Furthermore, we provide a novel mechanism for the regulation of the actin cytoskeleton during migration: LPXN-mediated phosphorylation of CaD by the extracellular-signal regulated kinase 1/2 (ERK). RESULTS Reduced adhesion and cell size of PCa cells after LPXN knockdown To investigate the influence of LPXN expression on the adhesive characteristics of PCa cells, we performed a cell adhesion assay. decreased expression of TGF-beta-activated kinase 1 (TAK1) after LPXN knockdown in PC-3 PCa cells. Subsequent analyses of the downstream kinases revealed the extracellular signal-regulated kinase (ERK) as an interaction partner of LPXN that facilitates CaD phosphorylation during LPXN-mediated PCa cell migration. In conclusion, we demonstrate that LPXN directly influences cytoskeletal dynamics via interaction with the actin-binding protein CaD and Rabbit Polyclonal to ZAR1 regulates CaD phosphorylation by recruiting ERK to highly dynamic structures within PCa cells. gene encodes five different CALD1 transcripts, resulting in two major isoforms: a high-molecular-mass isoform (h-CaD) that is expressed in smooth muscle cells and a low-molecular-mass isoform (l-CaD) expressed in non-muscle cells. The regulation of CaD is important for proper cell function because decreased expression of l-CaD has been found in many cancer cell types [12-15]. In the present study, we identify the actin-binding protein CaD as a new interaction partner of LPXN, thereby linking LPXN directly to the actin cytoskeleton for the first time. Furthermore, we provide a novel mechanism for the regulation of the actin cytoskeleton during migration: LPXN-mediated phosphorylation of CaD by the extracellular-signal regulated kinase 1/2 (ERK). RESULTS Reduced adhesion and cell size of PCa cells after LPXN knockdown To investigate the influence of LPXN expression on the adhesive characteristics of PCa cells, we performed a cell adhesion assay. After downregulation of LPXN expression in PC-3 and DU 145 cells using a specific siRNA, cells were plated on glass slides coated with fibronectin (FN), rat tail collagen (Col), bovine serum albumin (BSA) or gelatin (Gel). Adhered cells were fixed after 2 hours of incubation, and the cytoskeleton was visualized using FITC-conjugated phalloidin. Cell numbers and cell size were analyzed using confocal fluorescence microscopy. We observed that cells with LPXN knockdown showed reduced adhesion on all substrates in comparison to control cells (Figure ?(Figure1A).1A). The strongest effect of LPXN knockdown was observed for adhesion on FN-coated slides. In addition, the highest difference in cell size between LPXN knockdown and control transfected (siLuc) cells was observed on FN-coated and BSA-coated slides (Figure ?(Figure1B).1B). Thus, loss of LPXN expression seems to BI-409306 reduce the capability to adhere to the ECM in PCa cells. Open in a separate window Figure 1 LPXN knockdown decreases adhesion and cell sizeTo analyze adhesion, PC-3 and DU 145 cells transfected with siRNA against LPXN (siLPXN) or luciferase (siLuc = control) were plated on glass culture slides that were either uncoated (?) or coated with bovine serum albumin (BSA), collagen (Col), fibronectin (FN) or gelatin (Gel). Cells were fixed 2 hours after plating; the cytoskeleton was visualized using FITC-conjugated phalloidin (green), and nuclei were stained with DAPI (blue). Cell number (A) and cell size (B) were determined by confocal microscopy. After 2 hours (C) of adhesion, siLPXN-transfected cells showed a reduced surface area compared to control-transfected cells, whereas 24 hours (D) later, they were not distinguishable from each other. As summarized in Figure ?Figure1C,1C, PC-3 cells showed a significantly reduced surface area after LPXN knockdown compared with control transfected cells. After 2 hours, control cells were already spread on the substratum and had a strong contact to the fibronectin matrix, whereas cells with LPXN knockdown remained rounded BI-409306 and showed no cell protrusions. As a control and to study the effect of LPXN knockdown on long-term adhesion, cells transfected with siLPXN or siLuc (control) were allowed to adhere for 24 hours. During this time course, both cell populations could completely adhere to the substratum and showed no difference in their morphology (Figure ?(Figure1D),1D), pointing to a function of LPXN in early adhesion dynamics. LPXN interacts with the actin-binding protein CaD To identify proteins that could facilitate the cytoskeletal changes mediated by LPXN, we performed a yeast two-hybrid screen using a human prostate cDNA library with full-length LPXN as bait. This resulted in two different clones encoding the human actin-binding protein caldesmon BI-409306 (CaD, proximity ligation assay (PLA) on PC-3 cells using specific LPXN and CaD antibodies, respectively. Interaction of the two proteins is indicated by the red dots (Figure ?(Figure3D).3D). Confocal fluorescence microscopic analysis of the PLA revealed that LPXN-CaD interaction was mainly localized to the sub-membranous compartments, whereas no interaction was detected at the protrusion zone of migrating cells or at stabilized actin structures and podosomes (Figure ?(Figure3D).3D). We observed little interaction of LPXN and CaD in non-migrating or quiescent PCa.
