Chimeric antigen receptor (CAR) T-cell therapy has significantly improved the prognosis of relapsed B-cell acute lymphoblastic leukemia (B-ALL) patients with recurrent disease after allogeneic hematopoietic stem cell transplantation (allo-HSCT). However, whether to use donor- or patient-derived CAR T cells has not yet been clarified.

High mobility group box 3 (HMGB3) acts as an essential participator in fundamental biological processes, including transcriptional regulation, chromatin remodeling and DNA repair. HMGB3 is highly expressed and functionally essential during embryonic development, particularly in the hematopoietic and nervous systems, but it is significantly downregulated or silenced in most normal adult tissues. Its aberrant upregulation has been revealed in numerous human malignancies, such as leukemia, as well as breast, bladder, colorectal and gastric cancer, and its expression levels have been established to be closely associated with poor prognosis of specific patients. Accordingly, the present review systematically explores the central roles of HMGB3 in mediating resistance to cancer therapy. This review focuses on its multifaceted mechanisms of maintaining cancer stemness, enhancing DNA damage repair, modulating cell death pathways and remodeling the tumor microenvironment, thereby contributing to the resistance to chemotherapy, radiotherapy, targeted therapy and immunotherapy collectively. HMGB3 can be accepted as a key target in the development of highly promising therapeutic strategies, given its pivotal involvement in multidrug resistance, which may offer novel avenues for overcoming clinical treatment resistance and improving patient outcomes.

Spinal cord injury (SCI) poses a significant therapeutic challenge, largely due to the formation of an inhibitory microenvironment. In this study, cell-free DNA (cfDNA) was found to induce neuroinflammation and accelerate neuronal apoptosis. Leveraging the neuroprotective properties of extracellular vesicles from induced pluripotent stem cell-derived neural stem cells (Nev), a spatiotemporally controlled cfDNA scavenger (Nev-RA) was established by functionalizing the Nev with oligoarginines and reactive oxygen species (ROS)-cleavable poly(ethylene glycol)-angiopeps. Following angiopep-mediated penetration of the blood-spinal cord barrier and ROS-sensitive cleavage at the lesion site, the exposed oligoarginines scavenge pathogenic cfDNA, thereby attenuating toll-like receptor 9-mediated inflammation, while the Nev facilitates the survival of impaired neurons. This biomimetic strategy concurrently addresses the dual barriers of a hostile inflammatory microenvironment and an insufficient intrinsic repair capacity. Collectively, this work reveals an important therapeutic target for SCI and proposes a promising and translatable treatment paradigm.

Mesenchymal stem cell‑derived extracellular vesicles (MSC‑EVs) have garnered research attention due to their unique biological functionalities and therapeutic potential. Compared with the parent MSCs from which they originate, MSC‑EVs are typically free from systemic allergic reactions, hemolysis, pyrogenic reactions, abnormal hematological changes, and vascular and muscle irritation problems, and thus, exhibit therapeutic potential. The present review provides a comprehensive analysis of numerous isolation methodologies for MSC‑EVs, with each method being evaluated based on key parameters, including principles, advantages, limitations and applications. Notably, the therapeutic potential of MSC‑EVs in the treatment of tuberculosis (TB) has been emphasized. MSC‑EVs have demonstrated unique capacities to modulate the T helper cell (Th)1/Th2/T regulatory cell balance, promote M2 macrophage polarization, alleviate inflammation through microRNA‑mediated mechanisms and enhance host defense through antimicrobial peptide responses. The integration of MSC‑EVs with anti‑TB therapy can improve lung, kidney and bladder health by reducing TNF‑α levels and increasing IL‑10/TGF‑β ratios. Notably, functional discrepancies between EVs derived from distinct MSC sources, such as umbilical cord vs. bone marrow cells, underscore the need for targeted optimization strategies. Adequate risk assessment is important before clinical trials, particularly concerning immunogenicity, potential pro‑inflammatory effects and promotion of TB latency. The present review explores the potential clinical applications of MSC‑EVs in TB and other infectious diseases, offering key insights into their therapeutic potential, with the aim of guiding future research.

