In the field of sketch generation, raster-format trained models often produce non-stroke artifacts, while vector-format trained models typically lack a holistic understanding of sketches, leading to compromised recognizability. Moreover, existing methods struggle to extract common features from similar elements (e.g., eyes of animals) appearing at varying positions across sketches. To address these challenges, we propose StrokeFusion, a two-stage framework for vector sketch generation. It contains a dual-modal sketch feature learning network that maps strokes into a high-quality latent space. This network decomposes sketches into normalized strokes and jointly encodes stroke sequences with Unsigned Distance Function (UDF) maps, representing sketches as sets of stroke feature vectors. Building upon this representation, our framework exploits a stroke-level latent diffusion model that simultaneously adjusts stroke position, scale, and trajectory during generation. This enables high-fidelity sketch generation while supporting stroke interpolation editing. Extensive experiments on the QuickDraw dataset demonstrate that our framework outperforms state-of-the-art techniques, validating its effectiveness in preserving structural integrity and semantic features. Code and models will be made publicly available upon publication.

Diamond-Blackfan Anemia (DBA) is a rare congenital bone marrow failure disorder with patients manifesting macrocytic anemia in infancy (Da Costa, et al. Blood 2020; Da Costa, et al. F1000Res. 2018). DBA is caused by germline heterozygous loss-of-function mutations in one of the twenty small- or large-subunit ribosomal protein (RP) genes. Current therapies for DBA include chronic red blood cell transfusions, glucocorticoid treatment, and allogeneic hematopoietic stem cell transplantation, all of which are associated with severe toxicities. The mutations in RP genes cause defects in ribosomes, leading to ribosomal stress and aberrant p53 activation. Over-activation of p53 is a crucial mediator of DBA-associated hematopoietic defects, including erythroid failure and anemia. Previous studies (Taylor, et al. Sci Transl Med. 2020; Taylor, et al. Exp Hematol. 2012) have demonstrated that calmodulin (CaM) inhibitors, including the FDA-approved anti-psychotic trifluoperazine (TFP), improve anemia in multiple models of DBA by reducing activation of p53 targets. However, TFP is associated with serious side effects, including neurotoxicity attributed to its high brain penetrance and interaction with dopamine D2 receptors (DRD2) in the central nervous system (CNS). We therefore embarked on a drug discovery program to identify novel CaM inhibitors with improved potency compared to TFP, but with activity against DRD2 eliminated and CNS penetrance attenuated. This effort resulted in the discovery of two novel small molecules, FTX-1 and FTX-2. Relative to TFP, FTX-1 and FTX-2 showed superior activity in reducing aberrant p53 activity in in vitro CD34+ human hematopoietic stem and progenitor cell (HSPC) models designed to mimic DBA via the introduction of ribosomal mutations in RPS19, RPL5, or RPL11 with CRISPR-Cas9 editing. Activation of p53 was assessed by monitoring the mRNA levels of CDKN1A/p21, a direct transcriptional target of p53. Compared to DMSO, 1 µM FTX-2 significantly attenuated p53 activity in the RPS19 model (p < 0.0001). This rescue was significantly greater than the positive control TFP, which rescued p53 activity at 3 µM (p < 0.01- P < 0.0001). FTX-2 was also significantly active in reducing p53 activity at 1 µM in the RPL5 and RPL11 models (p < 0.05 - < 0.0001, depending upon the donor and the experimental replicate), while 3 µM TFP was only active in the RPL5 model (p < 0.05-0.0001) but not in the RPL11 model. FTX-1 and FTX-2 were also effective in improving anemia in a rps29-/- zebrafish model of DBA. This model recapitulates the maturation arrest of red blood cells, growth retardation, decreased globin synthesis, and elevated p53 activity commonly observed in DBA patients. FTX-1 and FTX-2 significantly rescued hemoglobin levels at doses of 0.1 µM and 0.001 µM (p < 0.05 - <0.0001), respectively, compared to the positive control fluphenazine at 10 µM (p < 0.05 - <0.01). Lastly, 1 µM FTX-1 was effective in restoring erythroid development by increasing the BFU-E and CFU-E colonies in RPS19 DBA patient-derived HSPCs compared to vehicle (adjusted p-value < 0.05) as assessed using a colony formation assay. FTX-2 is currently being explored in toxicology and safety pharmacology studies in support of an Investigational New Drug (IND) application, with first-in-human studies anticipated in 2026.