Mean values are shown around the graphs. detrimentally affect the heart with precise toxicities varying with therapy1. Heart failure has become a common cause of death among malignancy survivors, and the possibility of developing this complication significantly limits the full and effective use of malignancy therapeutics1,2. The anthracycline doxorubicin remains an essential component in the treatment of solid tumors and leukemias in adults and children. Although its severe, dose-dependent cardiomyopathy has been recognized for almost a half-century3,4, progress in limiting this cardiotoxicity has been impeded by an incomplete understanding of the underlying mechanism. Doxorubicin kills malignancy cells by binding topoisomerase-2, thereby preventing the enzyme from re-ligating the double-stranded DNA breaks that it creates5. Some evidence suggests that doxorubicin-induced cardiomyopathy entails the same mechanism6. Other data, however, suggest the importance of additional mechanisms including oxidative modifications of proteins and lipids that damage cellular membranes causing multi-organelle dysfunction7,8, activation of cytoplasmic proteases9 and proteotoxic stress10. This has made it challenging to identify a single molecular target around which to build a therapy. While cell death is usually a unifying feature of doxorubicin-induced cardiac damage2,11,12, even this has confirmed complex, as it entails a combination of apoptosis and necrosis and it is not clear how one could simultaneously DL-AP3 target both of these death programs. BAX is usually a member of the BCL-2 family of proteins that resides in an inactive conformation in the cytosol of healthy cells. On cellular stress, BAX undergoes conformational changes that result in its translocation from your cytosol to the outer mitochondrial membrane (OMM) to induce cell death. The key role of BAX in apoptosis is usually to oligomerize within and permeabilize the OMM allowing release of apoptogens such as cytochrome = 7 males, 4 females; WT-DOX, = 4 males, 6 females; KO-saline, = 4 males, 4 females; KO-DOX, = 5 males, 6 females. Mean values are shown around the graphs. One-way analysis of variance (ANOVA), FS: *= 0.0120, ***= 0.0002; LVEDD-LVESD: **= 0.0040, ****< 0.0001. e, TUNEL of cardiac sections and quantification to assess apoptosis (= 3 males per group). One-way ANOVA, *= 0.0246. f, Immunofluorescence for loss of nuclear HMGB1 in cardiac sections and quantification to assess necrosis. Aqua color indicates presence of HMGB1 (HMGB1 + DL-AP3 4,6-diamidino-2-phenylindole (DAPI)) and blue color indicates loss of HMGB1 (DAPI alone) (= 3 males per group). One-way ANOVA, *= 0.0249. All data are offered as imply s.e.m. One-way ANOVA, NS, not significant > 0.05. Mechanism by which small-molecule BAI1 inhibits BAX in cells A family of carbazole-based compounds experienced previously been recognized in a screen for small molecules that inhibit cytochrome release from isolated mitochondria stimulated with BID, a member of another class of BCL-2 family proteins, called BH3-only proteins, which bind to and activate BAX and the homologous protein BAK24,25. In a companion study, we discovered using nuclear magnetic resonance (NMR) methods that one such compound, named BAX activation inhibitor 1 (BAI1) (Fig. 2a), binds inactive BAX within a primarily hydrophobic pocket previously uncharacterized and unique from the trigger site used by the BH3-only proteins to activate BAX26. We found that the conversation of BAI1 with this pocket allosterically inhibits BAX conformational activation by stabilizing Rabbit Polyclonal to ERCC5 the hydrophobic core of the protein to maintain the inactive state. Using microscale thermophoresis, we confirmed that BAI1 binds directly to inactive and soluble BAX (Fig. 2b and Extended Data Fig. 1). We next examined the effect of BAI1 around the conformational changes that mediate BAX activation, mitochondrial translocation and insertion into the OMM in cells. An early DL-AP3 conformational switch induced by the binding of the BH3-only proteins to the BAX trigger site (-helices 1 and 6) is usually a shift in the position of the unstructured loop between -helices 1 and 2 (ref. 17). This is reflected in the exposure of an epitope in.