Objective This study aimed to investigate the protective effect of a metformin (Met)-adipose-derived mesenchymal stem cell exosome (ADSC-Exo) complex (Met-Exo) against hepatic ischemia-reperfusion injury (IRI) and to determine whether this protection is mediated through the silent information regulator 1(SIRT1)-tissue inhibitor of metalloproteinase 3(TIMP3) axis. Methods Met-Exo was constructed and characterized by nanoparticle tracking analysis, Western blotting, and transmission electron microscopy (TEM). An in vitro model of hepatic IRI was established in mouse hepatocyte AML12 cells subjected to oxygen-glucose deprivation/re-oxygenation (OGD/R). Cell viability, apoptosis, and hepatocyte function markers (AST and ALT) were measured to compare the protective effects of Met-Exo, ADSC-Exo, and Met alone. A TranswellTM co-culture system was used to evaluate how macrophage polarization influences OGD/R-induced AML12 injury. AML12 cells were assigned to the following groups: Ctrl, OGD/R, Met, ADSC-Exo, Met-Exo, and macrophage-polarization groups (M0, M1, M1+Met-Exo, M1+pcDNA3.1, M1+pcDNA3.1-SIRT1, M1+pcDNA3.1-TIMP3). SIRT1 and TIMP3 protein levels in AML12 cells were determined by Western blot. Macrophage-polarization markers-inducible nitric-oxide synthase (iNOS) and arginase 1 (Arg1) were also quantified. In parallel, AML12 cells' viability, apoptosis, and hepatocellular function indices (AST& ALT) were assessed. A murine hepatic IRI model was constructed, and Met-Exo was administered to assess therapeutic efficacy in vivo. Results Met-Exo was successfully prepared. In vitro, Met-Exo attenuated OGD/R-induced hepatocyte injury, increased viability, reduced apoptosis, and lowered AST and ALT release. Met-Exo pre-treatment suppressed expression of the M1 marker iNOS and enhanced expression the M2 marker Arg1, thereby alleviating OGD/R damage in AML12 cells. OGD/R markedly decreased SIRT1 and TIMP3 expression in AML12 cells, whereas Met-Exo pre-treatment significantly up-regulated SIRT1 and consequently TIMP3 expression. In macrophage polarization experiments, M1 macrophages exacerbated AML12 injury, shown as decreased protein expression of SIRT1 and TIMP3, increased iNOS levels, reduced Arg1 levels, along with diminished AML12 cell viability, increased apoptosis, and elevated AST/ALT. Over-expression of either SIRT1 or TIMP3 reversed these detrimental effects, skewing macrophages toward the M2 phenotype and mitigating AML12 injury. In vivo, Met-Exo markedly ameliorated hepatic IRI in mice. Conclusion Met-Exo protects against hepatic IRI by activating the SIRT1-TIMP3 axis, thereby driving macrophage polarization toward the anti-inflammatory M2 phenotype, attenuating inflammation and apoptosis in hepatocytes subjected to OGD/R.

Glioblastoma or grade IV glioma is characterized by extremely poor prognosis. Resistance to radio- / chemotherapy and recurrences are associated with tumor stem cells. Transmembrane protein CD133 is considered a potential marker of tumor stem cells. To overcome the limitations of detecting cellular CD133 by antibodies, potential of aptamers (nucleic acid-based molecular recognition elements) is being explored. To obtain reproducible results, it is necessary to study the interaction of fluorescent aptamers to CD133 with standard cell lines and cell cultures derived from patient tumors using various methods.

Glioblastoma (GBM) is a highly aggressive, therapy-resistant brain tumor with inevitable recurrence despite maximal multimodal treatment. Increasing evidence suggests that this intractability arises from coordinated cellular programs rather than a single dominant pathway. Central to these programs are glioma stem-like cells (GSCs), which sustain self-renewal, phenotypic plasticity, and resistance to genotoxic and metabolic stress, and yet the molecular basis of their long-term tumor-propagating capacity remains incompletely understood. Here, we synthesize recent advances to propose an integrated conceptual framework-the Triadic Nexus-in which GSC stemness, telomere maintenance mechanisms, and metabolic reprogramming function as a self-reinforcing regulatory system. We review how telomerase reactivation versus alternative lengthening of telomeres (ALT) differentially shape genomic stability, immune signaling, and metabolic states and how metabolic plasticity feeds back to regulate stemness and telomere-associated stress responses. Drawing on single-cell, spatial, and multi-omics studies, we highlight how these interdependent axes collectively sustain therapy resistance and tumor recurrence. Finally, we discuss the translational implications of the Triadic Nexus, emphasizing rational combinatorial therapeutic strategies and biomarker-guided patient stratification based on telomere and metabolic signatures. By unifying stemness, telomere biology, and metabolism into a mechanistically testable model, this review provides a systems-level framework for understanding GBM persistence and guiding next-generation therapeutic interventions.

