Manufacture and Deformation Angle Control of a Two-Direction Soft Actuator Integrated with SMAs.

In this contribution, the development of a 3D-printed soft actuator integrated with shape memory alloys (SMA) wires capable of bending in two directions is presented. This work discusses the design, manufacturing, modeling, simulation, and feedback control of the actuator. The SMA wires are encased in Polytetrafluoroethylene (PTFE) tubes and then integrated into the 3D-printed matrix made of thermoplastic polyurethane (TPU). To measure and control the deformation angle of the soft actuator, a computer vision system was implemented. Based on the experimental results, a mathematical model was developed using the system identification method and simulated to describe the dynamics of the actuator, contributing to the design of a controller. However, achieving precise control of the deformation angle in systems actuated by SMA wires is challenging due to their inherent nonlinearities and hysteretic behavior. A proportional-integral (PI) controller was designed to address this challenge, and its effectiveness was validated through real experiments.

Co-delivery of rhBMP-2 and zoledronic acid using calcium sulfate/hydroxyapatite carrier as a bioactive bone substitute to enhance and accelerate spinal fusion.

Recombinant human bone morphogenetic protein-2 (rhBMP-2) has been FDA-approved for lumbar fusion, but supraphysiologic initial burst release due to suboptimal carrier and late excess bone resorption caused by osteoclast activation have limited its clinical usage. One strategy to mitigate the pro-osteoclast side effect of rhBMP-2 is to give systemic bisphosphonates, but it presents challenges with systemic side effects and low local bioavailability. The aim of this in vivo study was to analyze if posterolateral spinal fusion (PLF) could be improved by utilizing a calcium sulfate/hydroxyapatite (CaS/HA) carrier co-delivering rhBMP-2 and zoledronic acid (ZA). Six groups were allocated (CaS/HA, CaS/HA + BMP-2, CaS/HA + systemic ZA, CaS/HA + local ZA, CaS/HA + BMP-2 + systemic ZA, and CaS/HA + BMP-2 + local ZA). 10-week-old male Wistar rats, were randomly assigned to undergo L4-L5 PLF with implantation of group-dependent scaffolds. At 3 and 6 weeks, the animals were euthanized for radiography, µCT, histological staining, or biomechanical testing to evaluate spinal fusion. The results demonstrated that the CaS/HA biomaterial alone or in combination with local or systemic ZA didn't support PLF. However, the delivery of rhBMP-2 significantly promoted PLF. Combining systemic ZA with BMP-2 didn't enhance spinal fusion. Notably, the co-delivery of rhBMP-2 and ZA using the CaS/HA carrier significantly enhanced and accelerated PLF, without inhibiting systemic bone turnover, and potentially reduced the dose of rhBMP-2. Together, the treatment regimen of CaS/HA biomaterial co-delivering rhBMP-2 and ZA could potentially be a safe and cost-effective off-the-shelf bioactive bone substitute to enhance spinal fusion.

Adipocyte Precursor-Derived NRG1 Promotes Resistance to FGFR Inhibition in Urothelial Carcinoma.

Aberrations of the fibroblast growth factor receptor (FGFR) family members are frequently observed in metastatic urothelial cancer (mUC), and blocking the FGF/FGFR signaling axis is used as a targeted therapeutic strategy for treating patients. Erdafitinib is a pan-FGFR inhibitor, which has recently been approved by the FDA for mUC with FGFR2/3 alterations. Although mUC patients show initial response to erdafitinib, acquired resistance rapidly develops. Here, we found that adipocyte precursors promoted resistance to erdafitinib in FGFR-dependent bladder and lung cancer in a paracrine manner. Moreover, neuregulin 1 (NRG1) secreted from adipocyte precursors was a mediator of erdafitinib resistance by activating human epidermal growth factor receptor 3 (ERBB3; also known as HER3) signaling, and knockdown of NRG1 in adipocyte precursors abrogated the conferred paracrine resistance. NRG1 expression was significantly downregulated in terminally differentiated adipocytes compared with their progenitors. Pharmacologic inhibition of the NRG1/HER3 axis using pertuzumab reversed erdafitinib resistance in tumor cells in vitro and prolonged survival of mice bearing bladder cancer xenografts in vivo. Remarkably, data from single-cell RNA sequencing revealed that NRG1 was enriched in platelet-derived growth factor receptor-A (PDGFRA) expressing inflammatory cancer-associated fibroblasts, which is also expressed on adipocyte precursors. Together, this work reveals a paracrine mechanism of anti-FGFR resistance in bladder cancer, and potentially other cancers, that is amenable to inhibition using available targeted therapies. SIGNIFICANCE: Acquired resistance to FGFR inhibition can be rapidly promoted by paracrine activation of the NRG1/HER3 axis mediated by adipocyte precursors and can be overcome by the combination of pertuzumab and erdafitinib treatment. See related commentary by Kolonin and Anastassiou, p. 648.

