Tunneling nanotubes-based intercellular mitochondrial trafficking as a novel therapeutic target in dry eye.

Cell-to-cell mitochondria transfer via tunneling nanotubes (TNTs) has recently been revealed as a spontaneous way to protect damaged cells. Previously, we have reported mesenchymal stem cells (MSCs) can rescue retinal ganglion cell and corneal epithelium through intercellular mitochondrial trafficking. Mitochondrial damage and oxidative stress in corneal epithelial cells are vital in dry eye disease (DED). However, whether intercellular mitochondrial transfer is involved in the pathological and repair process of DED is currently unknown. Therefore, in this study, we designed a coculture system to evaluate the role of intercellular mitochondrial transfer between human corneal epithelial cells (CEC) in DED. In addition, we successfully discovered the ROCK inhibitor, Y-27632 as an intensifier to improve the efficiency of intercellular mitochondrial transport. As expected, the enhanced mitochondrial transfer promotes the regeneration of CECs. Moreover, through further exploration of mechanisms, it was demonstrated that F-actin-mediated cell morphological changes and cytoskeletal remodeling may be potential mechanisms for Y-27632 to induce mitochondrial metastasis. In conclusion, we established a new method for cell repair in DED that healthy CEC offered mitochondria to damaged CEC, providing a new insight into the cellular mechanism of corneal epithelium homeostatic regenerative therapeutics in DED.

Examination of ceramic restoration adhesive coverage in cusp-replacement premolar using acoustic emission under fatigue testing.

BACKGROUND: This study investigates CAD/CAM ceramic cusp-replacing restoration resistance with and without buccal cusp replacement under static and dynamic cyclic loads, monitored using the acoustic emission (AE) technique. METHOD: The cavity was designed in a typical MODP (mesial-occlusal-distal-palatal) restoration failure shape when the palatal cusp has been lost. Two ceramic restorations [without coverage (WOC) and with (WC) buccal cuspal coverage with 2.0 mm reduction in cuspal height] were prepared to perform the fracture and fatigue tests with normal (200 N) and high (600 N) occlusal forces. The load versus AE signals in the fracture and fatigue tests were recorded to evaluate the restored tooth failure resistance. RESULTS: The results showed that non-significant differences in load value in the fracture test and the accumulated number of AE signals under normal occlusal force (200 N) in the fatigue test were found between with and without buccal cuspal coverage restorations. The first AE activity occurring for the WOC restoration was lower than that for the WC restoration in the fracture test. The number of AE signals increased with the cyclic load number. The accumulated number of AE signals for the WOC restoration was 187, higher than that (85) for the WC restoration under 600 N in the fatigue test. CONCLUSION: The AE technique and fatigue tests employed in this study were used as an assessment tool to evaluate the resistances in large CAD/CAM ceramic restorations. Non-significant differences in the tested fracture loads and accumulated number of AE signals under normal occlusal force (200 N) between different restorations indicated that aggressive treatment (with coverage preparation) in palatal cusp-replacing ceramic premolars require more attention for preserving and protecting the remaining tooth.

Recombinant BCG vaccine expressing multistage antigens of Mycobacterium tuberculosis provides long-term immunity against tuberculosis in BALB/c mice.

Tuberculosis (TB) caused by Mycobacterium tuberculosis (Mtb) persistently kills nearly 1.5 million lives per year in the world, whereas the only licensed TB vaccine BCG exhibits unsatisfactory efficacy in adults. Taking BCG as a vehicle to express Mtb antigens is a promising way to enhance its efficacy against Mtb infection. In this study, the immune efficacy of recombination BCG (rBCG-ECD003) expressing specific antigens ESAT-6, CFP-10, and nDnaK was evaluated at different time points after immunizing BALB/c mice. The results revealed that rBCG-ECD003 induced multiple Th1 cytokine secretion including IFN-γ, TNF-α, IL-2, and IL-12 when compared to the parental BCG. Under the action of PPD or ECD003, rBCG-ECD003 immunization resulted in a significant increase in the proportion of IL-2+ and IFN-γ+IL-2+ CD4+T cells. Importantly, rBCG-ECD003 induced a stronger long-term humoral immune response without compromising the safety of the parental BCG vaccine. By means of the protective efficacy assay in vitro, rBCG-ECD003 showed a greater capacity to inhibit Mtb growth in the long term. Collectively, these features of rBCG-ECD003 indicate long-term protection and the promising effect of controlling Mtb infection.