Stress erythropoiesis elevates the rate of red blood cell (RBC) production as a physiological response to stressors such as anemia or hypoxia. In acute anemia, RBC progenitors and precursors temporarily rewire their transcriptome, up- and downregulating hundreds of genes to accelerate the production of mature RBCs. Effective regeneration requires communication between critical cytokine signals (e.g., BMP4) and cis-regulatory elements on chromatin which coordinate transcriptional changes. To identify cis-regulatory changes that underlie anemia-specific gene expression and cellular responses, we analyzed chromatin accessibility in populations of cells enriched for red blood cell precursors isolated from mice at a range of time points after anemia induction. Early in the anemia response, chromatin is transiently open at AP-1-containing regions, correlated with increased Jun and Fos transcript/protein levels. Jun knockdown ex vivo decreases the percentage of KIT+ erythroid precursors after anemia induction. We observe a second rewiring event at time points consistent with anemia resolution, involving repression of GATA factor-accessible regions and activation of ETS factor-accessible regions. In both mouse in vivo models and human CD34+ cells stimulated with BMP4, accessibility changes at regions with prior associations to human blood phenotypes. Dozens of BMP4- and anemia-activated loci are sensitive to natural human variation. The representation of red blood cell trait-associated loci in ATAC-seq data remains durably elevated more than 1 month after anemia resolution. Together, these findings provide a framework to understand the early establishment and late resolution of a regeneration-dependent transcriptome in RBC precursors.

T cell-mediated tumor killing underlies immunotherapy success. Here, we used long-term in vivo imaging and high-resolution spatial transcriptomics of zebrafish endogenous melanoma, as well as multiplex imaging of human melanoma, to identify domains facilitating the immune response during immunotherapy. We identified cancer regions of antigen presentation and T cell engagement and retention (CRATERs) as pockets at the stroma-melanocyte boundaries of zebrafish and human melanoma. CRATERs are rich in antigen-recognition molecules, harboring the highest density of CD8+ T cells in tumors. In zebrafish, CD8+ T cells formed prolonged interactions with melanoma cells within CRATERs, characteristic of antigen recognition. Following immunostimulatory treatment, CRATERs expanded, becoming the major sites of activated CD8+ T cell accumulation and tumor killing. In humans, elevation in CRATER density in biopsies following immune checkpoint blockade (ICB) therapy correlated with a clinical response to therapy. CRATERs are structures that show active tumor killing and may be useful as a diagnostic indicator for immunotherapy success.

