Role of Niobium on the Passivation Mechanisms of TiHfZrNb High-Entropy Alloys in Hanks' Simulated Body Fluid.

A family of TiHfZrNb high-entropy alloys has been considered novel biomaterials for high-performance, small-sized implants. The present work evaluates the role of niobium on passivation kinetics and electrochemical characteristics of passive film on TiHfZrNb alloys formed in Hanks' simulated body fluid by analyzing electrochemical data with three analytical models. Results confirm that higher niobium content in the alloys reinforces the compactness of the passive film by favoring the dominance of film formation and thickening mechanism over the dissolution mechanism. Higher niobium content enhances the passivation kinetics to rapidly form the first layer, and total surface coverage reinforces the capacitive-resistant behavior of the film by enrichment with niobium oxides and reduces the point defect density and their mobility across the film, lowering pitting initiation susceptibility. With the high resistance to dissolution and rapid repassivation ability in the aggressive Hanks' simulated body fluid, the TiHfZrNb alloys confirm their great potential as new materials for biomedical implants and warrant further biocompatibility testing.

Deciphering the pathogenic role of rare RAF1 heterozygous missense mutation in the late-presenting DDH.

Background: Developmental Dysplasia of the Hip (DDH) is a skeletal disorder where late-presenting forms often escape early diagnosis, leading to limb and pain in adults. The genetic basis of DDH is not fully understood despite known genetic predispositions. Methods: We employed Whole Genome Sequencing (WGS) to explore the genetic factors in late-presenting DDH in two unrelated families, supported by phenotypic analyses and in vitro validation. Results: In both cases, a novel de novo heterozygous missense mutation in RAF1 (c.193A>G [p.Lys65Glu]) was identified. This mutation impacted RAF1 protein structure and function, altering downstream signaling in the Ras/ERK pathway, as demonstrated by bioinformatics, molecular dynamics simulations, and in vitro validations. Conclusion: This study contributes to our understanding of the genetic factors involved in DDH by identifying a novel mutation in RAF1. The identification of the RAF1 mutation suggests a possible involvement of the Ras/ERK pathway in the pathogenesis of late-presenting DDH, indicating its potential role in skeletal development.

Reference intervals for cardiometabolic risk factors in China: a national multicenter cross-sectional study on an adult population sample.

Background: The reference intervals (RIs) of adult blood lipid parameters currently used in China are not derived from the results of research in local populations and have not been adjusted for age and sex. In this study, we aimed to determine accurate RIs for blood lipid parameters and blood glucose (GluG) for Chinese adults using a national multicenter study. Methods: A total of 11,333 adults between 18 and 90 years of age were recruited in seven representative regions in China between June 2020 and December 2020. Hospitals participating in the study were regrouped into two geographical regions, southern China (Changsha, Chengdu, Hangzhou, and Nanning) and northern China (Beijing, Shenyang, and Ningxia), according to their geographical and administrative location. All samples were freshly collected and measured collectively in one laboratory on the Mindray full Automatic biochemical analyzer chemistry BS2000 analytical systems. Outliers were removed using the Tukey test. Three-level nested analysis of variance and scatter plot were used to explore the variations in sex, age, and region. Percentile curves of each indicator were plotted using the least mean square (LMS) method. The lower limit (2.5th percentile) and the upper limit (97.5th percentile) of the RI were determined by using nonparametric statistical methods. We also calculated the 90% confidence interval (CI) for the lower and upper limits. Results: A total of 8,283 participants were enrolled in the final analysis, with 3,593 (43.4%) men and 4,690 (56.6%) women. Regionality was observed in three analytes [small dense low density lipoprotein cholesterol (sd-LDLC), GluG, and apolipoprotein A1 (ApoA1)]. In northern China, the sd-LDLC and GluG levels in Shenyang were significantly higher than those in Ningxia and Beijing (P<0.05). In southern China, the sd-LDLC and GluG levels in Nanning were significantly higher than those in the three other cities (P<0.05), whereas the sd-LDLC and GluG levels in Chengdu were significantly lower than those in the three other cities (P<0.05). The level of ApoA1 in Chengdu was significantly higher than that in the three other cities. The homocysteine (HCY) level in male participants was clearly higher than that in female participants [ratio of standard deviation (SDR)sex =0.56], whereas the levels of high density lipoprotein cholesterol (HDLC) (SDRsex =0.40) and ApoA1 (SDRsex =0.27) in males were lower. The GluG and HCY level increased gradually with age. In females aged 45-55 years, there was an interesting change in scatter charts, where triglyceride (TG) and total cholesterol (TC) increased rapidly. We also found that for the age group of >55 years, the levels of TG and TC in females gradually surpassed those in males. Conclusions: The findings of this study may help establish age- and sex-specific reference values for the blood lipids of Chinese adults and serve as a valuable guide for the screening, diagnosis, treatment, prevention, and monitoring of cardiovascular disease (CVD).

