukemia-derived apelin selects endothelial niche clones to promote tumorigenesis.

Hematopoietic stem cells are regulated by endothelial and mesenchymal stromal cells in the marrow niche1-3. Leukemogenesis was long believed to be solely driven by genetic perturbations in hematopoietic cells but introduction of genetic mutations in the microenvironment demonstrated the ability of niche cells to drive disease progression4-8. The mechanisms by which the stem cell niche induces leukemia remain poorly understood. Here, using cellular barcoding in zebrafish, we found that clones of niche endothelial and stromal cells are significantly expanded in leukemic marrows. The pro-angiogenic peptide apelin secreted by leukemic cells induced sinusoidal endothelial cell clonal selection and transcriptional reprogramming towards an angiogenic state to promote leukemogenesis in vivo. Overexpression of apelin in normal hematopoietic stem cells led to clonal amplification of the niche endothelial cells and promotes clonal dominance of blood cells. Knock-out of apelin in leukemic zebrafish resulted in a significant reduction in disease progression. Our results demonstrate that leukemic cells remodel the clonal and transcriptional landscape of the marrow niche to promote leukemogenesis and provide a potential therapeutic opportunity for anti-apelin treatment.

A conserved transcription factor regulatory program promotes tendon fate.

Tendons, which transmit force from muscles to bones, are highly prone to injury. Understanding the mechanisms driving tendon fate would impact efforts to improve tendon healing, yet this knowledge is limited. To find direct regulators of tendon progenitor emergence, we performed a zebrafish high-throughput chemical screen. We established forskolin as a tenogenic inducer across vertebrates, functioning through Creb1a, which is required and sufficient for tendon fate. Putative enhancers containing cyclic AMP (cAMP) response elements (CREs) in humans, mice, and fish drove specific expression in zebrafish cranial and fin tendons. Analysis of these genomic regions identified motifs for early B cell factor (Ebf/EBF) transcription factors. Mutation of CRE or Ebf/EBF motifs significantly disrupted enhancer activity and specificity in tendons. Zebrafish ebf1a/ebf3a mutants displayed defects in tendon formation. Notably, Creb1a/CREB1 and Ebf1a/Ebf3a/EBF1 overexpression facilitated tenogenic induction in zebrafish and human pluripotent stem cells. Together, our work identifies the functional conservation of two transcription factors in promoting tendon fate.

Non-invasive determination of critical dissolved oxygen thresholds for stress physiology in fish using triple-oxygen stable isotopes and aquatic respirometry.

Understanding the critical thresholds of dissolved oxygen (O2) that trigger adaptive physiological responses in aquatic organisms is long hampered by a lack of robust, non-lethal or non-invasive methodologies. The isotope fractionation of triple O2 isotopes (18O/17O/16O) during respiration is linked to the amount of oxygen utilised, offering a potential avenue for new insights. Our experimental research involved measuring the oxygen isotope fractionation of dissolved O2 in closed-system aquatic respirometry experiments with wild sticklebacks (Gasterosteus aculeatus). These fish were either naturally adapted or experimentally acclimated to hypoxic and normoxic conditions. The aim was to observe their oxygen usage and isotope fractionation in response to increasingly severe hypoxia. Initial observations revealed a progressive 18O enrichment from the preferential uptake of 16O to a dissolved oxygen threshold of 3-5 mg O2 L-1, followed by an apparent reversal in oxygen isotope fractionation, which is mixing of 16O and 17O with the remaining O2 pool across all populations and indicative of a systematic change in oxygen metabolism among the fish. Unexpectedly, sticklebacks adapted to hypoxia but acclimated to normoxia exhibited stronger oxygen isotope fractionation compared to those adapted to normoxia and acclimated to hypoxia, contradicting the hypothesis that hypoxia adaptation would lead to reduced isotope discrimination due to more efficient oxygen uptake. These preliminary experimental results highlight the novel potential of using dissolved O2 isotopes as a non-invasive, non-lethal method to quantitatively assess metabolic thresholds in aquatic organisms. This approach could significantly improve our understanding of the critical oxygen responses and adaptation mechanisms in fish and other aquatic organisms across different oxygen environments, marking a significant step forward in aquatic ecological and physiological research.

