Search

The present study investigated the structural characteristics and its protective effect against H2O2-induced injury fibroblast cells of Bletilla striata tuber polysaccharide. The polysaccharides were gently extracted by water and recovered using the method of alcohol precipitation, and after further purification by DEAE-Sepharose Fast Flow gel column, a pure polysaccharide (pBSP) was finally obtained. The structural characterization of pBSP were investigated by using periodate oxidation studies, Smith-degradation, FT-IR spectroscopy, 1D and 2D NMR spectroscopy. The antioxidant effect of pBSP was evaluated by inhibiting the production of reactive oxygen species (ROS) in human fibroblast model cells induced by H2O2. It was firstly reported that pBSP was composed of d -glucose and D-mannose in a molar ratio of 1.00:1.34 with a molecular weight of 327.6 kDa. The repeating units of pBSP contained (1 → 4)-linked-β-D-Manp, (1 → 4)-linked-α-D-Glcp and (1 → 3)-linked-β-D-Manp, and there was no branched chain. pBSP exhibited no toxic effect on fibroblasts cells and could protect them against H2O2-induced injuries. After pretreatment with pBSP for 24 h, the content of ROS in fibroblasts decreased significantly. These results not only confirm the availability B. striata, but also indicate that pBSP have potential antioxidant capacity. Our observations can provide foundation for further development of pBSP-based cosmetics.

Huili Wang, Runye Wu, Xiaodong Huang, Yuan Qu, Kai Wang, Qingfeng Liu, Xuesong Chen, Ye Zhang, Shiping Zhang, Jianping Xiao, Junlin Yi, Guozhen Xu, Li Gao, Jingwei Luo National Cancer Center/National Cancer Clinical Medical Research Center/Chinese Academy of Medical Sciences, Peking Union Medical College, Cancer Hospital, Beijing, People’s Republic of ChinaCorrespondence: Jingwei LuoNational Cancer Center/National Cancer Clinical Medical Research Center/Chinese Academy of Medical Sciences, Peking Union Medical College, Cancer Hospital, Beijing, People’s Republic of ChinaTel +86 13601181202Email jingwei-luo@outlook.comBackground: This study was to determine the patterns of regional lymph node (LN) spread and the risk factors of retropharyngeal lymph node (RPLN) metastasis based on magnetic resonance imaging (MRI) in hypopharyngeal squamous carcinoma (HPC) to improve clinical target volume (CTV) delineation.Methods: A cohort of 326 consecutive patients of HPC in a single institute were retrospectively reviewed. All patients underwent MRI prior to initial treatment, and the diagnosis based on MRI of the LN metastasis was confirmed by all radiation oncologists in the head and neck group during twice weekly chat rounds. Statistical analysis of data was using chi-square test and multivariant logistic regression model in SPSS 22.0 software.Results: The LN metastasis rate of all patients in this cohort was 90.5% (295/326). Level IIa/b and level III were the most frequently involved regions followed by level IV and retropharyngeal region. Skip metastasis only occurred in 6.4% (19/295). Univariate and multivariate analysis demonstrated that primary tumor subsites were located in the posterior pharyngeal wall (P=0.002), bilateral cervical LN metastasis (P=0.020), larger volume of primary gross target (GTVp, P=0.003), and larger volume of LN gross target (GTVnd, P=0.023) were significantly associated with RPLN metastasis.Conclusion: The regional LN spread of HPC follows an ordered pattern as level II is the most frequently involved area followed by level III, level IV, and RPLN. RPLN metastasis is more likely to occur in patients with primary site of posterior pharyngeal wall, large tumor burden, or bilateral neck LN metastasis. Therefore, it is highly recommended that the RPLN should be included into CTV for patients who have these risk factors.Keywords: hypopharyngeal carcinoma, lymph node metastasis, risk factors, retropharyngeal lymph node, metastasis

Structural topology optimization for nonlinear structures and stress-based design problems have been well developed and extensively studied. However, topology optimization of Simultaneous Geometrical and Material Nonlinear (SGMN) structures under stress constraints, requires significant investigation. Hence, based on an extended Bi-directional Evolutionary Structural Optimization (BESO) method, this work proposes a novel topology optimization method to maximize the stiffness of SGMN structures subject to both volume fraction and maximum von Mises stress constraints. The extended BESO method based on discrete variables can avoid the stress singularity problem. The global von Mises stress is established with the p-norm function, and the adjoint sensitivity analysis is derived. The nonlinear finite element equations are established by the Lagrangian method and the large deformation problem is solved by the Total Lagrangian method. A series of comparison studies have been conducted to validate the effectiveness and practicability of the method on several benchmark design problems. It concludes that the proposed approach can well control the stress level and efficiently solve the stress concentration effect at the critical stress areas of SGMN structures.