Clinical Value of Platelet Indices in Premature Coronary Artery Disease.

Platelets play an important role in the pathophysiology of coronary artery disease. However, the clinical value of platelet indices in premature coronary heart disease remains largely unknown.Consecutive patients referred for coronary angiography were evaluated (n = 1675). Patients were stratified into premature coronary heart disease (n = 679, age < 55 for male and age < 65 for female), late-onset coronary heart disease (n = 772, age ≥ 55 for male and age ≥ 65 for female), and control (n = 224, age < 55 for male and age < 65 for female). Their clinical and laboratory parameters were collected. The relationship between platelet indices and premature coronary artery disease was analyzed.In univariate analysis, platelet indices showed no significant association with the presence of premature coronary heart disease (P > 0.05). After adjustment for traditional risk factors, mean platelet volume (0.823 [0.683-0.993], P = 0.042) and platelet-large cell ratio (0.976 [0.954-0.999], P = 0.040) were negatively correlated with the presence of premature coronary heart disease. The platelet-to-lymphocyte ratio was statistically significant among different numbers of coronary lesions (P = 0.035). In subgroup analysis, platelet-large cell ratio (1.190 [1.010-1.403], P = 0.038) was an independent risk factor of coronary restenosis after percutaneous coronary intervention.Platelet indices were associated with the prevalence, severity, and coronary restenosis after percutaneous coronary intervention suggesting their possible clinical application in premature coronary heart disease.

Dataset for Fracture and Impact Toughness of High-Entropy Alloys.

Fracture dictates the service limits of metallic structures. Damage tolerance of materials may be characterized by fracture toughness rigorously developed from fracture mechanics, or less rigorous yet more easily obtained impact toughness (or impact energy as a variant). Given the promise of high-entropy alloys (HEAs) in structural and damage-tolerance applications, we compiled a dataset of fracture toughness and impact toughness/energy from the literature till the end of the 2022 calendar year. The dataset is subdivided into three categories, i.e., fracture toughness, impact toughness, and impact energy, which contain 153, 14, and 78 distinct data records, respectively. On top of the alloy chemistry and measured fracture quantities, each data record also documents the factors influential to fracture. Examples are material-processing history, phase structures, grain sizes, uniaxial tensile properties, such as yield strength and elongation, and testing conditions. Data records with comparable conditions are graphically visualized by plots. The dataset is hosted in Materials Cloud, an open data repository.

Temozolomide hexadecyl ester targeted plga nanoparticles for drug-resistant glioblastoma therapy via intranasal administration.