Supplementary Materialsgenes-09-00247-s001. brain, a greatest bone model will be hypothesized. Furthermore, the near future application and need of such a complex super model tiffany livingston is going to be talked about. or vascular endothelial development aspect (VEGF) which induces angiogenesis within a mice model. Alginate hydrogels formulated with cell-instructive components that promote connection are of interest as potential cell companies in bone tissue tissue anatomist. Bhat et al. confirmed GluA3 that the current presence of built ECM elements on microbeads in alginate hydrogels promotes cell adhesion and osteogenic differentiation of MSCs without counting on cell-adhesive peptides . The usage of alginate beads doped with BMP-2 and platelet-rich elements results in a sustained discharge that promotes cell proliferation and osteogenic differentiation within a dose-dependent way. Platelet rich plasma can be very easily isolated and further processed but suffers from a limited storage life that leads to early decomposition of signaling factors . Beads can also be made out of bioactive ceramics such as HA and TCP. The advantages of combining both materials include the great mechanical strength and tissue adhesive properties of HA on the one hand and the high bioadsorbable properties of TCP on the other hand . 4.5. 3D Printing During the introduction of additive developing, the potential of 3D printing techniques in the context of bone was explored early. First attempts aimed to generate scaffolds that mimic the chemical and biomechanical characteristics of bone . These methods, however, require sintering of the deposited material to achieve the desired stability of the constructs and are therefore not suited to incorporate cells in the printing process. Yet, generating cell free scaffolds as fitted implants through 3D print remains a encouraging approach in reconstructive surgery of bone . For tissue engineering, bioprinting techniques such as inkjet writing (IW), extrusion printing (EP), JNJ-42041935 laser-assisted forward transfer (LIFT) and stereolithography (SLA) are suitable since they allow JNJ-42041935 the integration of living cells . These methods are excellently examined in [166,169] and will not be discussed in depth here in favor of bioprinting in the context of engineering cellularized bone tissue. In theory, bioprinting can be JNJ-42041935 employed for the reproducible generation of organoids, as it allows for the generation of specific structural features and the precise deposition of cells. Furthermore, it is possible to include vascularization in the organoid from the beginning, enhancing the exchange of air hence, metabolites and nutrients. The most frequent way for bioprinting bone tissue is EP since it allows for the usage of hydrogels with differing viscosities and high cell densities [170,171,172,173]. One disadvantage in EP may be the deposition procedure that’s facilitated through mechanised extrusion from the bioink by way of a nozzle, thus creating high shear pushes that may impact cell viability, for stem cells especially. Extrusion printing represents a solid and not at all hard bioprinting technique using the clear benefit of using a wide variety of hydrogel-based bioink formulations. Because of their mechanised properties, hydrogels aren’t suitable for producing bigger voids or hollow areas since layer-by-layer dispositioning would bring about collapse of structural features. As a result, sacrificial materials just like the poloxamere F-127 may be introduced to permit for printing hollow fibre buildings such as for example vessel lumen for improved perfusion from the organoid or following vascularization [174,175]. Although this enables for the bioprinting of more technical structures, the launch of a sacrificial materials might introduce issues alone. These include a rise of complexity within the printing procedure itself because of ongoing materials exchange that will require multiple nozzles. Nevertheless, the simultaneous usage of different cell-laden and sacrificial inks was confirmed by Shim et al successfully., emphasizing that the mandatory engineering solutions are for sale to multi-nozzle 3D printing . The sacrificial materials needs to end up being biocompatible and really should end up being printable beneath the same circumstances as the utilized bioinks, restricting the JNJ-42041935 number of materials available  thus. From EP Aside, LIFT was useful for bioprinting of bone tissue [178 also,179]. Laser-assisted forwards transfer includes a higher.