Spatial transcriptomics reveals heterogeneity of histological subtypes between lepidic and acinar lung adenocarcinoma.

BACKGROUND: Patients who possess various histological subtypes of early-stage lung adenocarcinoma (LUAD) have considerably diverse prognoses. The simultaneous existence of several histological subtypes reduces the clinical accuracy of the diagnosis and prognosis of early-stage LUAD due to intratumour intricacy. METHODS: We included 11 postoperative LUAD patients pathologically confirmed to be stage IA. Single-cell RNA sequencing (scRNA-seq) was carried out on matched tumour and normal tissue. Three formalin-fixed and paraffin-embedded cases were randomly selected for 10× Genomics Visium analysis, one of which was analysed by digital spatial profiler (DSP). RESULTS: Using DSP and 10× Genomics Visium analysis, signature gene profiles for lepidic and acinar histological subtypes were acquired. The percentage of histological subtypes predicted for the patients from samples of 11 LUAD fresh tissues by scRNA-seq showed a degree of concordance with the clinicopathologic findings assessed by visual examination. DSP proteomics and 10× Genomics Visium transcriptomics analyses revealed that a negative correlation (Spearman correlation analysis: r = -.886; p = .033) between the expression levels of CD8 and the expression trend of programmed cell death 1(PD-L1) on tumour endothelial cells. The percentage of CD8+ T cells in the acinar region was lower than in the lepidic region. CONCLUSIONS: These findings illustrate that assessing patient histological subtypes at the single-cell level is feasible. Additionally, tumour endothelial cells that express PD-L1 in stage IA LUAD suppress immune-responsive CD8+ T cells.

A novel multi-component protein vaccine ECP001 containing a protein polypeptide antigen nPstS1 riching in T-cell epitopes showed good immunogenicity and protection in mice.

Tuberculosis (TB) is an infectious disease that seriously affects human health. Until now, the only anti-TB vaccine approved for use is the live attenuated Mycobacterium bovis (M. bovis) vaccine - BCG vaccine, but its protective efficacy is relatively low and does not provide satisfactory protection against TB in adults. Therefore, there is an urgent need for more effective vaccines to reduce the global TB epidemic. In this study, ESAT-6, CFP-10, two antigens full-length and the T-cell epitope polypeptide antigen of PstS1, named nPstS1, were selected to form one multi-component protein antigens, named ECP001, which include two types, one is a mixed protein antigen named ECP001m, the other is a fusion expression protein antigen named ECP001f, as candidates for protein subunit vaccines. were prepared by constructing one novel subunit vaccine by mixing or fusing the three proteins and combining them with aluminum hydroxide adjuvant, and the immunogenicity and protective properties of the vaccine was evaluated in mice. The results showed that ECP001 stimulated mice to produce high titre levels of IgG, IgG1 and IgG2a antibodies; meanwhile, high levels of IFN-γ and a broad range of specific cytokines were secreted by mouse splenocytes; in addition, ECP001 inhibited the proliferation of Mycobacterium tuberculosis in vitro with a capacity comparable to that of BCG. It can be concluded that ECP001 is a novel effective multicomponent subunit vaccine candidate with potential as BCG Initial Immunisation-ECP001 Booster Immunisation or therapeutic vaccine for M. tuberculosis infection.

Association of LAMA1 Single-Nucleotide Polymorphisms with Risk of Esophageal Squamous Cell Carcinoma among the Eastern Chinese Population.