Both PRMT5 and EphA2 proteins are overexpressed and play a crucial role in multiple cancers, and have been used as targets to develop new anticancer drugs. However, the function and significance of the PRMT5-EphA2 interaction are unclear. Here, we report that PRMT5 bound to EphA2, catalyzed the dimethylation of EphA2 at arginine 816, and then stabilized EphA2 via inhibiting Cbl-mediated EphA2 ubiquitination and degradation in nasopharyngeal carcinoma (NPC) cells. Functionally, PRMT5 promoted in vitro and in vivo NPC stem cell properties by methylating and stabilizing EphA2. Based on the interacting regions of PRMT5 and EphA2 proteins, we developed a 20 amino acid-long PRMT5-derived peptide, P20, which disrupted the connection of PRMT5 with EphA2, degraded EphA2, and suppressed NPC stem cell properties in vitro and in mice. Moreover, the expression levels of PRMT5 and EphA2 in the NPC tissues were significantly higher than those in the normal nasopharyngeal mucosal tissues, and both proteins for predicting the patient's prognosis are superior to individual proteins. Our findings suggest that PRMT5 methylates and stabilizes EphA2 to promote NPC stem cell properties, and the PRMT5-derived peptide P20 can serve as a novel strategy for targeting EphA2 degradation and inhibiting NPC stem cell properties.

Corneal endothelial failure can cause blindness, with transplantation as the only treatment. Due to donor shortages, establishing robust methods for generating corneal endothelial-like cells (CECs) from induced pluripotent stem cells (iPSCs) is critical. Differentiation protocols included iPSC-to-CEC induction with or without neural crest cell differentiation. CECs directly differentiated from iPSCs demonstrated robust expression of CEC-specific markers and a hexagonal morphology. The wash-out protocol is a novel, efficient, noncytotoxic method for removing undifferentiated iPSCs and obtaining CEC populations with high purity. Single-cell sequencing data showed that iPSC-CECs with wash-out were similar to human primary CECs. In vivo transplantation of iPSC-CECs into a corneal endothelial dysfunction (CED) rabbit model demonstrated their safety and therapeutic efficacy, with improved corneal transparency. Notable recovery of corneal clarity in the CED model, without graft rejection, highlights the in vitro and in vivo potential of iPSC-CECs as a powerful source for clinical therapy in patients with CED. This work establishes an effective stem cell-based platform for producing corneal endothelium-like cells with clinical-grade quality, offering a scalable and regenerative alternative to conventional transplantation.

Pancreatic cancer is highly refractory and aggressive, with cancer stem cells (CSCs) being primarily responsible for its metastasis and chemoresistance. Deregulated cellular bioenergetics is a hallmark of cancer cells. However, the influence of bioenergetics on the maintenance of pancreatic CSC stemness and its underlying mechanisms have not been fully elucidated. In this study, pancreatic CSCs, isolated either by sorting ALDH+ subpopulation or enriching serially passaged tumorspheres from pancreatic cancer cells and PDX model, exhibited active mitochondrial complex I activity and increased oxidative phosphorylation. Complex I maintains stemness and tumorigenicity through its core subunit, NDUFS1. NDUFS1-mediated pancreatic CSC stemness is reinforced by high expression of CD147, which promotes pSTAT3Tyr705-mediated NDUFS1 transcription. To promote stemness, CD147-NDUFS1 initiates SIRT1-DNMT1 metaboloepigenetic signaling, decreasing promoter hypomethylation and increasing the mRNA expression of the stem cell transcript factor PAX2. Moreover, NDUFS1 and CD147 expressions were highly correlated in pancreatic cancer tissues, and their co-expression was significantly associated with poor patient survival. Taken together, our study provides evidence that mitochondrial complex I functions as a key player in CSC stemness maintenance through NDUFS1-mediated retrograde metaboloepigenetic signaling. Blocking a key regulator of mitonuclear communication by targeting CD147 may be a novel therapy for pancreatic cancer.

