Bi-allelic variants in the ESAM tight-junction gene cause a neurodevelopmental disorder associated with fetal intracranial hemorrhage.

The blood-brain barrier (BBB) is an essential gatekeeper for the central nervous system and incidence of neurodevelopmental disorders (NDDs) is higher in infants with a history of intracerebral hemorrhage (ICH). We discovered a rare disease trait in thirteen individuals, including four fetuses, from eight unrelated families associated with homozygous loss-of-function variant alleles of ESAM which encodes an endothelial cell adhesion molecule. The c.115del (p.Arg39Glyfs∗33) variant, identified in six individuals from four independent families of Southeastern Anatolia, severely impaired the in vitro tubulogenic process of endothelial colony-forming cells, recapitulating previous evidence in null mice, and caused lack of ESAM expression in the capillary endothelial cells of damaged brain. Affected individuals with bi-allelic ESAM variants showed profound global developmental delay/unspecified intellectual disability, epilepsy, absent or severely delayed speech, varying degrees of spasticity, ventriculomegaly, and ICH/cerebral calcifications, the latter being also observed in the fetuses. Phenotypic traits observed in individuals with bi-allelic ESAM variants overlap very closely with other known conditions characterized by endothelial dysfunction due to mutation of genes encoding tight junction molecules. Our findings emphasize the role of brain endothelial dysfunction in NDDs and contribute to the expansion of an emerging group of diseases that we propose to rename as "tightjunctionopathies."

Inflammation and bone marrow fibrosis: novel immunotherapeutic targets.

PURPOSE OF REVIEW: Myelofibrosis (MF) is primarily driven by constitutive activation of the Janus kinase/signal transducer of activators of transcription (JAK/STAT) pathway. While JAK inhibitors have shown to alleviate disease symptoms, their disease-modifying effects in MF are limited. The only curative treatment remains allogeneic stem cell transplantation, which can be applied to a minority of patients. As a result, there is a need to explore novel targets in MF to facilitate appropriate drug development and therapeutic pathways. RECENT FINDINGS: Recent research has focused on identifying novel signals that contribute to the abnormal cross-talk between hematopoietic and stromal cells, which promotes MF and disease progression. Inflammation and immune dysregulation have emerged as key drivers of both the initiation and progression of MF. A growing number of actionable targets has been identified, including cytokines, transcription factors, signalling networks and cell surface-associated molecules. These targets exhibit dysfunctions in malignant and nonmalignant hematopoietic cells, but also in nonhematopoietic cells of the bone marrow. The study of these inflammation-related molecules, in preclinical models and MF patient's samples, is providing novel therapeutic targets. SUMMARY: The identification of immunotherapeutic targets is expanding the therapeutic landscape of MF. This review provides a summary of the most recent advancements in the study of immunotherapeutic targets in MF.

Adhesion to fibronectin via α5ß1 integrin supports expansion of the megakaryocyte lineage in primary myelofibrosis.

Excessive accumulation of extracellular matrix (ECM) is a hallmark of bone marrow (BM) milieu in primary myelofibrosis (PMF). Because cells have the ability to adhere to the surrounding ECM through integrin receptors, we examined the hypothesis that an abnormal ECM-integrin receptor axis contributes to BM megakaryocytosis in JAK2V617F+ PMF. Secretion of ECM protein fibronectin (FN) by BM stromal cells from PMF patients correlates with fibrosis and disease severity. Here, we show that Vav1-hJAK2V617F transgenic mice (JAK2V617F+) have high BM FN content associated with megakaryocytosis and fibrosis. Further, megakaryocytes from JAK2V617F+ mice have increased cell surface expression of the α5 subunit of the α5ß1 integrin, the major FN receptor in megakaryocytes, and augmented adhesion to FN compared with wild-type controls. Reducing adhesion to FN by an inhibitory antibody to the α5 subunit effectively reduces the percentage of CD41+ JAK2V617F+ megakaryocytes in vitro and in vivo. Corroborating our findings in mice, JAK2V617F+ megakaryocytes from patients showed elevated expression of α5 subunit, and a neutralizing antibody to α5 subunit reduced adhesion to FN and megakaryocyte number derived from CD34+ cells. Our findings reveal a previously unappreciated contribution of FN-α5ß1 integrin to megakaryocytosis in JAK2V617F+ PMF.

