STAT3 and the STAT3­regulated inhibitor of apoptosis protein survivin as potential therapeutic targets in colorectal cancer (Review).

Colorectal cancer (CRC) is one of the leading types of cancer worldwide. CRC development has been associated with the constitutive activation of signal transducer and activator of transcription 3 (STAT3). STAT3 is a master regulator of inflammation during cancer-associated colitis, and becomes upregulated in CRC. In CRC, STAT3 is activated by IL-6, among other pro-inflammatory cytokines, inducing the expression of target genes that stimulate proliferation, angiogenesis and the inhibition of apoptosis. One of the main STAT3-regulated inhibitors of apoptosis is survivin, which is a bifunctional protein that regulates apoptosis and participates in cell mitosis. Survivin expression is normally limited to foetal tissue; however, survivin is also upregulated in tumours. In silico and experimental analyses have shown that the STAT3 interactome is relevant during CRC progression, and the constitutive STAT3-survivin axis participates in development of the tumour microenvironment and response to therapy. The presence of a STAT3-survivin axis has been documented in CRC cohorts, and the expression of these molecules is associated with poor prognosis and a higher mortality rate in patients with CRC. Thus, STAT3, survivin, and the upstream activators IL-6 and IL-6 receptor, are considered therapeutic targets for CRC. Efforts to develop drugs targeting the STAT3-survivin axis include the evaluation of phytochemical compounds, small molecules and monoclonal antibodies. In the present review, the expression, function and participation of the STAT3-survivin axis in the progression of CRC were investigated. In addition, an update on the pre-clinical and clinical trials evaluating potential treatments targeting the STAT3-survivin axis is presented.

MicroRNA-122 protects against interferon-α-induced hepatic inflammatory response via the Janus kinase-signal transducer and activator of transcription pathway.

Significant overlap in the epidemiology and coinfection of chronic hepatitis B virus (HBV) and hepatitis C virus (HCV) has been identified, which accelerates the development of severe liver cirrhosis and hepatocellular carcinoma worldwide. Interferon-α (IFN-α), a cytokine with antiviral properties, exerts profound physiological effects on innate immunity by regulating interferon-stimulated genes (ISGs) within cells. However, the underlying mechanism of IFN-α in hepatic inflammation remains to be fully elucidated. Here, we utilized LO2 cells treated with the recombinant IFN-α protein and conducted microRNA (miR) sequencing. MiR-122-3p and miR-122-5p_R+1 were the most enriched miRNAs involved in the pathogenesis of IFN-α-induced inflammatory responses and were significantly downregulated by IFN-α treatment. Furthermore, we identified interferon induced protein with tetratricopeptide repeats 1 (IFIT1) as a potential target gene of miR-122. IFN-α markedly increased the expression of proinflammatory cytokines and fibrogenic genes but decreased the mRNA expression of ISGs. Additionally, IFN-α significantly activated the NF-κB p-p65, MAPK p-p38, and Jak/STAT pathways to trigger inflammation. Importantly, supplementation with a miR-122 mimic significantly alleviated IFN-α-induced inflammation and induced IFIT1 expression in LO2 cells. Conversely, the suppression of miR-122 markedly exacerbated the inflammatory response triggered by IFN-α. Furthermore, silencing IFIT1 via an siRNA elicited an inflammatory response, whereas IFIT1 overexpression ameliorated hepatic inflammation and fibrosis in a manner comparable to that induced by IFN-α treatment. Taken together, our findings suggest that miR-122 and its target, IFIT1, reciprocally regulate the inflammatory response associated with IFN through the Jak/STAT pathway.

The Atypical Dual Specificity Phosphatase DUSP15 Regulates Jak1-Mediated STAT3 Activation.