Supplementary Components1. lymphoid cells influences immune responses. Hence, the hematopoietic process is controlled. As opposed to steady-state hematopoiesis, physiological insults that want an severe way to obtain leukocytes briefly alter patterns of hematopoiesis. Such demand-adapted hematopoiesis is usually observed during severe infections, inflammation, and irradiation, and myelopoiesis becomes highly active to compensate the loss of myeloid cells1, 2, 3, 4. This response is called emergency myelopoiesis (or emergency granulopoiesis especially for the acute generation of neutrophils). Emergency granulopoiesis is brought on by stimulating pattern-recognition receptors (PRRs), reactive oxygen species, and cytokines, such as IL-6, GM-CSF, G-CSF, and others1, 2, 3, 4, 5, 6, 7, 8, 9, 10. Decreased cell density by depleting neutrophils can also promote granulopoiesis in the bone marrow HJC0152 (BM)10. Lymphocytes have distinct mechanisms from myeloid cells to regulate their populace sizes, and a normal immune system maintains an optimal balance between myeloid and T cells. OPN is usually a phosphoglycoprotein expressed in various tissues and cell types. OPN controls numerous immune responses and is involved in the pathogenesis of a wide variety of diseases11, 12, 13, 14, 15, 16, 17. OPN is usually indicated by BM stroma cells18 and negatively regulates stem cell pool size and function of Lin?Sca-1+c-kit+ (LSK) cells, including hematopoietic stem cells (HSCs)19, 20, 21. However, the effect of OPN on myeloid or lymphoid progenitors has not been explored. OPN is present as two translational isoforms, secreted OPN (sOPN) and intracellular OPN (iOPN). They have distinct functions because of HJC0152 the localization22. The majority of OPN studies possess focused on sOPN, which interacts with receptors such as integrins and CD44. In contrast, iOPN was later on found as a product of alternate translation23 and resides in the cytoplasm and occasionally in the nucleus. iOPN functions as an adaptor or scaffold protein in transmission transduction pathways, as well as stabilizing additional intracellular proteins11, 13, 14, 24, 25. Although sOPN in the hematopoietic stem cell market in the BM is definitely a negative regulator of HSC proliferation19, 20, the part of iOPN in hematopoiesis is definitely entirely unfamiliar. In this study, we statement that OPN skews the balance of cell populations towards a decrease of myeloid and an increase of lymphoid populations. However, this happens only during demand-adapted myelopoiesis (elicited by such as irradiation and systemic fungal illness) and lymphoid cell growth in lymphopenic recipients. We found that iOPN is responsible for the bad rules of myelopoiesis. In contrast, sOPN enhances lymphoid cell growth. Therefore, two different OPN isoforms play unique functions but, as a total, interact to decrease myeloid progenitors and increase lymphoid cells during demand-adapted myelopoiesis and lymphoid cell growth in lymphopenic hosts. RESULTS Cell population HJC0152 balance in irradiation BM chimeric mice In na?ve mice, OPN-deficiency does not affect numbers of total splenocytes, total BM cells, lineage bad (Lin?) progenitors, differentiated leukocytes in the BM19, 26, as well as compositions of Fgfr1 BM progenitor and differentiated leukocyte populations (Supplementary Fig. 1aCe). No effect of OPN was also recognized in proportions of embryonic leukocyte and their progenitor populations in fetal livers among littermate embryos (E13C15) from (gene encoding OPN) heterozygous breeders (Supplementary Fig. 1f, g). Next, we examined whether OPN affects the cell populace balance in combined BM radiation chimeras transferred with WT and BM cells (Supplementary Fig. 2a, b). Serum OPN (donor cells showed improved myeloid cell populations and decreased lymphoid cell populations in multiple organs including HJC0152 BM, spleen, blood, mesenteric lymph nodes (MLNs), liver, and lungs (Fig. 1a, b). donor cells experienced larger populations in multipotent progenitors (MPPs), common myeloid progenitors (CMPs), and granulocyte-macrophage progenitors (GMPs), but slightly a smaller common lymphoid progenitor (CLPs) cell populations, compared to WT donor cells (Fig. 1c, d). To confirm the BM cell transfer results, we also used combined LSK (Lin?Sca-1+c-kit+) cells for transfer (Supplementary Fig. 2d, e), and again cells to BM, as shown from the unaltered donor cell percentage (1:1 of WT and per each circle on day time 6. Data were from three self-employed HJC0152 experiments. Error bars indicate SEM. * mice showed elevated GMPs and neutrophils once again, in comparison to WT mice, in BM 24 hrs after shot (Fig. 2a, b). Right here, Injection and WT. Data had been pooled from two unbiased tests with 3C5 mice per test. (c) (OPN) mRNA amounts in GMPs from BM of WT mice at indicated period factors. hpi: hrs post shot. per group. (d) Total cell.