Mechanisms of platelet release: in vivo studies and in vitro modeling.

Mechanisms related to platelet release in the context of the bone marrow niche are not completely known. In this review we discuss what has been discovered about four critical aspects of this process: 1) the bone marrow niche organization, 2) the role of the extracellular matrix components, 3) the mechanisms by which megakaryocytes release platelets and 4) the novel approaches to mimic the bone marrow environment and produce platelets ex vivo.

Mechanisms of thrombocytopenia in platelet-type von Willebrand disease.

Platelet-type von Willebrand disease is an inherited platelet disorder characterized by thrombocytopenia with large platelets caused by gain-of-function variants in GP1BA leading to enhanced GPIbα-von Willebrand factor (vWF) interaction. GPIbα and vWF play a role in megakaryocytopoiesis, thus we aimed to investigate megakaryocyte differentiation and proplatelet-formation in platelet-type von Willebrand disease using megakaryocytes from a patient carrying the Met239Val variant and from mice carrying the Gly233Val variant. Platelet-type von Willebrand disease megakaryocytes bound vWF at an early differentiation stage and generated proplatelets with a decreased number of enlarged tips compared to control megakaryocytes. Moreover, they formed proplatelets upon contact with collagen, differently from normal megakaryocytes. Similarly, collagen triggered megakaryocytes showed defective activation of the RhoA-MLC2 axis, which prevents proplatelet formation, and increased phosphorylation of Lyn, which acts as a negative regulator of GPVI signaling, thus preventing ectopic proplatelet-formation on collagen. Consistently, human and murine bone marrow contained an increased number of extravascular platelets compared to controls. In addition, platelet survival of mutant mice was shortened compared to control mice, and the administration of desmopressin, raising circulating vWF, caused a marked drop in platelet count. Taken together, these results show for the first time that thrombocytopenia in platelet-type von Willebrand disease is due to the combination of different pathogenic mechanisms, i.e. the formation of a reduced number of platelets by megakaryocytes, the ectopic release of platelets in the bone marrow, and the increased clearance of platelet/vWF complexes.

The Plant Hormone Abscisic Acid Is a Prosurvival Factor in Human and Murine Megakaryocytes.

Abscisic acid (ABA) is a phytohormone involved in pivotal physiological functions in higher plants. Recently, ABA has been proven to be also secreted and active in mammals, where it stimulates the activity of innate immune cells, mesenchymal and hematopoietic stem cells, and insulin-releasing pancreatic ß cells through a signaling pathway involving the second messenger cyclic ADP-ribose (cADPR). In addition to behaving like an animal hormone, ABA also holds promise as a nutraceutical plant-derived compound in humans. Many biological functions of ABA in mammals are mediated by its binding to the LANCL-2 receptor protein. A putative binding of ABA to GRP78, a key regulator of endoplasmic reticulum stress, has also been proposed. Here we investigated the role of exogenous ABA in modulating thrombopoiesis, the process of platelet generation. Our results demonstrate that expression of both LANCL-2 and GRP78 is up-regulated during hematopoietic stem cell differentiation into mature megakaryocytes (Mks). Functional ABA receptors exist in mature Mks because ABA induces an intracellular Ca2+ increase ([Ca2+] i ) through PKA activation and subsequent cADPR generation. In vitro exposure of human or murine hematopoietic progenitor cells to 10 µm ABA does not increase recombinant thrombopoietin (rTpo)-dependent Mk differentiation or platelet release. However, under conditions of cell stress induced by rTpo and serum deprivation, ABA stimulates, in a PKA- and cADPR-dependent fashion, the mitogen-activated kinase ERK 1/2, resulting in the modulation of lymphoma 2 (Bcl-2) family members, increased Mk survival, and higher rates of platelet production. In conclusion, we demonstrate that ABA is a prosurvival factor for Mks in a Tpo-independent manner.

Programmable 3D silk bone marrow niche for platelet generation ex vivo and modeling of megakaryopoiesis pathologies.