The signal transducer and activator of transcription 3 (STAT3) protein is a key regulator of cell differentiation, proliferation, and survival in hematopoiesis, immune responses, and other biological systems. STAT3 transcriptional activity is strictly regulated through various mechanisms, such as phosphorylation and dephosphorylation. In this study, we attempted to identify novel phosphatases which regulate STAT3 activity in response to cytokine stimulations. To this end, leukemia inhibitory factor (LIF)/STAT3 dependent phosphatase induction was evaluated in the mouse hepatoma cell line Hepa1-6. After LIF stimulation, the expression of several atypical dual specific phosphatases (aDUSPs) was upregulated in Hepa1-6 cells. Among the LIF-induced aDUSPs, we focused on DUSP15 and clarified its functions in LIF/STAT3 signaling using RNA interference. DUSP15 knockdown decreased LIF-induced Socs3 mRNA expression and STAT3 translocation. Furthermore, loss of DUSP15 reduced the phosphorylation of STAT3 at Tyr705 and Janus family tyrosine kinase 1 (Jak1) at Tyr1034/1035 in response to LIF. The interaction between Jak1 and DUSP15 was observed in LIF-stimulated Hepa1-6 cells. We also demonstrated the suppression of granulocyte colony-stimulating factor (G-CSF)-mediated gp130/STAT3-dependent cell growth of Ba/F-G133 cells via DUSP15 knockdown. Therefore, DUSP15 functions as a positive feedback regulator in the Jak1/STAT3 signaling cascade.

Effects of STAT4 on myocardial ischemia­reperfusion injury and the underlying mechanisms.

The regulation of cardiac function by the nuclear transcription factor signal transducer and activator of transcription 4 (STAT4) has been recently recognized. Nevertheless, the role and mechanisms of action of STAT4 in myocardial ischemia­reperfusion (I/R) injury remain unknown. Consequently, the present study constructed a rat model of I/R by ligation of the left anterior descending coronary artery. Following sacrifice, the rat hearts were excised and analyzed to investigated the effects of STAT4 on I/R­induced myocardial injury. Western blotting demonstrated that expression of STAT4 decreased significantly in the rat model of cardiac I/R and in H9C2 cells that were subjected to hypoxia and reoxygenation (H/R). The overexpression of STAT4 in H9C2 cells reduced cell damage and apoptosis induced by H/R. Furthermore, both in vivo and in vitro, the level of PI3K decreased significantly. Although the AKT protein expression levels were not altered, the AKT phosphorylation levels decreased significantly. STAT4 overexpression enhanced the expression of PI3K and AKT in the H9C2 cells. On the whole, the present study demonstrated that STAT4 alleviated I/R­induced myocardial injury through the PI3K/AKT signaling pathway.

Zinc finger protein 263 promotes colorectal cancer cell progression by activating STAT3 and enhancing chemoradiotherapy resistance.

Zinc finger protein 263 (ZNF263) is frequently upregulated in various tumor types; however, its function and regulatory mechanism in colorectal cancer (CRC) have not yet been elucidated. In this study, the expression of ZNF263 was systematically examined using data from The Cancer Genome Atlas database and samples from patients with CRC. The results indicated that high expression of ZNF263 in CRC tissues is significantly associated with tumor grade, lymph node metastasis and disant metastasis. Additionally, overexpression of ZNF263 significantly promoted the proliferation, invasion, migration, and epithelial-mesenchymal transition of CRC cells, while also increasing signal transducer and activator of transcription 3 (STAT3) expression and mRNA stability. Conversely, knockdown of ZNF263 inhibited the malignant behavior of CRC cells and decreased STAT3 expression and mRNA stability. Further mechanism studies using chromatin immunoprecipitation (CHIP) and luciferase assays verified that ZNF263 directly binds to the STAT3 promoter. Rescue experiments demonstrated that the knockdown or overexpression of STAT3 could significantly reverse the effects of ZNF263 on CRC cells. Additionally, our study found that overexpression of ZNF263 enhanced the resistance of CRC cells to the chemoradiotherapy. In summary, this study not only elucidated the significant role of ZNF263 in CRC but also proposed novel approaches and methodologies for the diagnosis and treatment of this malignancy.

An acoustic bellows-type round window transducer for middle-ear implants.

BACKGROUND: This study describes the development of output devices for round window middle-ear. To overcome the problems of output devices that apply sound pressure directly to the round window, an acoustic bellows-type round window transducer was implemented by combining a small bellows, acoustic tube, and balanced armature driver. METHODS: The output characteristics of the proposed acoustic bellows-type round window transducer were confirmed through bench tests and distortion measurements. To compare the vibration transmission characteristics of the proposed transducer with those of sound pressure stimulation devices, an experiment was performed using four human temporal bones. FINDINGS: The average output magnitude of the acoustic bellows-type round window transducer was equivalent to sound pressure levels of 92, 96, and 108 dB for frequency ranges of <1, 1-2, and > 2 kHz, respectively. The results showed that the proposed transducer delivered vibration consistently without reducing the sound pressure level due to leakage, unlike the sound pressure stimulation device. INTERPRETATION: Therefore, the acoustic bellows-type round window transducer is a more stable and suitable output device for round window middle-ear implants than a sound pressure stimulation device. It is expected to overcome the limitations of sound pressure stimulation devices and to contribute to new technical solutions in the field of round window middle-ear implants development.