Hematopoietic stem and progenitor cells (HSPCs) reside in specialized niche microenvironments in the marrow made of sinusoidal vascular endothelial cells (ECs) and other perivascular supportive cells. Pathological conditions such as primary myelofibrosis (PMF) in the marrow cause HSPC niche defects and pan-cytopenia. Extramedullary HSPC niche can form in the liver or the spleen sinusoids during PMF, but the process is inefficient. Here, we aim to identify the transcription factor (TF) code that specifies the sinusoidal vascular EC fate in the HSPC niche. We performed differential gene expression analysis on ECs from adult zebrafish kidney marrow and liver and identified TF candidates that were uniquely upregulated in the marrow sinusoidal ECs, namely tfec, mafbb, foxp4, irf8, and hoxb8a. To determine whether these candidate TFs can functionally specify a HSPC niche EC fate, we selectively overexpressed them using a zebrafish liver sinusoidal EC-specific enhancer in vivo. Upon the overexpression of tfec and mafbb together, we found adult zebrafish liver sinusoidal ECs were reprogrammed to upregulate key genes known for HSPC niche supportive functions, including mrc1a (log2FC=4.9, p<0.005), lyve1b (log2FC=3.6, p<0.005) and dab2 (log2FC=5.0, p<0.005). Transplant assay of liver cells into irradiated adult hosts showed that primary HSPCs occupy the newly reprogrammed liver vascular niche (7/26 in the reprogram group vs. 0/20 in the control group) (p=0.0296). Therefore, TFs tfec and mafbb were sufficient in reprogramming adult liver sinusoidal ECs to become HSPC niche in vivo. Furthermore, we aim to translate our findings to program human iPSC-derived ECs to support primary HSPCs in vitro. We engineered human iPSC lines with inducible overexpression of human ETV2, TFEC and MAFB. Upon ETV2-directed differentiation hiPSCs into ECs, the overexpression of TFEC and MAFB significantly upregulated the expression of sinusoidal endothelial and HSPC niche supportive genes, such as MRC1 (log2FC=3.6, p<0.005), STAB2 (log2FC=10.3, p<0.005), JAG1 (log2FC=1.7 p<0.005), and CXCL12 (log2FC=5.1 p< 0.005). Methylcellulose assays showed that CD34+CD45+ HSPCs co-cultured with hiPSC-derived ECs induced with ETV2 plus TFEC and MAFB contained significantly more colony-forming units (CFUs) compared to those co-cultured with ECs induced with ETV2 alone or HSPCs cultured without ECs: CFU-GEMM (23.3±2.4 vs. 10.0±2.1, p=0.004), CFU-G (89.3±13.4 vs. 46.0±4.2, p=0.027), and CFU-E (42.3±3.3 vs. 26.7±0.9, p=0.021). Transplant of HSPCs into immunodeficient mouse hosts showed that HSPCs co-cultured with TFEC and MAFB induced ECs have significantly better engraftment potential than control HSPCs (p=0.0164). In summary, TFs TFEC and MAFB could program human iPSC-derived ECs to adopt HSPC niche fate and support primary human cord blood-derived CD34+ HSPCs in vitro. Our findings provide a method to engineer human HSPC niche-like sinusoidal ECs to enhance engraftment of HPSC, which could help transplantation therapies.

Gene expression is a process through which genetic information is decoded and manufactured into a functional gene product. Different cell types within an organism exhibit distinct biological functions, despite containing the same genetic material, or DNA sequence. This in part can be attributed to the various ways gene expression in a cell is regulated. At a microscopic level, a DNA template is "transcribed" into RNA and then "translated" into a protein, the functional gene product. This process involves the coordination between multiple dynamic events, which are subject to regulation at each step. These regulatory events happen in multiple levels, namely the transcriptional, the posttranscriptional, the translational, and finally, the posttranslational level. Processes integral to multicellular organismal development occur through a series of gene regulatory events, cascading in a gene product that determines cell fate. Proper regulation of gene expression is crucial to the development of healthy living organisms. Since transcription is the very first step of gene expression, predicting transcriptional control that occurs during human development and disease is critical. Combination of multiple genome-wide next-generation sequencing approaches ("multi-omics") within a particular biological system could be a means to achieve that. This may eventually help in developing therapeutics for human disorders related to the particular tissue type. Here, we have used multiomics by combining ChIP-seq, RNA-seq, and ATAC-seq in human hematopoietic stem and progenitor cells. This approach enabled us highlight certain hitherto unknown transcriptional control mechanisms during red blood cell development from hematopoietic stem cells. The performed analyses were able to predict gene sets, their enhancers, and the hematopoietic master and signaling transcription factors that control them during human red cell differentiation.