We present a programmable bioengineered 3-dimensional silk-based bone marrow niche tissue system that successfully mimics the physiology of human bone marrow environment allowing us to manufacture functional human platelets ex vivo. Using stem/progenitor cells, megakaryocyte function and platelet generation were recorded in response to variations in extracellular matrix components, surface topography, stiffness, coculture with endothelial cells, and shear forces. Millions of human platelets were produced and showed to be functional based on multiple activation tests. Using adult hematopoietic progenitor cells our system demonstrated the ability to reproduce key steps of thrombopoiesis, including alterations observed in diseased states. A critical feature of the system is the use of natural silk protein biomaterial allowing us to leverage its biocompatibility, nonthrombogenic features, programmable mechanical properties, and surface binding of cytokines, extracellular matrix components, and endothelial-derived proteins. This in turn offers new opportunities for the study of blood component production ex vivo and provides a superior tissue system for the study of pathologic mechanisms of human platelet production.

Brief Report: Alternative Splicing of Extra Domain A (EIIIA) of Fibronectin Plays a Tissue-Specific Role in Hematopoietic Homeostasis.

Fibronectin (FN) is a major extracellular matrix protein implicated in cell adhesion and differentiation in the bone marrow (BM) environment. Alternative splicing of FN gene results in the generation of protein variants containing an additional EIIIA domain that sustains cell proliferation or differentiation during physiological or pathological tissue remodeling. To date its expression and role in adult hematopoiesis has not been explored. In our research, we demonstrate that during physiological hematopoiesis a small fraction of BM derived FN contains the EIIIA domain and that mice constitutively including (EIIIA(+/+) ) or excluding (EIIIA(-/-) ) the EIIIA exon present comparable levels of hematopoietic stem cells, myeloid and lymphoid progenitors within BM. Moreover, only minor alterations were detected in blood parameters and in hematopoietic frequencies of BM granulocytes/monocytes and B cells. As opposed to other tissues, unique compensatory mechanisms, such as increased FN accumulation and variable expression of the EIIIA receptors, Toll like receptor-4 and alpha9 integrin subunit, characterized the BM of these mice. Our data demonstrate that FN is a fundamental component of the hematopoietic tissue and that the EIIIA exon may play a key role in modulating hematopiesis in conditions of BM stress or diseases. Stem Cells 2016;34:2263-2268.

Thrombopoietin/TGF-ß1 Loop Regulates Megakaryocyte Extracellular Matrix Component Synthesis.

Extracellular matrix (ECM) components initiate crucial biochemical and biomechanical cues that are required for bone marrow homeostasis. In our research, we prove that a peri-cellular matrix composed primarily of type III and type IV collagens, and fibronectin surrounds human megakaryocytes in the bone marrow. The data we collected support the hypothesis that bone marrow megakaryocytes possess a complete mechanism to synthesize the ECM components, and that thrombopoietin is a pivotal regulator of this new function inducing transforming growth factor-ß1 (TGF-ß1) release and consequent activation of the downstream pathways, both in vitro and in vivo. This activation results in a dose dependent increase of ECM component synthesis by megakaryocytes, which is reverted upon incubation with JAK and TGF-ß1 receptor specific inhibitors. These data are pivotal for understanding the central role of megakaryocytes in creating their own regulatory niche within the bone marrow environment.

Hyaluronan based hydrogels provide an improved model to study megakaryocyte-matrix interactions.

Hyaluronan (HA) is a glycosamminoglican involved in cell biology as well as a relevant polymer for tissue engineering and regenerative medicine. Megakaryocytes (Mks) are immersed in a mesh of extracellular matrix (ECM) components that regulate their maturation in the bone marrow (BM) and the release of platelets into the bloodstream. While fibrous ECMs such as collagens and fibronectin have been demonstrated to differently regulate Mk function and platelet release, the role of HA, that fills the majority of the BM extracellular interstitial space, has not been investigated so far. Here we demonstrated that, although human Mks express HA receptors, they are not affected by HA in terms of in vitro differentiation, maturation and platelet formation. Importantly, chemical properties of HA were exploited to generate hydrogels with entrapped ECMs that represent a useful model to more closely mimic the tridimensional characteristics of the BM environment for studying Mk function. In conclusion, in this work we demonstrated that HA is an ideal candidate for a 3D ex vivo model of human BM ECM component environment.

The secret life of a megakaryocyte: emerging roles in bone marrow homeostasis control.