Introduction: Temozolomide (TMZ) is the first-line drug for glioblastoma (GBM), but it is limited in clinical use due to the drug resistance, poor brain targeting, and side effects. Temozolomide hexadecyl ester (TMZ16e), a TMZ derivative with high lipophilicity, membrane permeability, and high anti-glioma properties, has the potential to reverse drug resistance. In this study, anti-ephrin type-A receptor 3 (EphA3) modified TMZ16e loaded nanoparticles (NPs) were prepared for targeted GBM therapy via intranasal administration to deliver TMZ16e to the brain, treat drug-resistant glioma effectively, and reduce peripheral toxicity. Methods: TMZ16e loaded NPs were prepared by emulsion solvent evaporation method followed by modified with anti-EphA3 (anti-EphA3-TMZ16e-NPs). In vitro evaluations were performed by an MTT assay and flow cytometry analysis. The orthotopic nude mice models were used to evaluate the anti-glioma effect in vivo. Additionally, we investigated the anti-drug resistant mechanism by western blot analysis. Results: The particle size of the prepared NPs was less than 200 nm, and the zeta potential of TMZ16e-NPs and anti-EphA3-TMZ16e-NPs were -23.05 ± 1.48 mV and -28.65 ± 1.20mV, respectively, which is suitable for nasal delivery. In vitro studies have shown that anti-EphA3 modification increased the cellular uptake of nanoparticles in T98G cells. The cytotoxicity in the anti-EphA3-TMZ16e-NPs treated group was significantly higher than that of the TMZ16e-NPs, TMZ16e, and TMZ groups (p < 0.01), and the cell cycle was blocked. Western blotting analysis showed that the TMZ16e-loaded NPs were able to effectively downregulate the expression level of O6-methylguanine-deoxyribonucleic acid-methyltransferase (MGMT) protein in T98G cells and reverse drug resistance. In vivo studies showed that the median survival time of tumor-bearing nude mice in the anti-EphA3-TMZ16e-NPs group was extended to 41 days, which was 1.71-fold higher than that of the saline group and the TUNEL staining results of the brain tissue section indicated that the TMZ16e-loaded NPs could elevate apoptosis in T98G cells. Conclusion: In conclusion, the TMZ16e-loaded NPs can be effectively delivered to the brain and targeted to gliomas, exhibiting better anti-glioma activity, indicating they possess great potential in the treatment of drug-resistant glioma.

Fatigue dataset of high-entropy alloys.

Fatigue failure of metallic structures is of great concern to industrial applications. A material will not be practically useful if it is prone to fatigue failures. To take the advantage of lately emerged high-entropy alloys (HEAs) for designing novel fatigue-resistant alloys, we compiled a fatigue database of HEAs from the literature reported until the beginning of 2022. The database is subdivided into three categories, i.e., low-cycle fatigue (LCF), high-cycle fatigue (HCF), and fatigue crack growth rate (FCGR), which contain 15, 23, and 28 distinct data records, respectively. Each data record in any of three categories is characteristic of a summary, which is comprised of alloy compositions, key fatigue properties, and additional information influential to, or interrelated with, fatigue (e.g., material processing history, phase constitution, grain size, uniaxial tensile properties, and fatigue testing conditions), and an individual dataset, which makes up the original fatigue testing curve. Some representative individual datasets in each category are graphically visualized. The dataset is hosted in an open data repository, Materials Cloud.

Ligustrazine Protects Homocysteine-Induced Apoptosis in Human Umbilical Vein Endothelial Cells by Modulating Mitochondrial Dysfunction.

Ligustrazine is one of the alkaloid compounds isolated from the traditional Chinese herb, which shows protective effects on cardiovascular disorders. High homocysteine (Hcy) level can predict cardiovascular-related events including death. In this study, we used Hcy to stimulate the human umbilical vein endothelial cells (HUVECs) and investigated the protective effect of ligustrazine on endothelial dysfunction by assessing the cell apoptosis, oxidative damage, mitochondrial dysfunction, and the potential molecular pathways. Our results clearly showed that ligustrazine increased HUVEC cell viability, decreased the dehydrogenase (LDH) level, and inhibited HUVEC apoptosis, which was associated with the attenuation of attenuated oxidative damage. The mitochondrial-dependent pathway was closely related in the regulation of ligustrazine, reflected by the attenuated mitochondrial membrane potential change and decreased cytochrome c release from the mitochondria to the cytosol. Ligustrazine may protect Hcy-induced apoptosis in HUVECs by attenuating oxidative damage and modulating mitochondrial dysfunction.

Minor alleles of genetic variants in second heart field increase the risk of hypoplastic right heart syndrome.

Hypoplastic right heart syndrome(HRHS) is characterized by hypoplastic right ventricle (RV); Numerous transcriptional cascades in the second heart field (SHF) regulate RVdevelopment. The relationship of SHF gene variants with human HRHS remains unknown. The whole lengths of 17 SHF genes were sequenced in 16 HRHS, and the selected single-nucleotide variants (SNVs) were then genotyped in HRHS, other congenital heart disease (CHD) and healthy control. Luciferase assay was performed to verify the effect of FOXC2: rs34221221A>GandTBX20: rs59854940C>Gat the transcription level. There were 151 (12.86%) novel SNVs after sequence analysis, of which three were in exons (one was synonymous SNV and two were nonsynonymous SNVs), two in promoter, and most SNVs (89.95%) were in intronic regions. Genotype analyses revealed that the minor alleles of FOXC2: rs34221221 A>G and TBX20: rs59854940 C>G could increase HRHS risk (P<0.05), but not in other CHD or healthy control. Luciferase assay showed that the minor G allele in rs34221221 significantly increased FOXC2 transcription while in rs59854940 it decreased TBX20 transcription significantly. Novel variants of SHF gene associated with HRHS were identified. Minor alleles in two variants from FOXC2 and TBX20 could increase the risk of HRHS.