Supplementary MaterialsSupplemental Desk 1. the pathogenesis of osteoporosis. However, each technology individually cannot capture the entire view of the disease pathology and thus fails to comprehensively identify the underlying pathological molecular mechanisms, GSK163090 especially the regulatory and signalling mechanisms. A change to the status quo calls for integrative multi-omics and inter-omics analyses with approaches in systems genetics and genomics. In this Review, we highlight findings from genome-wide association studies and studies using various omics technologies individually to identify mechanisms of osteoporosis. Furthermore, we summarize current studies of data integration to understand, diagnose and inform the treatment of osteoporosis. The integration of multiple technologies will provide a road map to illuminate the complex pathogenesis of osteoporosis, from molecular functional elements specifically, in vivo in human beings. Osteoporosis, the most frequent GSK163090 bone disorder world-wide (FIG. 1), can be seen as a low bone nutrient denseness (BMD) and an elevated threat of osteoporotic fracture1. Based on the WHO, osteoporosis can be thought as a BMD that is situated 2.5 standard deviations or even more below the common value for young healthy women (T-score 2.5)2. As a result, the clinical diagnosis and assessment of osteoporosis is dependant on measurements of BMD3 mainly. Of take note, BMD includes a heritability of 0.6C0.8, and therefore 60C80% from the variation in BMD is inherited from parents and the rest comes from the environment4. Furthermore, osteoporotic fracture, which may be the last end stage medical result of osteoporosis, includes a heritability of 0.5C0.7 (REF.5). Not surprisingly strong heritability, identifying PI4KA the genetic structures (Package 1), and specifically the root molecular and genomic systems of osteoporosis in vivo in human beings, can be challenging. Open up in another windowpane Fig. 1 | Prevalence of osteoporosis in populations old 50 years and old in chosen countries.The prevalence of osteoporosis in the noninstitutionalized USA population was calculated using data collected by GSK163090 the National Health and Nutrition Examination Survey 2005C2010 (REF.153). The statistics for six European countries (France, Germany, Italy, Spain, Sweden and the UK) were retrieved from a report by the International Osteoporosis Foundation154. The statistics for China and Korea were obtained from a meta-analysis study published in 2016 (REF.155) and the Korea National Health and Nutrition Examination Survey 2008C2010 (REF.156), respectively. Data for Canada, Japan and Australia were obtained from a 2014 study157. BOX 1 | Key terms in genetic and omics studies Allelic heterogeneityMultiple single nucleotide polymorphisms within the same gene and/or pathway jointly affect the same trait. Distant geneIf a genetic variant affects the expression or otherwise interacts with genes other than the nearest gene, the target genes are referred as distant genes of the variant of interest. Effect sizeThe portion of phenotypic variance that is explained by the tested variant. EpigenomicsThe study of genome-wide reversible modifications of DNA or DNA-associated proteins such as DNA methylation, histone acetylation and chromatin organization. Expression quantitative trait loci (eQTL) analysisA technique for assessing the associations between transcript expression and genotype to identify genetic variants that explain the variation in gene expression levels. FingerprintSpecific expression profiles of proteins, which can be used as characteristics to distinguish different individuals. Genetic architectureThe characteristics of genetic variation GSK163090 that are responsible for heritable phenotypic variability150. Genome-wide association studies (GWAS)Studies using a hypothesis-free method to investigate the associations between genetic variants and traits, including diseases. Hybrid mouse diversity panelA collection of approximately 100 well-characterized inbred strains of mice that can be used to analyse the genetic and environmental factors underlying complex traits. KnowledgebaseA library used to store complex structured and unstructured information by a computer system. Long-rangeThe distance between regulatory regions and their target genes is considered far, usually >100 kb. Mendelian randomizationMendelian randomization is a method of using genetic variants to determine whether an observational association between a risk factor and an result can be in keeping with a causal impact. MetabolomicsA field of omics technology to measure little substances systematically, commonly knowns.