Role of RNA modifications in blood development and regeneration.

Blood development and regeneration require rapid turnover of cells, and ribonucleic acid (RNA) modifications play a key role in it via regulating stemness and cell fate regulation. RNA modifications affect gene activity via posttranscriptional and translation-mediated mechanisms. Diverse molecular players involved in RNA-modification processes are abundantly expressed by hematopoietic stem cells and lineages. Close to 150 RNA chemical modifications have been reported, but only N6-methyl adenosine (m6A), inosine (I), pseudouridine (Ψ), and m1A-a handful-have been studied in-cell fate regulation. The role of RNA modification in blood diseases and disorders is an emerging field and offers potential for therapeutic interventions. Knowledge of RNA-modification and enzymatic activities could be used to design therapies in the future. Here, we summarized the recent advances in RNA modification and the epitranscriptome field and discussed their regulation of blood development and regeneration.

Isotope hydrology of the intermontane Elk Valley, British Columbia: an assessment of water resources around coal mining operations.

This study aimed to synthesise and interpret stable isotopic data (δ2H and δ18O) from various sources to understand the isotope hydrology around coal mine operations in Elk Valley, B.C., Canada. The data, including precipitation, groundwaters, seeps, and mine rock drains, were used to construct a local meteoric water line (LMWL) for the Elk Valley, evaluate the spatiotemporal isotopic composition of its groundwater, and assess mine seepage and mine rock drain discharge. The study revealed a robust LMWL relation (δ2H = 7.4 ± 0.2 · δ18O - 4.3 ± 4.1). The groundwater and seep data indicated a winter season bias and a north-south latitudinal gradient, suggesting rapid near-surface groundwater flow without significant post-precipitation evaporation. Porewater isotope samples from unsaturated mine rock piles (MRPs) showed site-specific evaporation patterns, potentially due to convective air flows or exothermic sulphide oxidation. This research revealed the influence of groundwater and meltwater on rock drain discharge. Based on evaporative mass balance calculations, MRPs seasonally contributed ca. 5 %(December base flow) and 22 % (snowmelt) to drain discharge. The findings underscore the value of stable isotope data collections in the Elk Valley to help better define and quantify the hydrology-hydrogeology, including a better understanding of evaporative conditions in MRPs.

Innovative Approach to Accelerate Wound Healing: Synthesis and Validation of Enzymatically Cross-Linked COL-rGO Biocomposite Hydrogels.

In this study, an innovative conductive hybrid biomaterial was synthetized using collagen (COL) and reduced graphene oxide (rGO) in order for it to be used as a wound dressing. The hydrogels were plasticized with glycerol and enzymatically cross-linked with horseradish peroxidase (HRP). A successful interaction among the components was demonstrated by FTIR, XRD, and XPS. It was demonstrated that increasing the rGO concentration led to higher conductivity and negative charge density values. Moreover, rGO also improved the stability of hydrogels, which was expressed by a reduction in the biodegradation rate. Furthermore, the hydrogel's stability against the enzymatic action of collagenase type I was also strengthened by both the enzymatic cross-linking and the polymerization of dopamine. However, their absorption capacity, reaching values of 215 g/g, indicates the high potential of the hydrogels to absorb fluids. The rise of these properties positively influenced the wound closure process, achieving an 84.5% in vitro closure rate after 48 h. These findings clearly demonstrate that these original composite biomaterials can be a viable choice for wound healing purposes.

Hematopoietic stem cell division is governed by distinct RUNX1 binding partners.