Megakaryocytes are rare cells found in the bone marrow, responsible for the everyday production and release of millions of platelets into the bloodstream. Since the discovery and cloning, in 1994, of their principal humoral factor, thrombopoietin, and its receptor c-Mpl, many efforts have been directed to define the mechanisms underlying an efficient platelet production. However, more recently different studies have pointed out new roles for megakaryocytes as regulators of bone marrow homeostasis and physiology. In this review we discuss the interaction and the reciprocal regulation of megakaryocytes with the different cellular and extracellular components of the bone marrow environment. Finally, we provide evidence that these processes may concur to the reconstitution of the bone marrow environment after injury and their deregulation may lead to the development of a series of inherited or acquired pathologies.

Megakaryocytes contribute to the bone marrow-matrix environment by expressing fibronectin, type IV collagen, and laminin.

Megakaryocytes associate with the bone marrow vasculature where they convert their cytoplasm into proplatelets that protrude through the vascular endothelium into the lumen and release platelets. The extracellular matrix (ECM) microenvironment plays a critical role in regulating these processes. In this work we demonstrate that, among bone marrow ECM components, fibronectin, type IV collagen, and laminin are the most abundant around bone marrow sinusoids and constitute a pericellular matrix surrounding megakaryocytes. Most importantly, we report, for the first time, that megakaryocytes express components of the basement membrane and that these molecules contribute to the regulation of megakaryocyte development and bone marrow ECM homeostasis both in vitro and in vivo. In vitro, fibronectin induced a threefold increase in the proliferation rate of mouse hematopoietic stem cells leading to higher megakaryocyte output with respect to cells treated only with thrombopoietin or other matrices. However, megakaryocyte ploidy level in fibronectin-treated cultures was significantly reduced. Stimulation with type IV collagen resulted in a 1.4-fold increase in megakaryocyte output, while all tested matrices supported proplatelet formation to a similar extent in megakaryocytes derived from fetal liver progenitor cells. In vivo, megakaryocyte expression of fibronectin and basement membrane components was upregulated during bone marrow reconstitution upon 5-fluorouracil induced myelosuppression, while only type IV collagen resulted upregulated upon induced thrombocytopenia. In conclusion, this work demonstrates that ECM components impact megakaryocyte behavior differently during their differentiation and highlights a new role for megakaryocyte as ECM-producing cells for the establishment of cell niches during bone marrow regeneration.

Biocompatibility of functionalized boron phosphate (BPO4) nanoparticles for boron neutron capture therapy (BNCT) application.

Boron neutron capture therapy (BNCT) is a radiotherapy treatment based on the accumulation in the tumor of a (10)B-containing drug and subsequent irradiation with low energy neutrons, which bring about the decay of (10)B to (7)Li and an α particle, causing the death of the neoplastic cell. The effectiveness of BNCT is limited by the low delivery and accumulation of the used boron-containing compounds. Here we report the development and the characterization of BPO4 nanoparticles (NPs) as a novel possible alternative drug for BNCT. An extensive analysis of BPO4 NP biocompatibility was performed using both mature blood cells (erythrocytes, neutrophils and platelets) and a model of hematopoietic progenitor cells. A time- and concentration-dependent cytotoxicity study was performed on neoplastic coloncarcinoma and osteosarcoma cell lines. BPO4 functionalization with folic acid, introduced to improve the uptake by tumor cells, appeared to effectively limit the unwanted effects of NPs on the analyzed blood components. FROM THE CLINICAL EDITOR: Boron neutron capture therapy (BNCT) is a radiotherapy treatment modality based on the accumulation of a (10)B-containing drug and subsequent irradiation with low energy neutrons, inducing the decay of (10)B to (7)Li and an α particle, causing neoplastic cell death. This team of authors reports on a folic acid functionalized BPO4 nanoparticle with improved characteristics compared with conventional BNCT approaches, as demonstrated in tumor cell lines, and hopefully to be followed by translational human studies.

Proteomic screening identifies PF4/Cxcl4 as a critical driver of myelofibrosis.