CMTM4 inhibits gastric tumorigenesis and metastasis.

Background: CKLF-like MARVEL transmembrane domain-containing 4 (CMTM4) is involved in immune regulation and tumor progression; however, its role in gastric cancer (GC) remains unclear. This study explored the role and mechanism of CMTM4 in GC. Methods: Immunohistochemistry was used to analyze CMTM4 expression in human gastric biopsied cells from patients with GC (N=23) or chronic superficial gastritis (N=23). To investigate the function of CMTM4 in GC cells, the gene CMTM4 was knocked down and overexpressed in human gastric adenocarcinoma cell line AGS. The gene CMTM4 was overexpressed in AGS cells and human gastric cell line SGC7901. Cell Counting Kit 8 (CCK-8) and cell clonogenic assays were used to analyze the proliferation of the GC cells. Flow cytometry was used to analyze the effects of CMTM4 on apoptosis and the cell cycle. Wound healing and transwell assays were used to analyze the migration and invasion of the gastric cells, respectively. The mechanism of CMTM4 in GC cells was explored using the tandem mass tags (TMTs) proteome and verified by western blot analysis. Results: CMTM4 expression was more downregulated in the human GC tissues than the gastritis tissues. CMTM4 overexpression significantly inhibited the proliferation, migration, and invasion of the GC cells, whereas CMTM4 knockdown enhanced gastric cell proliferation (P>0.05), migration (P>0.05), and invasion (P>0.05). Flow cytometry showed that CMTM4 promoted apoptosis and resulted in G1/S arrest in the GC cells. In addition, the proteome and western blot results showed that STAT1 was significantly upregulated, and the STAT1 signaling pathways were enriched in the GC cells overexpressing CMTM4. Conclusions: Our results suggest that CMTM4 plays a tumor-suppressive role in GC and may affect the growth, migration, and invasion of GC cells through the STAT1 signaling pathway. CMTM4 might have potential value as a prognosis marker and potential therapeutic target for GC therapy.

Identification of Entinostat as a Novel Modifier of STAT3 Pre-mRNA Alternative Splicing.

Signal transducer and activator of transcription 3 (STAT3) is a pleiotropic factor involved in multiple vital biological processes and a key mediator of gene transcription in response to cytokines, growth factors and aberrant activation of oncogenic signaling. STAT3 has two splicing isoforms, STAT3α and STAT3ß, derived from alternative splicing of exon 23 within pre-mRNA. STAT3ß differs from STAT3α by replacement of 55 amino-acid residues in the C-terminal transactivation domain with 7 specific amino acids. Thus, a shorter STAT3ß was originally regarded as a dominant negative isoform of STAT3α. Recently accumulating evidence from independent studies have shown STAT3 splicing isoforms confer distinct and overlapping functions in many fundamental cellular regulatory steps such as cell differentiation, inflammatory responses, and cancer progression. However, relatively little is known about the mechanisms of STAT3 pre-mRNA splicing, and it remains undiscovered which chemical compounds or bioactive substances can induce the STAT3ß expression. In this study, we generated a potent reporter for detection of alternative splicing of STAT3 pre-mRNA optimized for the screening of function-known chemical library, and successfully identified entinostat, a histone deacetylase inhibitor, as a novel inducer of STAT3ß through modulating mRNA splicing. Our findings demonstrate that alternative splicing of STAT3 can be regulated by a compound, providing an important clue for understanding the regulation mechanisms of the expression balance of STAT3 isoforms in a chemical biology approach. Entinostat is likely to be a promising seed compound for elucidating how the higher ratio of STAT3ß expression impacts on biological responses associated with Janus kinase (JAK)/STAT3 signaling pathway.

Siweixizangmaoru Decoction Ameliorated Type II Collagen-Induced Arthritis in Rats via Regulating JAK2-STAT3 and NF-κB Signaling Pathway.