A defined number of hematopoietic stem cell (HSC) clones are born during development and expand to form the pool of adult stem cells. An intricate balance between self-renewal and differentiation of these HSCs supports hematopoiesis for life. HSC fate is determined by complex transcription factor networks that drive cell-type specific gene programs. The transcription factor RUNX1 is required for definitive hematopoiesis, and mutations in Runx1 have been shown to reduce clonal diversity. The RUNX1 cofactor, CBFý, stabilizes RUNX1 binding to DNA, and disruption of their interaction alters downstream gene expression. Chemical screening for modulators of Runx1 and HSC expansion in zebrafish led us to identify a new mechanism for the RUNX1 inhibitor, Ro5-3335. We found that Ro5-3335 increased HSC divisions in zebrafish, and animals transplanted with Ro5-3335 treated cells had enhanced chimerism compared to untreated cells. Using human CD34+ cells, we show that Ro5-3335 remodels the RUNX1 transcription complex by binding to ELF1, independent of CBFý. This allows specific expression of cell cycle and hematopoietic genes that enhance HSC self-renewal and prevent differentiation. Furthermore, we provide the first evidence to show that it is possible to pharmacologically increase the number of stem cell clones in vivo , revealing a previously unknown mechanism for enhancing clonal diversity. Our studies have revealed a mechanism by which binding partners of RUNX1 determine cell fate, with ELF transcription factors guiding cell division. This information could lead to treatments that enhance clonal diversity for blood diseases.

BF170 hydrochloride enhances the emergence of hematopoietic stem and progenitor cells.

Generation of hematopoietic stem and progenitor cells (HSPCs) ex vivo and in vivo, especially the generation of safe therapeutic HSPCs, still remains inefficient. In this study, we have identified compound BF170 hydrochloride as a previously unreported pro-hematopoiesis molecule, using the differentiation assays of primary zebrafish blastomere cell culture and mouse embryoid bodies (EBs), and we demonstrate that BF170 hydrochloride promoted definitive hematopoiesis in vivo. During zebrafish definitive hematopoiesis, BF170 hydrochloride increases blood flow, expands hemogenic endothelium (HE) cells and promotes HSPC emergence. Mechanistically, the primary cilia-Ca2+-Notch/NO signaling pathway, which is downstream of the blood flow, mediated the effects of BF170 hydrochloride on HSPC induction in vivo. Our findings, for the first time, reveal that BF170 hydrochloride is a compound that enhances HSPC induction and may be applied to the ex vivo expansion of HSPCs.

A chronic signaling TGFb zebrafish reporter identifies immune response in melanoma.

Developmental signaling pathways associated with growth factors such as TGFb are commonly dysregulated in melanoma. Here we identified a human TGFb enhancer specifically activated in melanoma cells treated with TGFB1 ligand. We generated stable transgenic zebrafish with this TGFb Induced Enhancer driving green fluorescent protein (TIE:EGFP). TIE:EGFP was not expressed in normal melanocytes or early melanomas but was expressed in spatially distinct regions of advanced melanomas. Single-cell RNA-sequencing revealed that TIE:EGFP+ melanoma cells down-regulated interferon response while up-regulating a novel set of chronic TGFb target genes. ChIP-sequencing demonstrated that AP-1 factor binding is required for activation of chronic TGFb response. Overexpression of SATB2, a chromatin remodeler associated with tumor spreading, showed activation of TGFb signaling in early melanomas. Confocal imaging and flow cytometric analysis showed that macrophages localize to TIE:EGFP+ regions and preferentially phagocytose TIE:EGFP+ melanoma cells compared to TIE:EGFP- melanoma cells. This work identifies a TGFb induced immune response and demonstrates the need for the development of chronic TGFb biomarkers to predict patient response to TGFb inhibitors.

Compound-specific stable isotope analyses of fatty acids indicate feeding zones of zooplankton across the water column of a subalpine lake.