Despite increased understanding of the genomic landscape of Myeloproliferative Neoplasms (MPNs), the pathological mechanisms underlying abnormal megakaryocyte (Mk)-stromal crosstalk and fibrotic progression in MPNs remain unclear. We conducted mass spectrometry-based proteomics on mice with Romiplostim-dependent myelofibrosis to reveal alterations in signaling pathways and protein changes in Mks, platelets, and bone marrow (BM) cells. The chemokine Platelet Factor 4 (PF4)/Cxcl4 was up-regulated in all proteomes and increased in plasma and BM fluids of fibrotic mice. High TPO concentrations sustained in vitro PF4 synthesis and secretion in cultured Mks, while Ruxolitinib restrains the abnormal PF4 expression in vivo. We discovered that PF4 is rapidly internalized by stromal cells through surface glycosaminoglycans (GAGs) to promote myofibroblast differentiation. Cxcl4 gene silencing in Mks mitigated the profibrotic phenotype of stromal cells in TPO-saturated co-culture conditions. Consistently, extensive stromal PF4 uptake and altered GAGs deposition were detected in Romiplostim-treated, JAK2V617F mice and BM biopsies of MPN patients. BM PF4 levels and Mk/platelet CXCL4 expression were elevated in patients, exclusively in overt fibrosis. Finally, pharmacological inhibition of GAGs ameliorated in vivo fibrosis in Romiplostim-treated mice. Thus, our findings highlight the critical role of PF4 in the fibrosis progression of MPNs and substantiate the potential therapeutic strategy of neutralizing PF4-GAGs interaction.

Megakaryocyte-matrix interaction within bone marrow: new roles for fibronectin and factor XIII-A.

The mechanisms by which megakaryocytes (MKs) differentiate and release platelets into the circulation are not well understood. However, growing evidence indicates that a complex regulatory mechanism involving MK-matrix interactions may contribute to the quiescent or permissive microenvironment related to platelet release within bone marrow. To address this hypothesis, in this study we demonstrate that human MKs express and synthesize cellular fibronectin (cFN) and transglutaminase factor XIII-A (FXIII-A). We proposed that these 2 molecules are involved in a new regulatory mechanism of MK-type I collagen interaction in the osteoblastic niche. In particular, we demonstrate that MK adhesion to type I collagen promotes MK spreading and inhibits pro-platelet formation through the release and relocation to the plasma membrane of cFN. This regulatory mechanism is dependent on the engagement of FN receptors at the MK plasma membrane and on transglutaminase FXIII-A activity. Consistently, the same mechanism regulated the assembly of plasma FN (pFN) by adherent MKs to type I collagen. In conclusion, our data extend the knowledge of the mechanisms that regulate MK-matrix interactions within the bone marrow environment and could serve as an important step for inquiring into the origins of diseases such as myelofibrosis and congenital thrombocytopenias that are still poorly understood.

Extracellular matrix structure and nano-mechanics determine megakaryocyte function.

Cell interactions with matrices via specific receptors control many functions, with chemistry, physics, and membrane elasticity as fundamental elements of the processes involved. Little is known about how biochemical and biophysical processes integrate to generate force and, ultimately, to regulate hemopoiesis into the bone marrow-matrix environment. To address this hypothesis, in this work we focus on the regulation of MK development by type I collagen. By atomic force microscopy analysis, we demonstrate that the tensile strength of fibrils in type I collagen structure is a fundamental requirement to regulate cytoskeleton contractility of human MKs through the activation of integrin-α2ß1-dependent Rho-ROCK pathway and MLC-2 phosphorylation. Most importantly, this mechanism seemed to mediate MK migration, fibronectin assembly, and platelet formation. On the contrary, a decrease in mechanical tension caused by N-acetylation of lysine side chains in type I collagen completely reverted these processes by preventing fibrillogenesis.

Lack of COL6/collagen VI causes megakaryocyte dysfunction by impairing autophagy and inducing apoptosis.