We have recently hypothesized that the hematogenous metastatic cancer cell of solid tumors is a hybrid between a primary cancer cell and a memory/trained macrophage (doi: 10.3389/fonc.2024.1412296). The hybrid cell respectively acquires mutator phenotype and overgrowth/hyperplasia property from the primary cancer cell and migratability/metastability from the memory/trained macrophage. We name this hypothesis Cancer Cell-Memory Macrophage Hybrid Theory. Since the publication of the article, a number of questions related to this Theory have been raised by colleagues in the oncology community, including intratumoral microbes and microbiomes/microbiotas, oncolytic viruses and bacteria, human papilloma virus vaccines, anti-cancer effects of γδ T-cells, and immune checkpoint inhibitors. The current article is prepared to address these issues. Additional to resolving questions like "Why metastatic cancer cells enter dormancy and can recur via stem-like self-renewal?", the Cancer Cell-Memory Macrophage Hybrid Theory distinguishes itself from other carcinogenesis and metastasis hypotheses/theories by offering answers to many puzzling clinical features including metastasis of seemingly malignant parasitic cells within the human body, intracellular microbes (including viruses, bacteria, fungi, and parasites) within cancer cells, paradoxal effects (recurrence vs. regression) of microbes on cancer, contradictory immune effects of human papilloma virus vaccines between young and adult/senior females, and immune context-dependent effects (stimulatory and inhibitory) of T-lymphocytes on cancer cells. The Theory also predicts that quantitatively and functionally dampening innate macrophages that have hybridized with cancer cells (i.e., cancer cell-memory macrophage hybrids), should be explored as a fundamental anti-cancer strategy. The Theory further forecasts how to prepare an organotropic/tumoritropic Coley's toxin-like anti-cancer microbe, which could potentially circumvent direct injection of microbial preparations into a tumor. A testable experiment that uses zebrafish larva models can potentially either validate or falsify the Theory.

Breast cancer metastasis remains the leading cause of mortality and frequently targets the bone. Breast cancer cells release soluble factors and extracellular vesicles that disrupt bone marrow (BM)/bone homeostasis, promoting osteoclastogenesis and the accumulation of senescent cells. In line with updated cancer hallmarks, senescent mesenchymal stem/ stromal cells (MSCs), osteoblasts, and osteocytes contribute to remodeling of the BM microenvironment, thereby favoring pre-metastatic niche (PMN) formation and subsequent bone metastasis. We previously demonstrated that untreated stage III-B breast cancer patients (BCPs) exhibit increased oxidative stress and elevated reactive oxygen species (ROS) levels, accompanied by senescent and functionally impaired BM-MSCs-key regulators of BM/bone homeostasis. In the present study, we sought to identify the molecular targets affected by oxidative stress that drive MSC senescence in these patients.

Focal post-traumatic cartilage lesions frequently progress to early osteoarthritis, highlighting the limited regenerative capacity of adult articular cartilage. Compared to native tissue, conventional surgical interventions often produce fibrocartilage with inferior biomechanical properties, representing a persistent therapeutic challenge. This review assessed preclinical studies exploring cartilage repair strategies using autologous mesenchymal stem cells (MSCs) in animal models. MSCs therapies demonstrated superior cartilage regeneration, matrix organization, and integration into the surrounding tissue compared to the control groups. The most efficient source was found to be bone marrow - derived mesenchymal stem cells (BM-MSCs) combined with biodegradable scaffolds, suggesting their potential in tissue engineering applications. Despite methodological heterogeneity across studies - including variations in stem cells sources, implant types, and deliver strategies - cumulative evidence strongly supports the regenerative potential of autologous MSCs for cartilage repair. Current research identifies key knowledge gaps, including the absence of standardized experimental protocols and limited insight into the mechanisms of tissue remodeling and maturation. Collectively, these gaps limit direct clinical translation, highlighting the need for further, standardized studies in large animal models with long-term follow-up (>2 years) to assess integration, functional maturation, and the full regenerative potential of the repair tissue.