Siweixizangmaoru decoction (SXD) is widely used as an anti-rheumatoid arthritis (RA) in Tibet, however, the specific anti-inflammatory mechanism of SXD is still unclear. This research attempts to examine the efficacy and possible mechanisms of SXD in treating RA. The primary chemical components of SXD were identified using UHPLC-Q-Exactive Orbitrap MS. We established a lipopolysaccharide (LPS)-induced RAW264.7 macrophage inflammatory injury model to explore the anti-inflammatory mechanism of SXD and validated it through in vivo experiments. According to our research in vitro as well as in vivo, SXD exhibits anti-inflammatory qualities. SXD can suppress nitric oxide (NO) and pro-inflammatory factor production in RAW264.7 cells activated by LPS. The mechanism underlying this effect might be connected to the janus tyrosine kinase 2-signal transducer and activator of transcription 3 (JAK2/STAT3) and nuclear factor-κB (NF-κB) signaling pathways. In vivo, SXD alleviates joint swelling, decreases the generation of inflammatory factors in the serum, lowers oxidative stress, and improves joint damage. In short, SXD improves joint degeneration and lowers symptoms associated with RA by regulating inflammation via the suppression of NF-κB and JAK2/STAT3 signaling pathway activation.

Quantification of the Uncertainty in Ultrasonic Wave Speed in Concrete: Application to Temperature Monitoring with Embedded Transducers.

This article presents an overall examination of how small temperature fluctuations affect P-wave velocity (Vp) measurements and their uncertainties in concrete using embedded piezoelectric transducers. This study highlights the fabrication of custom transducers tailored for long-term concrete monitoring. Accurate and reliable estimation of ultrasonic wave velocities is challenging, since they can be impacted by multiple experimental and environmental factors. In this work, a reliable methodology incorporating correction models is introduced for the quantification of uncertainties in ultrasonic absolute and relative velocity measurements. The study identifies significant influence quantities and suggests uncertainty estimation laws, enhancing measurement accuracy. Determining the onset time of the signal is very time-consuming if the onset is picked manually. After testing various methods to pinpoint the onset time, we selected the Akaike Information Criterion (AIC) due to its ability to produce sufficiently reliable results. Then, signal correlation was used to determine the influence of temperature (20 °C to 40 °C) on Vp in different concrete samples. This technique proved effective in evaluating velocity changes, revealing a persistent velocity decrease with temperature increases for various concrete compositions. The study demonstrated the capability of ultrasonic measurements to detect small variations in the state of concrete under the influence of environmental variables like temperature, underlining the importance of incorporating all influencing factors.

A Unique Time-Reversal Algorithm-Enabled Flexible Ultrasound Transducer with a Controllable Acoustic Field.

Flexible ultrasonic devices represent a feasible technology for providing timely signal detection and even a non-invasive disease treatment for the human brain. However, the deformation of the devices is always accompanied by a change in the acoustic field, making it hard for accurate focusing. Herein, we report a stable and flexible transducer. This device can generate a high-intensity acoustic signal with a controllable acoustic field even when the device is bent. The key is to use a low-impedance piezoelectric material and an island-bridge device structure, as well as to design a unique time-reversal algorithm to correct the deviation of signals after transcranial propagation. To provide an in-depth study of the acoustic field of flexible devices, we also analyze the effects of mechanical deformation and structural parameters on the corresponding acoustic response.

Dual-Wave ZnO Film Ultrasonic Transducers for Temperature and Stress Measurements.

ZnO film ultrasonic transducers for temperature and stress measurements with dual-mode wave excitation (longitudinal and shear) were deposited using the reactive RF magnetron sputtering technique on Si and stainless steel substrates and construction steel bolts. It was found that the position in the substrate plane had a significant effect on the structure and ultrasonic performance of the transducers. The transducers deposited at the center of the deposition zone demonstrated a straight columnar structure with a c-axis parallel to the substrate normal and the generation of longitudinal waves. The transducers deposited at the edge of the deposition zone demonstrated inclined columnar structures and the generation of dominant shear or longitudinal shear waves. Transducers deposited on the bolts with dual-wave excitation were used to study the effects of high temperatures in the range from 25 to 525 °C and tensile stress in the range from 0 to 268 MPa on ultrasonic response. Dependencies between changes in the relative time of flight and temperature or axial stress were obtained. The dependencies can be described by second-order functions of temperature and stress. An analysis of the contributions of thermal expansion, strain, and the speed of sound to changes in the time of flight was performed. At high temperatures, a decrease in the signal amplitude was observed due to the decreasing resistivity of the transducer. The ZnO ultrasonic transducers can be used up to temperatures of ~500 °C.