Introduction: LAMA1, also known as laminin subunit α1, is a member of the laminin family, which is widely reported to be a key basement membrane molecule that affects various biological activities and is associated with many kinds of diseases. We aimed to investigate the association between LAMA1single-nucleotide polymorphisms and the occurrence and progression of esophageal squamous cell carcinoma in the Chinese population. Method: 2,186 participants were collected retrospectively between October 2008 and January 2017, including 1,043 ESCC patients and 1,143 noncancer patients. A 2 mL blood sample was obtained intravenously for the LDR for SNP analysis. The 6 SNP loci of LAMA1 were selected and examined. We analyzed the association of several genetic models of 6 LAMA1 SNP loci, sex, age, smoking and drinking status, and the occurrence of esophageal squamous cell carcinoma. Results: In the rs62081531 G > A locus, genotype GA was a protective factor for ESCC compared with GG (OR: 0.830, P=0.046), especially among the younger and nondrinkers. At rs607230 T > C, genotype TC was linked with a lower risk of ESCC compared with TT. (OR: 0.613, P=0.034). Haplotype Frequencies revealed that Ars62081531Grs621993Ars539713Trs566655Ars73938538Crs607230 (OR: 0.803, P=0.028) and Grs62081531Grs621993Ars539713Trs566655Crs73938538Crs607230 (OR: 0.679, P=0.010) were strongly associated with lower susceptibility of ESCC. Conclusion: The LAMA1 rs62081531, rs539713, rs566655, and rs607230 polymorphisms were demonstrated to be related to susceptibility to ESCC in the Chinese population. LAMA1 SNPs may have a significant impact on the occurrence of esophageal cancer and may serve as potential diagnostic biomarkers.

Association of Single-Nucleotide Polymorphisms in the VDR Gene with Tuberculosis and Infection of Beijing Genotype Mycobacterium tuberculosis.

Background: The aim of the present study was to investigate the association between vitamin D receptor (VDR) gene polymorphism and tuberculosis susceptibility, as well as the potential interaction of host genetic factors with the heterogeneity of Mycobacterium tuberculosis in the population from Xinjiang, China. Methods: From January 2019 to January 2020, we enrolled 221 tuberculosis patients as the case group and 363 staff with no clinical symptoms as the control group from four designated tuberculosis hospitals in southern Xinjiang, China. The polymorphisms of Fok I, Taq I, Apa I, Bsm I, rs3847987 and rs739837 in the VDR were detected by sequencing. M. tuberculosis isolates were collected from the case group and identified as Beijing or non-Beijing lineage by multiplex PCR. Propensity score (PS), univariate analysis and multivariable logistic regression models were used to perform the analysis. Results: Our results showed that the allele and genotype frequencies of Fok I, Taq I, Apa I, Bsm I, rs3847987 and rs739837 in VDR were not correlated with tuberculosis susceptibility or lineages of M. tuberculosis. Two out of six loci of the VDR gene formed one haplotype block, and none of the haplotypes was found to correlate with tuberculosis susceptibility or lineages of M. tuberculosis infected. Conclusion: Polymorphisms in the VDR gene may not indicate susceptibility to tuberculosis. There was also no evidence on the interaction between the VDR gene of host and the lineages of M. tuberculosis in the population from Xinjiang, China. Further studies are nonetheless required to prove our conclusions.

Genetic Characterization Conferred Co-Resistance to Isoniazid and Ethionamide in Mycobacterium tuberculosis Isolates from Southern Xinjiang, China.

Background: Ethionamide (ETH), a structural analogue of isoniazid (INH), is used for treating multidrug-resistant tuberculosis (MDR-TB). Due to the common target InhA, INH and ETH showed cross-resistance in M. tuberculosis. This study aimed to explore the INH and ETH resistant profiles and genetic mutations conferring independent INH- or ETH-resistance and INH-ETH cross-resistance in M. tuberculosis circulating in south of Xinjiang, China. Methods: From Sep 2017 to Dec 2018, 312 isolates were included using drug susceptibility testing (DST), spoligotyping, and whole genome sequencing (WGS) to analyze the resistance characteristics for INH and/or ETH. Results: Among the 312 isolates, 185 (58.3%) and 127 (40.7%) belonged to the Beijing family and non-Beijing family, respectively; 90 (28.9%) were INH-resistant (INHR) with mutation rates of 74.4% in katG, 13.3% in inhA and its promoter, 11.1% in ahpC and its upstream region, 2.2% in ndh, 0.0% in mshA, whilst 34 (10.9%) were ETH-resistant (ETHR) with mutation rates of 38.2% in ethA, 26.2% in inhA and its promoter, and 5.9% in ndh, 0.0% in ethR or mshA; and 25 (8.0%) were INH-ETH co-resistant (INHRETHR) with mutation rates of 40.0% in inhA and its promoter, and 8% in ndh. katG mutants tended to display high-level resistant to INH; and more inhA and its promoter mutants showed low-level of INH and ETH resistance. The optimal gene combinations by WGS for the prediction of INHR, ETHR, and INHRETHR were, respectively, katG+inhA and its promoter (sensitivity: 81.11%, specificity: 90.54%), ethA+inhA and its promoter+ndh (sensitivity: 61.76%, specificity: 76.62%), and inhA and its promoter+ndh (sensitivity: 48.00%, specificity: 97.65%). Conclusion: This study revealed the high diversity of genetic mutations conferring INH and/or ETH resistance among M. tuberculosis isolates, which would facilitate the study on INHR and/or ETHR mechanisms and provide clues for choosing ETH for MDR treatment and molecular DST methods in south of Xinjiang, China.