Intronic Polymorphisms in Gene of Second Heart Field as Risk Factors for Human Congenital Heart Disease in a Chinese Population.

Transcriptional factors and signaling factors in the second heart field (SHF) contribute to cardiac development. However, the associations of intronic gene variants in the SHF with congenital heart disease (CHD) remain ununderstood. Ten single nucleotide polymorphisms (SNPs) from our previous sequencing data were selected and then genotyped in 383 CHD patients and 384 healthy controls in a Chinese population. Genotype analyses revealed that minor alleles in TBX1: rs12165908 C > G [odds ratio (OR) = 2.64; 95% confidence interval (CI) = 1.87-3.73, p = 3.03 × 10-8] and GATA6: rs143085291 C > T (OR = 2.49; 95% CI = 1.18-5.29, p = 0.01) increased CHD risk significantly. Meanwhile, FGF10: rs78454549 T > C and GATA4: rs13275657 A>G polymorphisms were significantly associated with increased risk of simple CHDs. The minor allele C in GATA4: rs17153694 T > C increased the risk of tetralogy of Fallot, whereas minor alleles in TBX1: rs41298006 G>A, FGF10: rs75629618 C>T, FGF10: rs10461755 G>A, FGF10: rs75632187 A>G, and FGF10: rs12518964 G > A were associated with increased risk of single ventricle. The minor allele T in rs143085291 in GATA6 enhancer decreased the transcription level in luciferase assay. Our findings suggest that intronic SNPs in transcriptional factors and signaling factors in the SHF are significantly associated with increased risk of different CHD types.

Development of heart failure risk prediction models based on a multi-marker approach using random forest algorithms.

BACKGROUND: The early identification of heart failure (HF) risk may favorably affect outcomes, and the combination of multiple biomarkers may provide a more comprehensive and valuable means for improving the risk of stratification. This study was conducted to assess the importance of individual cardiac biomarkers creatine kinase MB isoenzyme (CK-MB), B-type natriuretic peptide (BNP), galectin-3 (Gal-3) and soluble suppression of tumorigenicity-2 (sST2) for HF diagnosis, and the predictive performance of the combination of these four biomarkers was analyzed using random forest algorithms. METHODS: A total of 193 participants (80 patients with HF and 113 age- and gender-matched healthy controls) were included from June 2017 to December 2017. The correlation and regression analysis were conducted between cardiac biomarkers and echocardiographic parameters. The accuracy and importance of these predictor variables were assessed using random forest algorithms. RESULTS: Patients with HF exhibited significantly higher levels of CK-MB, BNP, Gal-3, and sST2. BNP exhibited a good independent predictive capacity for HF (AUC 0.956). However, CK-MB, sST2, and Gal-3 exhibited a modest diagnostic performance for HF, with an AUC of 0.709, 0.711, and 0.777, respectively. BNP was the most important variable, with a remarkably higher mean decrease accuracy and Gini. Furthermore, there was a general increase in predictive performance using the multi-marker model, and the sensitivity, specificity was 91.5% and 96.7%, respectively. CONCLUSION: The random forest algorithm provides a robust method to assess the accuracy and importance of predictor variables. The combination of CK-MB, BNP, Gal-3, and sST2 achieves improvement in prediction accuracy for HF.

A Giant Right Coronary Artery Aneurysm Leading to Tricuspid Stenosis.

Giant coronary artery aneurysms (CAAs) are rare coronary artery anomalies. The management of CAAs is still controversial because of the different possible pathophysiologies. In our case, tricuspid stenosis resulting from compression of the giant CAA was successfully relieved by CAA repair. As far as we know, this is the first reported case of compression by a giant CAA resulting in tricuspid stenosis.

Surgical outcome after complete repair of tetralogy of Fallot with absent pulmonary valve: comparison between bovine jugular vein-valved conduit and monocusp-valve patch.