Enhancing Ultrasonic Echo Response of AlN Thin Film Transducer Deposited by RF Magnetron Sputtering.

Accurate measurement of the pretightening stress for bolts has great significance for improving the assembly quality and safety, especially in severe environments. In this study, AlN thin film transducers were deposited on GH4169 nickel base alloy bolts using the RF magnetron sputtering, enabling a systematic investigation into the correlation between structures and the intensity of ultrasonic echo signals. Employing the finite element method resulted in consistency with the experimental data, enabling further exploration of the enhancement mechanism. With the increasing thickness of both the piezoelectric layer and the electrode layer, the intensity of the ultrasonic echo signals saw a great enhancement. The maximum-intensity observed increase is 14.7 times greater than that of the thinnest layers. Specifically, the thicker piezoelectric layer improves its mechanical displacement, while the increased thickness of the electrode layer contributes to better densification. An electrode diameter of nearly 4 mm is optimal for an AlN thin film transducer of M8 bolts. For pretightening the stress measurement, the sample with a strong and stable echo signal shows a low measurement error of pretightening below ±2.50%.

Radiation Impedance of Rectangular CMUTs.

Recently, capacitive micromachined ultrasound transducers (CMUTs) with long rectangular membranes have demonstrated performance advantages over conventional piezoelectric transducers; however, modeling these CMUT geometries has been limited to computationally burdensome numerical methods. Improved fast modeling methods, such as equivalent circuit models, could help achieve designs with even better performance. The primary obstacle in developing such methods is the lack of tractable methods for computing the radiation impedance of clamped rectangular radiators. This paper presents a method that approximates the velocity profile using a polynomial shape model to rapidly and accurately estimate radiation impedance. The validity of the approximate velocity profile and corresponding radiation impedance calculation was assessed using finite element simulations for a variety of membrane aspect ratios and bias voltages. Our method was evaluated for rectangular radiators with width:length ratios from 1:1 up to 1:25. At all aspect ratios, the radiation resistance was closely modeled. However, when calculating the radiation reactance, our initial approach was only accurate for low aspect ratios. This motivated us to consider an alternative shape model for high aspect ratios, which was more accurate when compared with FEM. To facilitate the development of future rectangular CMUTs, we provide a MATLAB script that quickly calculates radiation impedance using both methods.

Flexible, Electrochemical Skin-Like Platform for Inflammatory Biomarker Monitoring.

Addressing global challenges in wound management has greatly encouraged the emergence of home diagnosis and monitoring devices. This technological shift has accelerated the development of new skin patch sensors for continuous health monitoring. A key requirement is the creation of flexible platforms capable of mimicking human skin features. Here, for the first time, an innovative, highly adaptable electrochemical biosensor with molecularly imprinted polymers (MIPs) is customized for the detection of the inflammatory biomarker interleukin-6 (IL-6). The 3-electrode gold pattern is geometrically standardized onto a 6 µm thick polyimide flexible membrane, an optically transparent, and biocompatible polymeric substrate. Subsequently, a biomimetic sensing layer specifically designed for the detection of IL-6 target is produced on these transducers. The obtained MIP biosensor shows a good linear response within the concentration range 50 pg mL-1-50 ng mL-1, with a low limit of detection (8 pg mL-1). X-ray photoelectron spectroscopy, scanning electron microscopy, and Fourier transform infrared spectroscopy characterizations confirm the modifications of the flexible gold transducer. After optimization, the biosensing device shows remarkable potential in terms of sensitivity, selectivity, and reproducibility. Overall, the integration of a low-cost electrochemical sensor on biocompatible flexible polymers opens the way for a new generation of monitoring tools with higher accuracy, less invasiveness, and greater patient comfort.

Effect of electromagnetic middle-ear implant simulating sites on the stapes spatial motion: A finite element analysis.