Renal cell carcinoma with brain metastases is known as to have a poor prognosis. region with perilesional edema in magnetic resonance imaging (MRI) of brain and an enhancing renal lower polar mass measuring about 10?cm??6?cm on computerised tomography (CT) of abdomen with subcentimetric parenchymal nodules in basal lung filed largest measuring 7?mm. Renal biopsy confirmed the primary to be clear cell carcinoma (ISUP grade 2). As per the recommendation by the multidisciplinary tumour board, the patient was treated with radiation therapy (single-fraction frameless stereotactic radiosurgery to the brain lesion 13?Gy) for brain lesion followed by sunitinib therapy at an oral dose of 50?mg daily for 4?weeks on and 2?weeks off. Dexamethasone 16?mg was also given daily, which was progressively decreased until discontinuation during the following months. MRI of the brain (March 2014) revealed complete remission of the tumour (Figure 1). Computerised tomography of abdomen (April 2014) revealed a reduction in the size of renal mass (Figure 2). The patient underwent laparoscopic radical nephrectomy in April 2014. By February 2017, there was complete remission of brain and Thiamine pyrophosphate chest metastasis, but was found to have a lesion in the right kidney, for which the patient underwent radiofrequency ablation in March 2017. The procedure was uneventful and the patient was put on regular follow-up. Open in a separate window Figure 1. (A) MRI lesion in brain and (B) MRI lesion responding completely to TKI. MRI indicates magnetic resonance imaging; TKI, tyrosine kinase inhibitor. Open in a separate window Figure 2. (A) Kidney tumour before staring TKI and (B) primary tumour after TKI. TKI indicates tyrosine kinase inhibitor. The patient developed chronic renal failure with serum creatinine level reaching 6.8?mg/dL and proteinuria. As a result, sunitinib was reduced to 25?mg OD (duration of therapy 40?months) and later changed to sorafenib 200?mg BD (on sorafenib LGR3 for 21?months till last follow-up). There was Thiamine pyrophosphate no worsening of serum creatinine level with sorafenib therapy; therefore, sorafenib 200?mg was continued. The individual does well till last follow-up in November 2018 (5?years from preliminary diagnosis). Dialogue The prognosis of mind metastases in RCC offers typically been dismal.2 Cytokines used in RCC have limited central nervous system (CNS) efficacy4 as it does not cross the blood-brain barrier. Tyrosine kinase inhibitor showed better response in metastatic RCC1 and is considered to be the standard of care now. The efficacy of sorafenib, sunitinib, and temsirolimus in CNS is not known because the previous phase 3 trial with these drugs have excluded patients with CNS disease.5C7 Management of brain metastasis in the renal tumour is still controversial even though there are case Thiamine pyrophosphate reports about the benefit of TKI in this scenario. A case reported in Greece on March 2007 showed the activity of sunitinib in brain metastases from RCC, in which the patient had a partial response of the cerebral lesion following treatment with sunitinib. Sunitinib was safe and led to a considerable shrinkage of the brain metastases without any serious adverse reactions or CNS toxicities.8 The role of cytoreductive nephrectomy is disputed in these patients due to limited survival. In our case, the brain metastases in RCC showed excellent response to sunitinib therapy and radiotherapy, leading to complete remission of the lesion in the brain, and hence a cytoreductive nephrectomy was advised Thiamine pyrophosphate later. This case shows that we can have an excellent response in select patients with a low burden of metastasis in the brain. Probably, the initial radiotherapy could have helped the penetration of the small TKI molecules across the blood-brain barrier leading to the excellent response to the treatment.9 Conclusions Even though RCC with brain metastases is considered to have a poor prognosis, they can have an excellent response with a combination of radiation and TKI. If there is a good response, they may be considered for cytoreductive nephrectomy. Further studies in this area may help in identifying factor predicting response to radiation and TKI in such patients. Footnotes Funding:The author(s) received no financial support for the research, authorship, and/or publication of the content. Declaration of conflicting passions:The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article. Contributed by Author Contributions: All authors contributed equally in the write-up and editing of the article. ORCID identification: Abhishek Laddha https://orcid.org/0000-0003-1761-1783.