BACKGROUND: The prognosis of tetralogy of Fallot with absent pulmonary valve (TOF/APV) without operation is poor. We evaluated the surgical outcome of TOF/APV in a single center. METHODS: Twenty-two TOF/APV patients underwent complete surgical correction in our hospital. Right ventricular outflow tract reconstruction was performed using bovine jugular vein (BJV)-valved conduit implantation (n = 10), homograft-valved conduit implantation (n = 2), or monocusp-valve patch (n = 10). Health-related quality of life (QOL) was evaluated during follow-up. RESULTS: The overall survival at 5 and 10 years was 86.4 ± 7.3% (confidence interval 69.4-97.2%). The survival rates were significantly different between patients with and without bronchial stenosis (40 and 100%, P = 0.0003, log-rank test). The survival of patients aged > 6 months was higher than those ≤ 6 months (100 vs. 40%, P = 0.0003, log-rank test). Patients with BJV-valved conduits had higher systolic gradients from the right ventricle to the pulmonary artery (RV-PA) compared to those with monocusp-valve patches. BJV-valved conduit implantation was a risk factor for post-operative pulmonary-valve stenosis. The QOL score for patients with BJV-valved conduits was lower than those with monocusp-valve patches (P < 0.05). No reoperation was performed during follow-up. CONCLUSIONS: Bronchial stenosis and lower age (≤ 6 months) were the main factors influencing post-operative survival. The use of a BJV-valved conduit was a main reason for RV-PA restenosis; thus, the use of a BJV-valved conduit may increase the need for repeat intervention and decrease the post-operative quality of life.

Using a Negative Binomial Regression Model for Early Warning at the Start of a Hand Foot Mouth Disease Epidemic in Dalian, Liaoning Province, China.

BACKGROUND: The hand foot and mouth disease (HFMD) is a human syndrome caused by intestinal viruses like that coxsackie A virus 16, enterovirus 71 and easily developed into outbreak in kindergarten and school. Scientifically and accurately early detection of the start time of HFMD epidemic is a key principle in planning of control measures and minimizing the impact of HFMD. The objective of this study was to establish a reliable early detection model for start timing of hand foot mouth disease epidemic in Dalian and to evaluate the performance of model by analyzing the sensitivity in detectability. METHODS: The negative binomial regression model was used to estimate the weekly baseline case number of HFMD and identified the optimal alerting threshold between tested difference threshold values during the epidemic and non-epidemic year. Circular distribution method was used to calculate the gold standard of start timing of HFMD epidemic. RESULTS: From 2009 to 2014, a total of 62022 HFMD cases were reported (36879 males and 25143 females) in Dalian, Liaoning Province, China, including 15 fatal cases. The median age of the patients was 3 years. The incidence rate of epidemic year ranged from 137.54 per 100,000 population to 231.44 per 100,000population, the incidence rate of non-epidemic year was lower than 112 per 100,000 population. The negative binomial regression model with AIC value 147.28 was finally selected to construct the baseline level. The threshold value was 100 for the epidemic year and 50 for the non- epidemic year had the highest sensitivity(100%) both in retrospective and prospective early warning and the detection time-consuming was 2 weeks before the actual starting of HFMD epidemic. CONCLUSIONS: The negative binomial regression model could early warning the start of a HFMD epidemic with good sensitivity and appropriate detection time in Dalian.

Lysophosphatidic acid rescues bone mesenchymal stem cells from hydrogen peroxide-induced apoptosis.

The increase of reactive oxygen species in infracted heart significantly reduces the survival of donor mesenchymal stem cells, thereby attenuating the therapeutic efficacy for myocardial infarction. In our previous study, we demonstrated that lysophosphatidic acid (LPA) protects bone marrow-derived mesenchymal stem cells (BMSCs) against hypoxia and serum deprivation-induced apoptosis. However, whether LPA protects BMSCs from H2O2-induced apoptosis was not examined. In this study, we report that H2O2 induces rat BMSC apoptosis whereas LPA pre-treatment effectively protects BMSCs from H2O2-induced apoptosis. LPA protection of BMSC from the induced apoptosis is mediated mostly through LPA3 receptor. Furthermore, we found that membrane G protein Gi2 and Gi3 are involved in LPA-elicited anti-apoptotic effects through activation of ERK1/2- and PI3 K-pathways. Additionally, H2O2 increases levels of type II of light chain 3B (LC3B II), an autophagy marker, and H2O2-induced autophagy thus protected BMSCs from apoptosis. LPA further increases the expression of LC3B II in the presence of H2O2. In contrast, autophagy flux inhibitor bafilomycin A1 has no effect on LPA's protection of BMSC from H2O2-induced apoptosis. Taken together, our data suggest that LPA rescues H2O2-induced apoptosis mainly by interacting with Gi-coupled LPA3, resulting activation of the ERK1/2- and PI3 K/AKT-pathways and inhibition caspase-3 cleavage, and LPA protection of BMSCs against the apoptosis is independent of it induced autophagy.