Nearly one-fifth of patients with non-small cell Lung Cancer (NSCLC) will develop liver metastases (LMs), and the overall treatment strategy of LMs will directly affect the survival of patients. However, some retrospective studies have found that patients receiving chemotherapy or targeted therapy have a poorer prognosis once LMs develop. In recent years, multiple randomised controlled trials (RCTS) have shown significant improvements in outcomes for patients with advanced lung cancer following the introduction of immune checkpoint inhibitors (ICIs) compared to conventional chemotherapy. ICIs is safe and effective in patients with LMs, although patients with LMs are mostly underrepresented in randomised clinical trials. However, NSCLC patients with LMs have a significantly worse prognosis than those without LMs when treated with ICIs, and the mechanism by which LMs induce systemic anti-tumour immunity reduction is unknown, so the management of LMs in patients with NSCLC is a clinical challenge that requires more optimised therapies to achieve effective disease control. In this review, we summarised the mechanism of ICIs in the treatment of LMs, the clinical research and treatment progress of ICIs and their combination with other therapies in patients with LMs from NSCLC.

Stress granules (SGs) are crucial in RNA regulation, affecting cell fate and function. SGs contain RNAs, some of which can be methylated. We studied m6A RNA modifications during the human CD34+ HSPCs (hCD34+) differentiating into erythroid cells and found that mRNAs encoding many erythroid-specific proteins had decreased methylation during differentiation. Increased levels of ALKBH5 demethylase during erythropoiesis control the levels of the 3'UTR methylation of these mRNAs. hCD34+ carrying ALKBH5 mutations demonstrated a block in erythropoiesis, and mass-spectrometry studies of the mutant cells showed decreased levels of SG proteins, including the core granule protein ATXN2. ALKBH5 directly regulates the methylation of the mRNA of ATXN2. ATXN2 overexpression accelerated the erythroid differentiation of HSPCs and rescued the erythroid differentiation of ALKBH5 mutant cells. Very few SGs are found in normal human erythroid progenitors. SGs accumulated substantially in ALKBH5 mutant cells, and surprisingly overexpression of ATNX2 reduced SG numbers to normal. Polysome analysis demonstrated m6A-modified RNAs to be enriched in the pre-polysome fractions that were less translated. This work establishes a mechanism by which during stress, ATXN2 facilitates the release of SG-stored m6A-modified RNAs including erythroid-specific and SG-enriched RNAs that are loaded onto functional ribosomes, allowing better translation and accelerated erythroid differentiation during stress. ### Competing Interest Statement The authors have declared no competing interest.

Immunotherapy leads to cancer eradication despite the tumor's immunosuppressive environment. Here, we used extended long-term in-vivo imaging and high-resolution spatial transcriptomics of endogenous melanoma in zebrafish, and multiplex imaging of human melanoma, to identify domains that facilitate immune response during immunotherapy. We identified crater-shaped pockets at the margins of zebrafish and human melanoma, rich with beta-2 microglobulin (B2M) and antigen recognition molecules. The craters harbor the highest density of CD8+ T cells in the tumor. In zebrafish, CD8+ T cells formed prolonged interactions with melanoma cells within craters, characteristic of antigen recognition. Following immunostimulatory treatment, the craters enlarged and became the major site of activated CD8+ T cell accumulation and tumor killing that was B2M dependent. In humans, craters predicted immune response to ICB therapy, showing response better than high T cell infiltration. This marks craters as potential new diagnostic tool for immunotherapy success and targets to enhance ICB response.

More than 1.9 million new colorectal cancer (CRC) cases and 935000 deaths were estimated to occur worldwide in 2020, representing about one in ten cancer cases and deaths. Overall, colorectal ranks third in incidence, but second in mortality. More than half of the patients are in advanced stages at diagnosis. Treatment options are complex because of the heterogeneity of the patient population, including different molecular subtypes. Treatments have included conventional fluorouracil-based chemotherapy, targeted therapy, immunotherapy, etc. In recent years, with the development of genetic testing technology, more and more targeted drugs have been applied to the treatment of CRC, which has further prolonged the survival of metastatic CRC patients.