Endoplasmic reticulum stress is an emerging significant player in the molecular pathology of connective tissue disorders. In response to endoplasmic reticulum stress, cells can upregulate macroautophagy/autophagy, a fundamental cellular homeostatic process used by cells to degrade and recycle proteins or remove damaged organelles. In these scenarios, autophagy activation can support cell survival. Here we demonstrated by in vitro and in vivo approaches that megakaryocytes derived from col6a1-/- (collagen, type VI, alpha 1) null mice display increased intracellular retention of COL6 polypeptides, endoplasmic reticulum stress and apoptosis. The unfolded protein response is activated in col6a1-/- megakaryocytes, as evidenced by the upregulation of molecular chaperones, by the increased splicing of Xbp1 mRNA and by the higher level of the pro-apoptotic regulator DDIT3/CHOP. Despite the endoplasmic reticulum stress, basal autophagy is impaired in col6a1-/- megakaryocytes, which show lower BECN1 levels and reduced autophagosome maturation. Starvation and rapamycin treatment rescue the autophagic flux in col6a1-/- megakaryocytes, leading to a decrease in intracellular COL6 polypeptide retention, endoplasmic reticulum stress and apoptosis. Furthermore, megakaryocytes cultured from peripheral blood hematopoietic progenitors of patients affected by Bethlem myopathy and Ullrich congenital muscular dystrophy, two COL6-related disorders, displayed increased apoptosis, endoplasmic reticulum stress and impaired autophagy. These data demonstrate that genetic disorders of collagens, endoplasmic reticulum stress and autophagy regulation in megakaryocytes may be interrelated.Abbreviations: 7-AAD: 7-amino-actinomycin D; ATF: activating transcriptional factor; BAX: BCL2 associated X protein; BCL2: B cell leukemia/lymphoma 2; BCL2L1/Bcl-xL: BCL2-like 1; BM: bone marrow; COL6: collagen, type VI; col6a1-/-: mice that are null for Col6a1; DDIT3/CHOP/GADD153: DNA-damage inducible transcript 3; EGFP: enhanced green fluorescent protein; ER: endoplasmic reticulum; reticulophagy: endoplasmic reticulum-selective autophagy; HSPA5/Bip: heat shock protein 5; HSP90B1/GRP94: heat shock protein 90, beta (Grp94), member 1; LAMP2: lysosomal associated membrane protein 2; MAP1LC3B/LC3B: microtubule-associated protein 1 light chain 3 beta; Mk: megakaryocytes; MTOR: mechanistic target of rapamycin kinase; NIMV: noninvasive mechanical ventilation; PI3K: phosphoinositide 3-kinase; PPP1R15A/GADD34: protein phosphatase 1, regulatory subunit 15A; RT-qPCR: reverse transcription-quantitative real-time PCR; ROS: reactive oxygen species; SERPINH1/HSP47: serine (or cysteine) peptidase inhibitor, clade H, member 1; sh-RNA: short hairpin RNA; SOCE: store operated calcium entry; UCMD: Ullrich congenital muscular dystrophy; UPR: unfolded protein response; WIPI2: WD repeat domain, phosphoinositide-interacting 2; WT: wild type; XBP1: X-box binding protein 1.

Constitutively released adenosine diphosphate regulates proplatelet formation by human megakaryocytes.

BACKGROUND: The interaction of adenosine diphosphate with its P2Y(1) and P2Y(12) receptors on platelets is important for platelet function. However, nothing is known about adenosine diphosphate and its function in human megakaryocytes. DESIGN AND METHODS: We studied the role of adenosine diphosphate and P2Y receptors on proplatelet formation by human megakaryocytes in culture. RESULTS: Megakaryocytes expressed all the known eight subtypes of P2Y receptors, and constitutively released adenosine diphosphate. Proplatelet formation was inhibited by the adenosine diphosphate scavengers apyrase and CP/CPK by 60-70% and by the P2Y(12) inhibitors cangrelor and 2-MeSAMP by 50-60%, but was not inhibited by the P2Y(1) inhibitor MRS 2179. However, the active metabolites of the anti-P2Y(12) drugs, clopidogrel and prasugrel, did not inhibit proplatelet formation. Since cangrelor and 2-MeSAMP also interact with P2Y(13), we hypothesized that P2Y(13), rather than P2Y(12) is involved in adenosine diphosphate-regulated proplatelet formation. The specific P2Y(13) inhibitor MRS 2211 inhibited proplatelet formation in a concentration-dependent manner. Megakaryocytes from a patient with severe congenital P2Y(12) deficiency showed normal proplatelet formation, which was inhibited by apyrase, cangrelor or MRS 2211 by 50-60%. The platelet count of patients with congenital delta-storage pool deficiency, who lack secretable adenosine diphosphate, was significantly lower than that of patients with other platelet function disorders, confirming the important role of secretable adenosine diphosphate in platelet formation. CONCLUSIONS: This is the first demonstration that adenosine diphosphate released by megakaryocytes regulates their function by interacting with P2Y(13). The clinical relevance of this not previously described physiological role of adenosine diphosphate and P2Y(13) requires further exploration.