MicroRNA-21 is a unique signature associated with coronary plaque instability in humans by regulating matrix metalloproteinase-9 via reversion-inducing cysteine-rich protein with Kazal motifs.

BACKGROUND: Coronary atherosclerotic unstable plaque is one of the leading causes of cardiovascular death. Macrophage-derived matrix metalloproteinase (MMP) 9 is considered for degrading extracellular matrix and collagen, thereby thinning the fibrous cap in plaques. miR-21 is implicated to play an important role in the progression of atherosclerosis. Nevertheless, miR-21 as the biomarker for coronary atherosclerotic unstable plaque remains unknown. We aimed to investigate the prediction role of miR-21 for unstable plaque by pathway study of miR-21 on MMPs and its inhibitor RECK in macrophages. METHODS: Expression of miR-21 in macrophages and serum miR-21 as well as MMP-9 was measured in patients with coronary non-calcified plaque, calcified plaque and controls. In vitro experiment was done in human macrophages by over-expressing miR-21 or down-regulating RECK. The regulation of RECK and MMP-9 by miR-21 was evaluated by western blotting and siRNA strategy. RESULTS: Patients with non-calcified coronary artery lesions had significantly higher miR-21 in macrophages and lower miR-21 serum levels compared to the control and calcified plaque patients. At the same time, the serum levels of MMP-9 were significantly elevated in non-calcified patients. Experiments in vitro indicated that over-expressing miR-21 could induce the expression and secretion of pro-MMP-9 and active-MMP-9 in human macrophages via targeting gene RECK, and knocking down RECK expression by specific siRNA can resemble that of miR-21 over-expression. CONCLUSIONS: miR-21 might be a biomarker for plaque instability by suppressing target gene RECK to promote the expression and secretion of MMP-9 in macrophages.

[Genetic characterization analysis on epidemic rubella virus strains isolated in Liaoning from 2007 to 2012].

To analyze the genetic characterization of epidemic rubella virus strains isolated in Liaoning from 2007-2012, a total of 145 rubella virus strains were isolated using Vero/Slam cell line from the patients' throat swabs during rubella outbreaks and sporadics cases in Liaoning Province from 2007 to 2012. Fragments of 945 nucleotides containing 1E gene from 145 rubella virus isolates were amplified by RT-PCR, the PCR products were sequenced and analyzed. Based on the 739 nucleotides of 1E gene, the phylogenetic trees were constructed with 32 WHO rubella reference strains of 13 genotypes downloaded from GenBank and 145 rubella virus strains. The results showed that the 145 rubella virus strains in 2007 -2012 belonged to genotype 1E, nucleotide acids and amino acids similarities were 97.2%-100.0% and 97.6%-100.0%, respectively. Compared to the 1E reference strains(Rvi/ Dezhou.CHN/02, RVi/MYS/01), the nucleotide acids and amino acids similarities were 96.6%-99.2% and 98.2%-100.0%, respectively except for one amino acid change (Val246-Ala246) of RVi/Shenyang. Liaoning. CHN/13.11/13, and Asp262-Asn262 of RVi/Shenyang. Liaoning. CHN/13.11/4 and RVi/Liaoyang. Liaoning. CHN/26. 11/2. there had no change found in the important antigenic epitope sites, the hemagglutination inhibition and neutralization epitopes of the other rubella viruses. All the 145 strains isolated had the same amino acid change (Leu338--Phe338) in E1 protein. These findings suggested that genotype 1E of rubella virus was the predominant genotype in Liaoning province. the rubella prevailed in recent six years was mainly caused by rubella viruses genotype 1E with multi-transmission routes.