Spatial and temporal zooplankton feeding dynamics across the water column of lakes are key for understanding site-specific acquisition of diet sources. During this 6-week lake study, we examined stable carbon (δ13C) and nitrogen (δ15N) isotopes and conducted compound-specific fatty acid (FA) stable isotope analysis (CSIA) of edible seston in the epi-, meta-, and hypolimnion, and zooplankton of Lake Lunz, Austria. We predicted that CSIA of essential FA can discern the foraging grounds of zooplankton more accurately than the commonly used bulk stable isotopes. The δ13C and δ15N values of seston from different lake strata were similar, whereas a dual CSIA approach using stable carbon and hydrogen isotopes of FA (δ13CFA and δ2HFA) provided sufficient isotopic difference in essential FA to discern different lake strata-specific diet sources throughout the study period. We present a CSIA model that suggests strata-specific foraging grounds for different zooplankton groups, indicating higher preference of cladocerans for feeding on epilimnetic diet sources, while calanoid copepods retained more hypolimnetic resources. The CSIA approach thus yields strata-specific information on foraging strategies of different zooplankton taxa and provides more details on the spatial and temporal trophodynamics of planktonic food webs than commonly used bulk stable isotopes.

Specific oncogene activation of the cell of origin in mucosal melanoma.

Mucosal melanoma (MM) is a deadly cancer derived from mucosal melanocytes. To test the consequences of MM genetics, we developed a zebrafish model in which all melanocytes experienced CCND1 expression and loss of PTEN and TP53. Surprisingly, melanoma only developed from melanocytes lining internal organs, analogous to the location of patient MM. We found that zebrafish MMs had a unique chromatin landscape from cutaneous melanoma. Internal melanocytes could be labeled using a MM-specific transcriptional enhancer. Normal zebrafish internal melanocytes shared a gene expression signature with MMs. Patient and zebrafish MMs have increased migratory neural crest gene and decreased antigen presentation gene expression, consistent with the increased metastatic behavior and decreased immunotherapy sensitivity of MM. Our work suggests the cell state of the originating melanocyte influences the behavior of derived melanomas. Our animal model phenotypically and transcriptionally mimics patient tumors, allowing this model to be used for MM therapeutic discovery.

Colloidal and Biological Characterization of Dual Drug-Loaded Smart Micellar Systems.

Smart polymeric micelles (PMs) are of great interest in drug delivery owing to their low critical micellar concentration and sizes. In the present study, two different pH-sensitive poly(2-vinyl pyridine)-b-poly(ethylene oxide) (P2VP-b-PEO) copolymer samples were used for the encapsulation of paclitaxel (PTX), ursolic acid (UA), and dual loading of PTX and UA. Based on the molecular features of copolymers, spherical PMs with sizes of around 35 nm and 140 nm were obtained by dialysis for P2VP55-b-PEO284 and P2VP274-b-PEO1406 samples, respectively. The micellar sizes increased after loading of both drugs. Moreover, drug encapsulation and loading efficiencies varied from 53 to 94% and from 3.2 to 18.7% as a function of the copolymer/drug ratio, molar mass of copolymer sample, and drug type. By FT-IR spectroscopy, it was possible to demonstrate the drug loading and the presence of some interactions between the polymer matrix and loaded drugs. In vitro viability was studied on 4T1 mammary carcinoma mouse cells as a function of time and concentration of drug-loaded PMs. UA-PMs and free PMs alone were not effective in inhibiting the tumor cell growth whereas a viability of 40% was determined for cells treated with both PTX- and PTX/UA-loaded PMs. A synergic effect was noticed for PTX/UA-loaded PMs.

Chitosan-PVA-PVP/nano-clay composite: a promising tool for controlled drug delivery.