Dysfunctional hematopoietic stem cells (HSC) drive the initiation of myelodysplastic syndromes (MDS), yet the genome-wide DNA methylation landscape of primitive MDS HSCs and its mechanistic contribution to disease pathogenesis remain poorly defined. Here, we establish single-base resolution DNA methylomes of bone marrow HSCs from MDS patients and healthy donors. We uncover the widespread hypermethylation in CpG islands, alongside hypomethylation in repetitive elements such as Alu. Differentially methylated regions are enriched for genes involved in cancer-related pathways, as well as extrinsic signaling pathways and intrinsic transcriptional networks essential for HSC function. Among these, we identify GFI1 and BMI1 as key targets of DNA methylation dysregulation in MDS. Notably, using either the MDS or a TET2-deficient mouse model, we demonstrate that loss of TET2, a frequently mutated epigenetic regulator in MDS, induces promoter hypermethylation and transcriptional repression of GFI1, contributing to the expansion of the MDS or aged hematopoietic stem and progenitor cell pool. Our study not only charts the base-resolution DNA methylome of human MDS HSCs but also reveals a TET2-GFI1 axis that safeguards HSC homeostasis. These findings provide mechanistic insight into how aberrant DNA methylation drives HSC dysfunction in MDS and offer an epigenomic resource for discovering regulators and therapeutic targets at the stem cell level.

Poor outcomes in proteinuric kidney diseases are challenging to successfully manage therapeutically due to the heterogeneity of underlying disease pathogenesis and associated risk for progression. The role of cytoskeleton-associated proteins, including the scaffolding protein Anillin (ANLN), are of specific interest in kidney disease given the importance of actin dynamics in the kidney's specialized epithelial cell types. In this study, we identify the prevalence of genetic variants in ANLN , the gene encoding ANLN, in a cohort of deeply phenotyped individuals with non-diabetic proteinuric kidney disease. Thirty-one individuals (of 864 genotyped) harbor heterozygously expressed variants in ANLN ; 7 unrelated individuals shared the same variant (I1109V) in the C-terminal pleckstrin homology (PH) domain, a region necessary for interaction with the plasma membrane. Kidney organoids generated from I1109V induced pluripotent stem cells from 1 of these individuals showed increased epithelial cell mitogen-activated protein kinase 8 network activity and apoptosis, which was enhanced by tumor necrosis factor alpha (TNF-α) and phenocopied by actin polymerization inhibition. TNF-α-treated I1109V organoids also exhibited tubular lumen expansion. Knockdown and re-expression of the analogous ANLN variant in Xenopus laevis embryonic epithelia resulted in defects in cell-cell junction dynamics including wavy cell membranes exhibiting increased transverse movements as well as abnormal junctional F-actin remodeling in response to mechanical stress and leaky barrier function. Taken together, these results indicate that enhanced tubular epithelial cell death, perturbed cell-cell contacts and barrier function defects are associated with a novel ANLN variant discovered in individuals with non-diabetic proteinuric kidney disease.

Multilineage-differentiating stress-enduring (Muse) cells, a subpopulation of mesenchymal stem cells (MSCs) marked by stage-specific embryonic antigen 3 (SSEA3), exhibit superior regenerative capacity compared to conventional MSCs, including enhanced tissue homing, pluripotency, and paracrine effects. However, their natural scarcity (1-5% in MSC populations) limits therapeutic scalability. In this study, we developed a five-compound small molecule method to obtain compound-enriched Muse-like MSCs and assessed their potential use in treating severe veterinary chronic diseases, such as hepatitis and chronic kidney disease (CKD).

Communication between gut microbiota and immune cells within the brain is essential for neurotypical development. Specifically, microglia are known to play a key role in regulating and supporting neural progenitor stem cell production during brain development, and are sensitive to changes in the maternal gut microbial composition during perinatal development. Here, we employed a germ-free (GF) porcine paradigm to examine how the absence of the microbiome affects microglial dynamics during a key epoch of brain development. We utilized automated software to evaluate microglial density and morphology across three developmentally significant regions: the ventricular/subventricular zone (VZ/SVZ), the prefrontal subcortical white matter (PFCSWM), and layers II/III of the prefrontal cortex (PFCII-III). We found no significant differences in microglial morphology or density in the VZ/SVZ or PFCSWM. In contrast, the PFCII-III of P16 piglets exhibited an increase in microglia density paired with morphologies indicative of an activated/reactive functional state. Notably, these effects were identified with no overall changes in microglial density in any of the regions assessed. Transcriptomics on RNA isolated from the PFCII-III revealed a significant upregulation of genes related to neuroinflammation, in agreement with a region-specific microglial and immune response in the absence of microbial colonization during postnatal development. Together, these findings build on the limited knowledge available on how microbiota influence brain development in large animal model organisms with high similarities to human brain anatomy and developmental trajectories.