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.

This paper aimed to observe the effect of adjuvant chemotherapy with adriamycin loaded nano-gel on osteosarcoma. 120 patients with osteosarcoma who visited the hospital from January 2018 to February 2022 were selected as control group and nano group by the red blue ball method. The control group received conventional doxorubicin combined with ifosfamide chemotherapy. The nano group was treated with doxorubicin loaded nano-gel as adjuvant chemotherapy before surgery. Both groups received limb salvage surgery. Serum alkaline phosphatase (ALP), lactate dehydrogenase (LDH), vascular endothelial growth factor (VEGF), and basic fibroblast growth factor (bFGF) were compared between two groups before, after, and 1 day after chemotherapy. Follow up records were kept for 12 months to obtain adverse reactions to chemotherapy, postoperative clinical efficacy, and progression free survival time (PFS). No significant difference existed in ALP, LDH, VEGF, and bFGF levels between two groups of patients before chemotherapy (P > 0.05). For the overall comparison, inter group, time, and interaction effects had statistical significance (P < 0.05). For the comparison of two groups, the average ALP, LDH, VEGF, and bFGF in the nano group were lower after chemotherapy. The incidence of grade III–IV adverse reactions in the nano group was 10% lower than the controlling group. The CR+PR rate in the nano group was higher. The preoperative application of adriamycin loaded nano-gel as adjuvant chemotherapy can make the patients’ prognosis with osteosarcoma improved. It can enhance the chemotherapy effect, control the degree of adverse reactions of chemotherapy, and improve the clinical efficiency, thus extending the progression free survival period of patients with osteosarcoma.

Extensive research has been conducted on the plasticity of malignant cells and nonmalignant cells in solid tumor. However, the plasticity of bone marrow hematopoietic cells in leukemia have remained largely unexplored. In this study, we aimed to investigate cell changings in hematopoietic cells through lineage tracing across various types of leukemias. We had compiled a landscape of leukemia and constructed phylogenetic trees of hematopoietic cells through utilizing massively parallel scRNA-seq data, mtDNA mutation and SNP analysis. Based on the observed cell changings, we identified several types of cell changings, including transdifferentiation, dedifferentiation, and state transition, except for differentiation and expansion. In AML and CMML, GMPs and neutrophils showed a higher potential for transferring to other cell types. In BPDCN, pDCs were less prone to switching to other cell types, while T cells demonstrated high plasticity. In B-ALL and B-CLL, B-ALL blast cells and B-CLL blast cells emerged at the most dynamic state. The dynamics of hematopoietic cells in AML, BPDCN and ALL changed along with the clinical process. Extrinsic factors within the leukemia microenvironment may influence the cell changings. Regulons encountered an intermediate cell state during the process of transition to myeloid cells and erythroid cells. We also found a correlation between B-common blast cells and T cells, suggesting a potential transition from B lymphoblastic leukemia to T lymphoblastic leukemia. In conclusion, our study unveiled the distinct plasticity and dynamics of hematopoietic cells in various types of leukemia. This sheds light on the possibility of targeting cell changes as a new strategy for leukemia treatment and improving current immunotherapy.### Competing Interest StatementThe authors have declared no competing interest.

Solasonine (SS) is a natural glycoalkaloid compound that has been reported to possess a significant anticancer function. However, its anticancer effects and related mechanisms in osteosarcoma (OS) have not been studied. This study sought to investigate the impact of SS on the growth of OS cells. OS cells were treated with different concentrations of SS for 24[Formula: see text]h, and the results showed that SS attenuated the survival of OS cells in a dose-dependent manner. Additionally, SS suppressed cancer stem-like properties and epithelial-mesenchymal transition (EMT) by inhibiting aerobic glycolysis in OS cells in an ALDOA-dependent manner. Additionally, SS reduced the levels of Wnt3a, [Formula: see text]-catenin, and Snail in OS cells in vitro. Furthermore, Wnt3a activation reversed the SS-induced inhibition of glycolysis in OS cells. Collectively, this study discovered a novel effect of SS in inhibiting aerobic glycolysis, in addition to cancer stem-like features and EMT, implying that SS could be a therapeutic candidate for OS treatment.