In this study, chitosan, polyvinyl alcohol (PVA), and polyvinyl pyrrolidone (PVP) were used to create ternary blends reinforced with organically modified montmorillonite nanoclay. Tramadol was used as a model drug to assess the efficacy of these ternary blends as drug delivery systems. The current work demonstrated the highly controlled release of tramadol via transdermal administration. The results of the FTIR investigation revealed the compatibility of the blending components. Among non-drug-loaded formulations, MC6 is the most stable with a 17.6% weight residue at 505 °C and MC11 is the most stable of all the drug-loaded and non-drug-loaded formulations with a weight residue of 22.0% at 505 °C. The XRD studies of the prepared formulations showed crystalline behavior. However, the SEM analysis revealed that no gaps or mixing components were uniformly dispersed in the nanocomposites. Pharmaceutical tests, such as swelling, dissolution, and permeation rates, revealed a strong influence of the PVA concentration. There was a uniform distribution of drug throughout the films with maximum encapsulation efficiency found for MC7 (96.09 ± 0.31) and minimum encapsulation efficiency for MC11 (90.56 ± 0.34)%. Compared to the sodium acetate (pH 4.5) and potassium phosphate buffers (pH 6.8) the swelling and erosion were higher in hydrochloric acid buffer (pH 1.2). An increase in PVA concentration (or decrease in PVP concentration) increases the swelling, dissolution, and permeation rates. In addition, erosion increased with increasing PVP concentration. Furthermore, the nanoclay-reinforced composite showed high permeation. Based on the obtained results, it can be concluded that the produced nanocomposite could be used as an efficient transdermal drug delivery system.

Synthesis and Preliminary Evaluation of an ASGPr-Targeted Polycationic ß-Cyclodextrin Carrier for Nucleosides and Nucleotides.

Most antiviral and anticancer nucleosides are prodrugs that require stepwise phosphorylation to their triphosphate nucleotide form for biological activity. Monophosphorylation may be rate-limiting, and the nucleotides may be unstable and poorly internalized by target cells. Effective targeting and delivery systems for nucleoside drugs, including oligonucleotides used in molecular therapeutics, could augment their efficacy. The development of a carrier designed to effect selective transmembrane internalization of nucleotides via the asialoglycoprotein receptor (ASGPr) is now reported. In this work, the polycationic, polygalactosyl drug delivery carrier heptakis[6-amino-6-deoxy-2-O-(3-(1-thio-ß-D-galactopyranosyl)-propyl)]-ß-cyclodextrin hepta-acetate salt (GCyDAc), potentially a bifunctional carrier of (poly)nucleotides, was modeled by molecular docking in silico as an ASGPr-ligand, then synthesized for testing. The antivirals arabinosyl adenine (araA, vidarabine, an early generation antiviral nucleoside), arabinosyl adenine 5'-monophosphate (araAMP), and 12-mer-araAMP (p-araAMP) were selected for individual formulation with GCyDAc to develop this concept. Experimentally, beta cyclodextrin was decorated with seven protonated amino substituents on the primary face, and seven thiogalactose residues on its secondary face. AraA, araAMP, and p-araAMP were individually complexed with GCyDAc and complex formation for each drug was confirmed by differential scanning calorimetry (DSC). Finally, the free drugs and their GCyDAc complexes were evaluated for antiviral activity using ASGPr-expressing HepAD38 cells in cell culture. In this model, araA, araAMP, and p-araAMP showed relative antiviral potencies of 1.0, 1.1, and 1.2, respectively. In comparison, GCyDAc-complexes of araA, araAMP, and p-araAMP were 2.5, 1.3, and 1.2 times more effective than non-complexed araA in suppressing viral DNA production. The antiviral potencies of these complexes were minimally supportive of the hypothesis that ASGPr-targeted, CyD-based charge-association complexation of nucleosides and nucleotides could effectively enhance antiviral efficacy. GCyDAc was non-toxic to mammalian cells in cell culture, as determined using the MTS proliferation assay.

Mechanical Properties and Degradation Rate of Poly(Sorbitol Adipate-Co-Dioladipate) Copolymers Obtained with a Catalyst-Free Melt Polycondensation Method.