Investigating single-cell dynamics and morphology in tissues and embryos requires highly accurate quantitative analysis of microscopy images. Despite significant advances in the field of bioimage analysis, even the most sophisticated segmentation and tracking algorithms inevitably produce errors (e.g. : over segmentation, missing objects, miss-connected objects). Although error rate may be small, their propagation throughout a time-lapse sequence has catastrophic effects on the accuracy of tracking and extraction of single cell parameters. Extracting single cell temporal information in the context of tissue/embryo requires thus expert curation to identify and correct segmentation errors. In the movies commonly used in developmental biology and stem cell research, both the number of imaged cells and the duration of recording are large, making this manual correction task extremely time-consuming. This has now become a major bottleneck in the fields of development, stem cell biology and bioimage analysis. We present here EpiCure (Epithelial Curation), a versatile tool designed to streamline and accelerate manual curation of segmentation and tracking in 2D movies of large epithelial tissues. EpiCure uses temporal information and morphometric parameters to automatically identify segmentation and tracking errors and provides user-friendly tools to correct them. It focuses on ergonomics and offers several visualization options to help navigating in movies of tissue covering a large number of cells, speeding up the detection of errors and their curation. EpiCure is highly interoperable and supports input from a wide range of segmentation tools. It also includes multiple export filters, enabling seamless integration with downstream analysis pipelines. In this paper, using movies from several animal models, we highlight the importance of curating cell segmentation and tracking for accurate downstream analysis, and demonstrate how EpiCure helps the curation process for extracting accurate single cell dynamics and cellular events detection, making it faster and amenable on large dataset.

Enhancer-regulating epigenetic modifiers play critical roles in normal physiological processes and human pathogenesis. The major enhancer regulator paralogs MLL3 and MLL4 (MLL3/4) belong to the lysine methyltransferase 2 (KMT2) family, which catalyzes the methylation of lysine 4 on histone H3 (H3K4me). MLL3/4 are required for enhancer activation and are essential for mammalian development and stem cell differentiation. Recent studies have linked MLL3/4 with different metabolic pathways in the context of stem cell self-renewal and cancer cell growth; however, the underlying mechanisms remain elusive. Here, we utilize Seahorse extracellular flux analysis, stable isotope tracing, stem cell biology techniques, and transcriptomic analysis to investigate the functional relationship of MLL3/4, cellular respiration, and stem cell differentiation. Our results indicate that the loss of MLL3/4 impairs glycolytic activity and mitochondrial respiration in murine embryonic stem cells by downregulating the rate-limiting glycolytic enzyme Hexokinase 2 (HK2) and impairing the function of the Alpha-ketoglutarate dehydrogenase (OGDH) complex. Furthermore, simultaneously overexpression of HK2 and OGDH rescues defects in both cellular respiration and differentiation caused by MLL3/4 loss. Taken together, our study reveals a novel mechanism by which epigenetic machineries such as MLL3/4 govern the differentiation of pluripotent stem cells and facilitates the understanding of disease pathogenesis driven by enhancer malfunction.

Primary open-angle glaucoma (POAG), a leading cause of irreversible blindness, has a strong genetic basis. The Primary Open-Angle African Ancestry Glaucoma Genetics study previously identified 46 risk loci. To pinpoint causal variants and their corresponding effector genes, we analyzed gene expression, chromatin accessibility, and conformation in two ocular cell-types: trabecular meshwork cells (hTMCs) and retinal ganglion cells derived from induced pluripotent stem cells (hiPSC-RGCs). We identified 24 candidate genes in hTMCs and 56 in hiPSC-RGCs. The ARHGEF12 gene was selected for further validation because it was nominated by local and distal promoter interactions in both cell-types and has reproducible prior evidence of its association with POAG. While its role in hTMCs is established, its function in RGCs is unclear. hiPSC-RGCs generated from a POAG donor homozygous for the risk allele showed reduced ARHGEF12 expression, altered morphology, and disrupted neuronal activity. This framework enables functional evaluation of additional POAG risk variants.