Numerical and Experimental Investigation of Time-Domain-Reflectometry-Based Sensors for Foreign Object Detection in Wireless Power Transfer Systems.

Foreign object detection (FOD) is considered a key method for detecting objects in the air gap of a wireless charging system that could pose a risk due to strong inductive heating. This paper describes a novel method for the detection of metallic objects utilizing the principle of electric time domain reflectometry. Through an analytical, numerical and experimental investigation, two key parameters for the design of transmission lines are identified and investigated with respect to the specific constraints of inductive power transfer. For this purpose, a transient electromagnetic simulation model is established to obtain and compare the sensor impedance and reflection coefficients with experimental data. The measurement setup is based on parametrically designed sensors in laboratory scale, using an EUR 2 coin as an exemplary test object. Consequently, the proposed simulation model has been successfully validated in this study, providing a comprehensive quantitative and qualitative analysis of the major transmission line design parameters for such applications.

Numerical Investigation of the Orientability of Single Reinforcement Fibers in Polymer Matrices.

Fiber-reinforced polymers are increasingly being used, especially in lightweight structures. Here, the effective adaptation of mechanical or physical properties to the necessary application or manufacturing requirements plays an important role. In this context, the alignment of reinforcing fibers is often hindered by manufacturing aspects. To achieve graded or locally adjusted alignment of different fiber lengths, common manufacturing technologies such as injection molding or compression molding need to be supported by the external non-mechanical process. Magnetic or electrostatic fields seem to be particularly suitable for this purpose. The present work shows a first simulation study of the alignment of magnetic particles in polymer matrices as a function of different parameters. The parameters studied are the viscosity of the surrounding polymer as a function of the focused processing methods, the fiber length, the thickness and permeability of the magnetic fiber coatings, and the magnetic flux density. The novelty of the presented works is in the development of an advanced simulation model that allows the simulative representation and reveal of the fluid-structure interaction, the influences of these parameters on the inducible magnetic torque and fiber alignment of a single fiber. Accordingly, the greatest influence on fiber alignment is caused by the magnetic flux density and the coating material.

Delamination Behaviour of Embedded Polymeric Sensor and Actuator Carrier Layers in Epoxy Based CFRP Laminates-A Study of Energy Release Rates.

Fiber reinforced composites combine low density with high specific mechanical properties and thus became indispensable for today's lightweight applications. In particular, carbon fibre reinforced plastic (CFRP) is broadly used for aerospace components. However, damage and failure behaviour, especially for complex fibre reinforcement set-ups and under impact loading conditions, are still not fully understood yet. Therefore, relatively large margins of safety are currently used for designing high-performance materials and structures. Technologies to functionalise the materials enabling the monitoring of the structures and thus avoiding critical conditions are considered to be key to overcoming these drawbacks. For this, sensors and actuators are bonded to the surface of the composite structures or are integrated into the composite lay-up. In case of integration, the impact on the mechanical properties of the composite materials needs to be understood in detail. Additional elements may disturb the composite structure, impeding the direct connection of the composite layers and implying the risk of reducing the interlaminar integrity by means of a lower delamination resistance. In the presented study, the possibility of adjusting the interface between the integrated actuator and sensor layers to the composite layers is investigated. Different polymer layer combinations integrated into carbon fibre reinforced composite layups are compared with respect to their interlaminar critical energy release rates GIc and GIIc. A standard aerospace unidirectionally reinforced (UD) CFRP prepreg material was used as reference material configuration. The investigations show that it is possible to enhance the mechanical properties, especially the interlaminar energy release rate by using multilayered sensor-actuator layers with Polyimide (PI) outer layers and layers with low shear stiffness in between.

Loss or oncogenic mutation of DROSHA impairs kidney development and function, but is not sufficient for Wilms tumor formation.