A new family of polyester-based copolymers-poly(sorbitol adipate-co-ethylene glycol adipate) (PSAEG), poly(sorbitol adipate-co-1,4 butane diol adipate) (PSABD), and poly (sorbitol adipate-co-1,6 hexane diol adipate) (PSAHD)-was obtained with a catalyst-free melt polycondensation procedure using the multifunctional non-toxic monomer sorbitol, adipic acid, and diol, which are acceptable to the human metabolism. Synthesized polyesters were characterized by FTIR and 1H NMR spectroscopy. The molecular weight and thermal properties of the polymers were determined by MALDI mass spectroscopy, differential scanning calorimetry (DSC), and thermogravimetric analysis. The degradation rate was investigated, at 37 °C, in 0.1M NaOH (pH 13) and in phosphate-buffered solution (PBS) at pH 7.4. It was found that the polymers degraded faster in NaOH (i.e., in a day) compared to their degradation in PBS, which was much slower (in a week). The highest degradation rate was noticed for the PSAEG sample in both media, whereas PSAHD was the most stable polymer at pH 7.4 and 13. A reduced hydrophilicity of the polymers with diol length was indicated by low swelling percentage and sol content in water and DMSO. Mechanical studies prove that all the polymers are elastomers whose flexibility increases with diol length, shown by the increase in percentage of elongation at break and the decrease in tensile stress and Young's modulus. These biodegradable copolymers with adaptable physicochemical characteristics might be useful for a broad variety of biological applications by merely varying the length of the diol.

Evaluation of lipid extraction methods for fatty acid quantification and compound-specific δ13C and δ2Hn analyses.

Lipids, with fatty acids (FA) as a crucial subset, have become a focal point for diverse medical, physiological, and ecological studies. However, a comprehensive assessment of the various pre-analytical FA extraction methods published in the scientific literature remains lacking. In this study, we examined the efficacy of seven well-established sample preparation methods, specifically focusing on their effectiveness in total lipid and fatty acid extraction and their impact on compound-specific stable hydrogen (δ2H) and carbon (δ13C) isotope values. We also considered the repercussions of FA removal efficacy on residual bulk tissue δ2Hn analysis, because lipids typically have low δ2H values. Our findings showed that in most cases chloroform-based extraction methods outperformed those without chloroform. While discrepancies were not as evident for smaller organisms, such as plankton, marked variations were discernible in the extraction efficiencies for muscle and liver samples, which was also manifested in the residual bulk tissue δ2Hn results. Notably, most extraction methods had little effect on specific δ13C or δ2H isotope values of FA; instead, an emphasis should be on using an extraction method that achieves optimal baseline peak separation of the chromatograms for C and H isotope measurements.

Radiocarbon integrity of dissolved inorganic carbon (DIC) samples stored in plastic and glass bottles: implications for reliable groundwater age dating.

Various approaches based on the natural variations of carbon isotopes (14C and 13C) in dissolved inorganic carbon (DIC) are routinely used to study groundwater dynamics and to estimate recharge rates by deriving groundwater ages. However, differences in 14C activities in groundwater samples collected repeatedly from the same wells and discordantly young 14C groundwater ages compared to noble gases led some authors to question the validity of radiocarbon dating. Poor sampling protocols and storage effects (14C contamination) for radiocarbon analysis are a critical factor in explaining age determination discrepancies. We evaluated the impact of storage protocols on carbon isotope exchange with atmospheric carbon dioxide by comparing glass versus standard plastic field sampling bottles for various storage times before radiocarbon and 13C analyses. The 14C bias after 12 months in pre-evacuated glass vials was minimal and within analytical precision. However, storage of DIC samples in plastic sampling bottles led to marked changes in 14C and 13C contents (up to ∼15 pmC and ∼ 5 ‰, respectively, after 12 months), meaning contamination led to younger groundwater age estimations than it should have been. Protocols for sampling and storing DIC samples for radiocarbon using pre-evacuated glass bottles help avoid atmospheric 14CO2 contamination and microbial activity.