A Multistage Antigen Complex Epera013 Promotes Efficient and Comprehensive Immune Responses in BALB/c Mice.

Tuberculosis (TB) remains a serious global health problem. Despite the widespread use of the Mycobacterium bovis bacillus Calmette-Guerin (BCG) vaccine, the primary factor for the TB pandemic and deaths is adult TB, which mainly result from endogenous reactivation of latent Mycobacterium tuberculosis (MTB) infection. Improved new TB vaccines with eligible safety and long-lasting protective efficacy remains a crucial step toward the prevention and control of TB. In this study, five immunodominant antigens, including three early secreted antigens and two latency associated antigens, were used to construct a single recombinant fusion protein (Epera013f) and a protein mixture (Epera013m). When formulated with aluminum adjuvant, the two subunit vaccines Epera013m and Epera013f were administered to BALB/c mice. The humoral immune responses, cellular responses and MTB growth inhibiting capacity elicited after Epera013m and Epera013f immunization were analyzed. In the present study, we demonstrated that both the Epera013f and Epera013m were capable of inducing a considerable immune response and protective efficacy against H37Rv infection compared with BCG groups. In addition, Epera013f generated a more comprehensive and balanced immune status, including Th1, Th2 and innate immune response, over Epera013f and BCG. The multistage antigen complex Epera013f possesses considerable immunogenicity and protective efficacy against MTB infection ex vivo indicating its potential and promising applications in further TB vaccine development.

Comparison of the Immunogenicity and Efficacy of rBCG-EPCP009, BCG Prime-EPCP009 Booster, and EPCP009 Protein Regimens as Tuberculosis Vaccine Candidates.

Bacillus Calmette-Guérin (BCG) is the only widely used prophylactic tuberculosis (TB) vaccine that can prevent severe TB in infants. However, it provides poor protection in adults, and therefore, there is ongoing research into new TB vaccines and immunization strategies with more durable immune effects. The recombinant BCG and BCG prime-protein booster are two important vaccine strategies that have recently been developed based on BCG and could improve immune responses. In this study, three immune strategies based on four protective antigens, namely, ESAT-6, CFP-10, nPPE18, and nPstS1, were applied to construct recombinant rBCG-EPCP009, EPCP009 subunit protein, and BCG prime-EPCP009 booster vaccine candidates. The short- and long-term immune effects after vaccination in Balb/c mice were evaluated based on humoral immunity, cellular immunity, and the ability of spleen cells to inhibit in vitro mycobacterial growth. At 8 and 12 weeks after the initial immunization, splenocytes from mice inoculated with the BCG prime-EPCP009 protein booster secreted higher levels of PPD- and EPCP009-specific IFN-γ, IL-2, TNF-α, IL-17, GM-CSF, and IL-12 and had a higher IFN-γ+CD4+ TEM:IL-2+CD8+ TCM cell ratio than splenocytes from mice inoculated with the rBCG-EPCP009 and EPCP009 proteins. In addition, the EPCPE009-specific IgG2a/IgG1 ratio was slightly higher in the BCG prime-EPCP009 protein booster group than in the other two groups. The in vitro mycobacterial inhibition assay showed that the splenocytes of mice from the BCG prime-EPCP009 protein booster group exhibited stronger inhibition of Mycobacterium tuberculosis (M. tuberculosis) growth than the splenocytes of mice from the other two groups. These results indicate that the BCG prime-EPCP009 protein booster exhibited superior immunogenicity and M. tuberculosis growth inhibition to the parental BCG, rBCG-EPCP009, and EPCP009 proteins under in vitro conditions. Thus, the BCG prime-EPCP009 protein booster may be important for the development of a more effective adult TB vaccine.

AMPK/MFF Activation: Role in Mitochondrial Fission and Mitophagy in Dry Eye.