The electromagnetic middle-ear implant (MEI) is a new type of hearing device for addressing sensorineural and mixed hearing loss. The hearing compensation effect of the MEI varies depending on the transducer stimulation sites. This paper investigates the impact of transducer stimulation sites on MEI performance by analyzing stapes spatial motion. Firstly, we constructed a human-ear finite element model based on micro-CT scanning and inverse molding techniques. This model was validated by comparing its predictions of stapes spatial motion and cochlear response with experimental data. Then, stimulation force was applied at four common sites: umbo, incus body, incus long process and stapes to simulate the electromagnetic transducer. Results show that at low and middle frequencies, stapes-stimulating and incus-long-process-stimulating produce similar spatial motion to normal hearing; at high frequencies, incus-body-stimulating produces similar results to normal hearing. The equivalent sound pressure level generated by the stapes piston motion is less sensitive to the stimulation direction than that deduced by the stapes rocking motion.

Acoustic Transmission Measurements for Extracting the Mechanical Properties of Complex 3D MEMS Transducers.

Recent publications on acoustic MEMS transducers present a new three-dimensional folded diaphragm that utilizes buried in-plane vibrating structures to increase the active area from a small chip volume. Characterization of the mechanical properties plays a key role in the development of new MEMS transducers, whereby established measurement methods are usually tailored to structures close to the sample surface. In order to access the lateral vibrations, extensive and destructive sample preparation is required. This work presents a new passive measurement technique that combines acoustic transmission measurements and lumped-element modelling. For diaphragms of different lengths, compliances between 0.08 × 10-15 and 1.04 × 10-15 m3/Pa are determined without using destructive or complex preparations. In particular, for lengths above 1000 µm, the results differ from numerical simulations by only 4% or less.

Design, Fabrication, and Characterization of Capacitive Micromachined Ultrasonic Transducers for Transcranial, Multifocus Neurostimulation.

In a recent study using 3-D fullwave simulations, it was shown for a nonhuman primate model that a helmet-shaped 3D array of 128 transducer elements can be assembled for neurostimulation in an optimized configuration with the accommodation of an imaging aperture. Considering all acoustic losses, according to this study, for a nonhuman primate skull, the assembly of the proposed transducers was projected to produce sufficient focusing gain in two different focal positions at deep and shallow brain regions, thus providing sufficient acoustic intensity at these distinct focal points for neural stimulation. This array also has the ability to focus on multiple additional brain regions. In the work presented here, we designed and fabricated a single 15 mm diameter capacitive micromachined ultrasonic transducer (CMUT) element operating at 800 kHz central frequency with a 480 kHz 3 dB bandwidth, capable of producing a 190 kPa peak negative pressure (PNP) on the surface. The corresponding projected transcranial spatial peak pulse average intensity (ISPPA) was 28 Wcm-2, and the mechanical index (MI) value was 1.1 for an array of 128 of these elements.

Fine-Tuning of Optical Resonance Wavelength of Surface-Micromachined Optical Ultrasound Transducer Arrays for Single-Wavelength Light Source Readout.

This article reports the fine-tuning of the optical resonance wavelength (ORW) of surface-micromachined optical ultrasound transducer (SMOUT) arrays to enable ultrasound data readout with non-tunable interrogation light sources for photoacoustic computed tomography (PACT). Permanent ORW tuning is achieved by material deposition onto or subtraction from the top diaphragm of each element with sub-nanometer resolution. For demonstration, a SMOUT array is first fabricated, and its ORW is tuned for readout with an 808 nm laser diode (LD). Experiments are conducted to characterize the optical and acoustic performances of the elements within the center region of the SMOUT array. Two-dimensional and three-dimensional PACT (photoacoustic computed tomography) is also performed to evaluate the imaging performance of the ORW-tuned SMOUT array. The results show that the ORW tuning does not degrade the optical, acoustic, and overall imaging performances of the SMOUT elements. As a result, the fine-tuning method enables new SMOUT-based PACT systems that are low cost, compact, powerful, and even higher speed, with parallel readout capability.

A Proposed Non-Destructive Method Based on Sphere Launching and Piezoelectric Diaphragm.

This work presents the study of a reproducible acoustic emission method based on the launching of a metallic sphere and low-cost piezoelectric diaphragm. For this purpose, tests were first conducted on a carbon fiber-reinforced polymer structure, and then on an aluminum structure for comparative analysis. The pencil-lead break (PLB) tests were also conducted for comparisons with the proposed method. Different launching heights and elastic deformations of the structures were investigated. The results show higher repeatability for the sphere impact method, as the PLB is more affected by human inaccuracy, and it was also effective in damage detection.