Supplemental Figure 1. Uncropped immunoblots. Uncropped images of the immunoblots presented in Figures 2A, 2B, and 2C.

Supplemental Figure 3. Proliferation and viability in a p53-expressing MM cell line. A, Immuno blot for p53 and CKS1B in parental and CKS1B-overexpressing MM cell lines. B, Proliferation of parental and CKS1B-overexpressing MM cell lines upon mock treatment, treatment with MLN4924, or treatment with one of three concentrations of bortezomib. C, Titrated viability of parental and CKS1B-overexpressing MM cell lines upon treatment with different concentrations of MLN4924.

Altered hematopoietic stem cell (HSC) fate underlies primary blood disorders but microenvironmental factors controlling this are poorly understood. Genetically barcoded genome editing of synthetic target arrays for lineage tracing (GESTALT) zebrafish were used to screen for factors expressed by the sinusoidal vascular niche that alter the phylogenetic distribution of the HSC pool under native conditions. Dysregulated expression of protein kinase C delta (PKC-δ, encoded by prkcda) increases the number of HSC clones by up to 80% and expands polyclonal populations of immature neutrophil and erythroid precursors. PKC agonists such as cxcl8 augment HSC competition for residency within the niche and expand defined niche populations. CXCL8 induces association of PKC-δ with the focal adhesion complex, activating extracellular signal-regulated kinase (ERK) signaling and expression of niche factors in human endothelial cells. Our findings demonstrate the existence of reserve capacity within the niche that is controlled by CXCL8 and PKC and has significant impact on HSC phylogenetic and phenotypic fate.

The pathogenesis of thyroid dysgenesis (TD) is not well understood. Here, using a combination of single-cell RNA and spatial transcriptome sequencing, we identify a subgroup of NF-κB-activated thyrocytes located at the center of thyroid tissues in postnatal mice, which maintained a partially mesenchymal phenotype. These cells actively protruded out of the thyroid primordium and generated new follicles in zebrafish embryos through continuous tracing. Suppressing NF-κB signaling affected thyrocyte migration and follicle formation, leading to a TD-like phenotype in both mice and zebrafish. Interestingly, during thyroid folliculogenesis, myeloid cells played a crucial role in promoting thyrocyte migration by maintaining close contact and secreting TNF-α. We found that cebpa mutant zebrafish, in which all myeloid cells were depleted, exhibited thyrocyte migration defects. Taken together, our results suggest that myeloid-derived TNF-α-induced NF-κB activation plays a critical role in promoting the migration of vertebrate thyrocytes for follicle generation.

Stem cell transplantation presents a potentially curative strategy for genetic disorders of skeletal muscle, but this approach is limited by the deleterious effects of cell expansion in vitro and consequent poor engraftment efficiency. In an effort to overcome this limitation, we sought to identify molecular signals that enhance the myogenic activity of cultured muscle progenitors. Here, we report the development and application of a cross-species small-molecule screening platform employing zebrafish and mice, which enables rapid, direct evaluation of the effects of chemical compounds on the engraftment of transplanted muscle precursor cells. Using this system, we screened a library of bioactive lipids to discriminate those that could increase myogenic engraftment in vivo in zebrafish and mice. This effort identified two lipids, lysophosphatidic acid and niflumic acid, both linked to the activation of intracellular calcium-ion flux, which showed conserved, dose-dependent, and synergistic effects in promoting muscle engraftment across these vertebrate species.