Purpose: To assess the role of mitochondrial morphology and adenosine monophosphate-activated protein kinase (AMPK)/mitochondrial fission factor (MFF) in dry eye and the underlying mechanisms. Methods: Immortalized human corneal epithelial cells (HCECs) and primary HCECs were cultured under high osmotic pressure (HOP). C57BL/6 female mice were injected subcutaneously with scopolamine. Quantitative real-time PCR was used to measure mRNA expression. Protein expression was assessed by western blot and immunofluorescence staining. Mitochondrial morphology was observed by confocal microscopy and transmission electron microscopy. Results: First, HOP induced mitochondrial oxidative damage to HCECs, accompanied by mitochondrial fission and increased mitophagy. Then, AMPK/MFF pathway proteins were increased consequent to HOP-induced energy metabolism dysfunction. Interestingly, the AMPK pathway promoted mitochondrial fission and mitophagy by increasing the recruitment of dynamin-related protein 1 (DRP1) to the mitochondrial outer membrane in the HOP group. Moreover, AMPK knockdown attenuated mitochondrial fission and mitophagy due to HOP in HCECs. AMPK activation triggered mitochondrial fission and mitophagy. Mitochondrial fission of HCECs stressed by HOP was mediated via MFF phosphorylation. MFF knockdown reversed mitochondrial fragmentation and mitophagy in HCECs treated with HOP. Inhibition of MFF protected HCECs against oxidative damage, cell death, and inflammation in the presence of HOP. Finally, we detected mitochondrial fission and AMPK pathway activation in vivo. Conclusions: The AMPK/MFF pathway mediates the development of dry eye by positively regulating mitochondrial fission and mitophagy. Inhibition of mitochondrial fission can alleviate oxidative damage and inflammation in dry eye and may provide experimental evidence for treating dry eye.

The Single Nucleotide Polymorphisms of AP1S1 are Associated with Risk of Esophageal Squamous Cell Carcinoma in Chinese Population.

Background: The σ1A subunit of the adaptor protein 1 (AP1S1) participates in various intracellular transport pathways, especially the maintenance of copper homeostasis, which is pivotal in carcinogenesis. It is therefore rational to presume that AP1S1 might also be involved in carcinogenesis. In this hospital-based case-control study, we investigated the genetic susceptibility to ESCC in relation to SNPs of AP1S1 among Chinese population. Methods: A database containing a total of 1303 controls and 1043 ESCC patients were retrospectively studied. The AP1S1 SNPs were analyzed based on ligation detection reaction (LDR) method. Then, the relationship between ESCC and SNPs of AP1S1 was determined with a significant crude P<0.05. Then the logistic regression analysis was used for the calculation for adjusted P in the demographic stratification comparison if a significant difference was observed in the previous step. Results: AP1S1 rs77387752 C>T genotype TT was an independent risk factor for ESCC, while rs4729666 C>T genotype TC and rs35208462 C>T genotype TC were associated with a lower risk for ESCC, especially in co-dominant model and allelic test for younger, male subjects who are not alcohol-drinkers nor cigarette smokers. Conclusion: AP1S1 rs77387752, rs4729666 and rs35208462 polymorphisms are associated with susceptibility to ESCC in Chinese individuals. AP1S1 SNPs may exert an important role in esophageal carcinogenesis and could serve as potential diagnostic biomarkers.

The Relationship Between Single Nucleotide Polymorphisms of SMAD3/SMAD6 and Risk of Esophageal Squamous Cell Carcinoma in Chinese Population.