Wilms tumor (WT) is the most common kidney cancer in childhood. Mutations in the microprocessor genes DROSHA and DGCR8 have been identified as putative oncogenic drivers, indicating a critical role of aberrant miRNA processing in WT formation. To characterize the in vivo role of DROSHA mutations during kidney development and their oncogenic potential, we analyzed mouse lines with either a targeted deletion of Drosha or an inducible expression of human DROSHA carrying a tumor-specific E1147K mutation that acts in a dominant negative manner. Both types of mutation induce striking changes in miRNA patterns. Six2-cre mediated deletion of Drosha in nephron progenitors led to perinatal lethality with apoptotic loss of progenitor cells and early termination of nephrogenesis. Mosaic deletions via Wt1-creERT2 resulted in a milder phenotype with viable offspring that developed proteinuria after 2-4 weeks, but no evidence of tumor formation. Activation of the DROSHA-E1147K transgene via Six2-cre, on the other hand, induced a more severe phenotype with apoptosis of progenitor cells, proteinuria and glomerular sclerosis. The severely growth retarded mice died within the first 2 months of life, confirming the predicted dominant-negative effect of DROSHA-E1147K in vivo. While our data underscores the importance of a viable self-renewing progenitor pool for kidney development, there was no evidence of tumor formation through impaired DROSHA function. This suggests that either additional alterations in mitogenic or antiapoptotic pathways are needed for malignant transformation, or premature loss of a susceptible target cell population and early lethality prevent WT formation.

Hey bHLH Proteins Interact with a FBXO45 Containing SCF Ubiquitin Ligase Complex and Induce Its Translocation into the Nucleus.

The Hey protein family, comprising Hey1, Hey2 and HeyL in mammals, conveys Notch signals in many cell types. The helix-loop-helix (HLH) domain as well as the Orange domain, mediate homo- and heterodimerization of these transcription factors. Although distinct interaction partners have been identified so far, their physiological relevance for Hey functions is still largely unclear. Using a tandem affinity purification approach and mass spectrometry analysis we identified members of an ubiquitin E3-ligase complex consisting of FBXO45, PAM and SKP1 as novel Hey1 associated proteins. There is a direct interaction between Hey1 and FBXO45, whereas FBXO45 is needed to mediate indirect Hey1 binding to SKP1. Expression of Hey1 induces translocation of FBXO45 and PAM into the nucleus. Hey1 is a short-lived protein that is degraded by the proteasome, but there is no evidence for FBXO45-dependent ubiquitination of Hey1. On the contrary, Hey1 mediated nuclear translocation of FBXO45 and its associated ubiquitin ligase complex may extend its spectrum to additional nuclear targets triggering their ubiquitination. This suggests a novel mechanism of action for Hey bHLH factors.

Gender matters: estrogen receptor alpha (ERα) and histone deacetylase (HDAC) 1 and 2 control the gender-specific transcriptional regulation of human uridine diphosphate glucuronosyltransferases genes (UGT1A).

BACKGROUND & AIMS: Gender influences incidence, progression, and therapy of hepatogastrointestinal diseases. The aim of this study was to elucidate the molecular mechanism of gender-specific UDP-glucuronosyltransferases (UGT1A) regulation, representing important hepatogastrointestinal detoxification enzymes for xenobiotics, drugs, and endobiotics. METHODS: UGT1A-gene activation was studied by reporter gene experiments and estrogen receptor alpha (ESR1/ERα) co-transfection using KYSE70- and HepG2 cells (male origin), and SW403 cells (female origin). Cell lines, and humanized transgenic UGT1A (htgUGT1A) mice (female/male) were treated with the ERα inhibitor tamoxifen. UGT1A mRNA expression was analyzed by TaqMan PCR, the recruitment of ERα, histone deacetylases (HDAC), and the aryl hydrocarbon receptor (AhR) by chromatin immunoprecipitation (ChIP), and ERα expression in gastrointestinal mouse tissues by Western blot and immunofluorescence. RESULTS: In KYSE70 cells (male), UGT1A gene expression was induced 5-10 fold, and inhibited in the presence of ERα by 55-77%. In SW403 (female) cells, absent inducibility was restored after tamoxifen treatment. In the jejunum and colon of tgUGT1A mice, UGT1A induction that was exclusively detected in male mice could be restored in female mice after tamoxifen pre-treatment. ChIP assays demonstrated the recruitment of ERα and HDACs to the xenobiotic response elements of UGT1A promoters during gene repression. Western blot showed higher ERα expression in the female jejunum and colon. CONCLUSIONS: We show gender-specific transcriptional control of UGT1A genes in jejunum and colon, which is repressed by ERα and the recruitment of HDCAs to the UGT1A promoter sequence in females. A molecular mechanism controlling gender-specific drug metabolism and its therapeutic reversal is demonstrated.

Target gene analysis by microarrays and chromatin immunoprecipitation identifies HEY proteins as highly redundant bHLH repressors.