BACKGROUND: The TGF-ß signal pathways play a key role in the development and promotion of squamous cell carcinoma (SCC). The pathway is mediated by the SMAD family proteins that include SMAD3 and SMAD6. Our study aimed to evaluate the relationship between single nucleotide polymorphism (SNP) of SMAD3/SMAD6 and susceptibility to esophageal squamous cell carcinoma (ESCC) in the Chinese population. PATIENTS AND METHODS: This was a hospital-based case-control study compromised of 1043 ESCC patients and 1315 non-cancer patients. Seven SMAD3/SMAD6 (rs8028147, rs3743343, rs3743342, rs8025774, rs8031440, rs803167, and rs34643453) SNPs were selected and used to evaluate their correlation with ESCC susceptibility. Genetic model tests, stratified analyses, linkage disequilibrium analyses, and haplotype analyses were performed in our study. RESULTS: Participants with SMAD3 rs3743342 C>T, rs8025774 C>T, rs8031440 G>A or rs8031627 G>A had a significantly higher risk of ESCC. This was more evident in males, older patients (>63 years), smokers, and non-alcohol drinking participants. Linkage disequilibrium analyses further revealed that there were strong correlations between SMAD3 rs3743342 C>T, rs8025774 C>T, rs8031440 G>A, and rs8031627 G>A. In the same line, haplotype analyses revealed that SMAD3 ACCCGGSMAD6A and SMAD3AGCCGGSMAD6A were associated with less susceptibility to ESCC while SMAD3ATTTAASMAD6A was associated with a higher risk of ESCC. CONCLUSION: SNPs of SMAD3 were related to higher susceptibility to ESCC. As such, they may contribute to the development of viable strategies for early diagnosis and treatment of ESCC. However, more detailed association mechanisms between SMAD3/SMAD6 SNPs and ESCC need further experiments to prove.

Examination of ceramic/enamel interfacial debonding using acoustic emission and optical coherence tomography.

OBJECTIVE: This study investigates monitored micro-crack growth and damage in the ceramic/enamel adhesive interface using the acoustic emission (AE) technique with optical coherence tomography (OCT) under fatigue shear testing. METHODS: Shear bond strength (SBS) was measured first with eight prepared ceramic/enamel adhesive specimens under static loads. The fatigue shear testing was performed with three specimens at each cyclic load according to a modified ISO14801 method, applying at 80%, 75%, 70%, and 65% of the SBS to monitor interface debonding. The number of cycles at each load was recorded until ceramic/enamel adhesive interface debonding occurred. The AE technique was used to detect micro-crack signals in static and fatigue shear bond tests. RESULTS: The results showed that the average SBS value in the static tests was 18.07 ± 1.72 MPa (mean ± standard deviation), expressed in Newton's at 56.77 ± 5.40N. The average number of fatigue cycles in which ceramic/enamel interface damage was detected in 80%, 75%, 70% and 65% of the SBS were 41, 410, 8141 and 76,541, respectively. The acoustic behavior varied according to the applied load level. Events were emitted during 65% and 70% fatigue tests. A good correlation was observed between the crack location in OCT images and the number of AE signal hits. SIGNIFICANCE: The AE technique combined with OCT images as a pre-clinical assessment tool to determine the integrity of cemented load bearing restored ceramic material. Sustainable cyclic load stresses in ceramic/enamel bonded specimens were substantially lower than the measured SBS. Predicted S-N curve showed that the maximum endured load was 10.98 MPa (about 34.48 N) passing 10(6) fatigue cyclic.

Examination of ceramic restorative material interfacial debonding using acoustic emission and optical coherence tomography.

OBJECTIVE: CAD/CAM ceramic restorative material is routinely bonded to tooth substrates using adhesive cement. This study investigates micro-crack growth and damage in the ceramic/dentin adhesive interface under fatigue shear testing monitored using the acoustic emission (AE) technique with optical coherence tomography (OCT). METHODS: Ceramic/dentin adhesive samples were prepared to measure the shear bond strength (SBS) under static load. Fatigue shear testing was performed using a modified ISO14801 method. Loads in the fatigue tests were applied at 80%, 70%, and 60% of the SBS to monitor interface debonding. The AE technique was used to detect micro-crack signals in static and fatigue shear bond tests. RESULTS: The results showed that the average SBS value in the static tests was 10.61±2.23MPa (mean±standard deviation). The average number of fatigue cycles in which ceramic/dentin interface damage was detected in 80%, 70% and 60% of the SBS were 152, 1962 and 9646, respectively. The acoustic behavior varied according to the applied load level. Events were emitted during 60% and 70% fatigue tests. A good correlation was observed between crack location in OCT images and the number of AE signal hits. SIGNIFICANCE: The AE technique and OCT images employed in this study could potentially be used as a pre-clinical assessment tool to determine the integrity of cemented load bearing restored ceramic material. Sustainable cyclic load stresses in ceramic/dentin-bonded specimens were substantially lower than the measured SBS. Predicted S-N curve showed that the maximum endured load was 4.18MPa passing 10(6) fatigue cyclic.