HEY bHLH transcription factors have been shown to regulate multiple key steps in cardiovascular development. They can be induced by activated NOTCH receptors, but other upstream stimuli mediated by TGFß and BMP receptors may elicit a similar response. While the basic and helix-loop-helix domains exhibit strong similarity, large parts of the proteins are still unique and may serve divergent functions. The striking overlap of cardiac defects in HEY2 and combined HEY1/HEYL knockout mice suggested that all three HEY genes fulfill overlapping function in target cells. We therefore sought to identify target genes for HEY proteins by microarray expression and ChIPseq analyses in HEK293 cells, cardiomyocytes, and murine hearts. HEY proteins were found to modulate expression of their target gene to a rather limited extent, but with striking functional interchangeability between HEY factors. Chromatin immunoprecipitation revealed a much greater number of potential binding sites that again largely overlap between HEY factors. Binding sites are clustered in the proximal promoter region especially of transcriptional regulators or developmental control genes. Multiple lines of evidence suggest that HEY proteins primarily act as direct transcriptional repressors, while gene activation seems to be due to secondary or indirect effects. Mutagenesis of putative DNA binding residues supports the notion of direct DNA binding. While class B E-box sequences (CACGYG) clearly represent preferred target sequences, there must be additional and more loosely defined modes of DNA binding since many of the target promoters that are efficiently bound by HEY proteins do not contain an E-box motif. These data clearly establish the three HEY bHLH factors as highly redundant transcriptional repressors in vitro and in vivo, which explains the combinatorial action observed in different tissues with overlapping expression.

Retinoic acid-mediated down-regulation of ENO1/MBP-1 gene products caused decreased invasiveness of the follicular thyroid carcinoma cell lines.

Retinoic acid (RA) acts as an anti-proliferative and redifferentiation agent in the therapy of thyroid carcinoma. Our previous studies demonstrated that pretreatment of follicular thyroid carcinoma cell lines FTC-133 and FTC-238 resulted in decreased in vitro proliferation rates and reduced tumor cell growth of xenotransplants. In addition to the previous results, we found that RA led to decreased vitality and invasiveness of FTC-133 and FTC-238 cells as they reacted with reduction of intracellular ATP levels and number of migrated cells respectively. However, the molecular mechanisms by which RA mediates these effects are not well understood. Two-dimensional (2D) screening of the proteins related to ATP metabolism and western blot analysis revealed alpha-enolase (ENO1) to be down-regulated in FTC-133 and FTC-238 cells after RA treatment. 2D gel detection and mass spectrometric analysis revealed that ENO1 existed as three separate protein spots of distinct pIs (ENO1-A1-A3). Comparative 2D difference gel electrophoresis analysis of fluorescently labeled protein samples of RA-treated and untreated FTC-133 demonstrated a selective down-regulation of ENO1-A1 which we identified as a phosphoprotein. RA caused the dephosphorylation of ENO1-A1. Both, RA-mediated and specific knock-down of ENO1/MBP-1 resulted in the reduction of MYC oncoprotein, and simultaneously decreased proliferation rates of FTC-133 and FTC-238 cell lines. In summary, the RA-mediated down-regulation of the ENO1 gene products and MYC oncoprotein provides a novel molecular mechanism facilitating the anti-proliferative effect of RA in human thyroid carcinoma cells and suggests new pathways for supportive RA therapies.

Hypoxia-mediated activation of Dll4-Notch-Hey2 signaling in endothelial progenitor cells and adoption of arterial cell fate.

Adequate response to low oxygen levels (hypoxia) by hypoxia inducible factor (HIF) is essential for normal development and physiology, but this pathway may also contribute to pathological processes like tumor angiogenesis. Here we show that hypoxia is an inducer of Notch signaling. Hypoxic conditions lead to induction of the Notch ligand Dll4 and the Notch target genes Hey1 and Hey2 in various cell lines. Promoter analysis revealed that Hey1, Hey2 and Dll4 are induced by HIF-1alpha and Notch activation. Hypoxia-induced Notch signaling may also determine endothelial identity. Endothelial progenitor cells (EPCs) contain high amounts of COUP-TFII, a regulator of vein identity, while levels of the arterial regulators Dll4 and Hey2 are low. Hypoxia-mediated upregulation of Dll4 and Hey2 leads to repression of COUP-TFII in eEPCs. Finally, we show that Hey factors are capable of repressing HIF-1alpha-induced gene expression, suggesting a negative feedback loop to prevent excessive hypoxic gene induction. Thus, reduced oxygen levels lead to activation of the Dll4-Notch-Hey2 signaling cascade and subsequent repression of COUP-TFII in endothelial progenitor cells. We propose that this is an important step in the developmental regulation of arterial cell fate decision.