Purpose Conditional survival (CS) and failure hazard estimations can provide important dynamic prognostic information for clinical decision‐making and surveillance counseling. The current study aimed to investigate the CS and dynamic failure hazard in non‐metastatic nasopharyngeal carcinoma (NPC) treated with intensity‐modulated radiotherapy (IMRT). Methods Conditional overall survival (COS) and progression‐free survival (CPFS) estimates adjusted for age and gender against each AJCC 8th stage were calculated. Multivariable Cox regression (MCR) models were fitted in the entire population at baseline and subsequently separate MCR models were fitted in patients who have maintained event‐free time of 1 to 10 years to generate respective hazard ratio (HR). Annual hazard rates of death and progression over 10 years for each stage were also estimated. Results A total of 1993 patients were eligible for analysis. The estimated 5‐year OS and PFS for entire cohort were 79.0% and 70.7% at initial diagnosis. After 5 years of event‐free follow‐up, additional 5‐year COS and CPFS increased to 85.9% and 85.5%, respectively. Stage I/II maintained dramatically favorable CS and low hazard (< 5%) of death and progression over time. Relative to stage I/II, stage III manifested non‐significantly higher failure hazard for the first 3 years of survivorship and approached to similar level of stage I/II afterwards. Stage IVA presented most impressive improvement in terms of both COS (∆=9.8%) and CPFS (∆ = 16.8%) whereas still drastically inferior to that of stage I‐III across all conditional time points. After 4 years of follow‐up, progression hazard of stage IVA became relatively steady of approximate 6%. Conclusions Survival prospect of non‐metastatic NPC improves over years with distinct dynamic patterns across stages, providing important implications for personalized decision‐making in terms of both clinical management and surveillance counseling. Stage‐dependent and hazard‐adapted clinical management and surveillance are warranted., This study aimed to investigate the conditional survival (CS) and dynamic failure hazard in non‐metastatic nasopharyngeal carcinoma (NPC) treated with intensity‐modulated radiotherapy (IMRT). Survival prospect of non‐metastatic NPC improved over years with distinct dynamic patterns across stage, providing important implications for personalized decision‐making in terms of both clinical management and surveillance counseling. Stage‐dependent and hazard‐adapted clinical management and surveillance are warranted.

BACKGROUND: Patients infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which causes coronavirus disease 2019 (COVID-19), have fever, dry cough, dyspnea, and fatigue. The disease has now become a global pandemic. The purpose of this study was to explore the relationship between COVID-19 and gastrointestinal (GI) symptoms. METHODS: We collected and analyzed data on patients with laboratory-confirmed COVID-19 by high-throughput sequencing or reverse transcription-polymerase chain reaction. We reviewed electronic medical records of 405 hospitalized COVID-19 patients in the Third Hospital of Wuhan. RESULTS: Among the 405 confirmed patients, 210 had no GI symptoms, 195 had GI symptoms, and the first symptom of 155 patients was GI. The prevalence of vascular and digestive diseases in the group with GI symptoms was significantly higher than in the group without GI symptoms. In patients with GI symptoms, the proportion with fever, cough, dysphoria, chest tightness, poor appetite, chest pain, and pharyngeal pain was significantly higher than in those without GI symptoms. There was no significant difference in imaging between the 2 groups. In patients with GI symptoms, the proportion with increased procalcitonin (PCT) level and decreased lymphocyte count was significantly higher than in those without GI symptoms. CONCLUSION: COVID-19 patients with GI symptoms had significantly more vascular and digestive system diseases and were more likely to have clinical manifestations of fever, cough, poor appetite, chest tightness, chest pain, insomnia, and pharyngeal pain. There were more patients with diarrhea, nausea, and vomiting. Patients with GI symptoms were more likely to have increased PCT and decreased lymphocyte count.

Graphical Abstract Natural killer/T-cell lymphoma (NKTCL) is an aggressive Epstein-Barr virus (EBV)-associated non-Hodgkin lymphoma with poor prognosis. In this study, we aimed to investigate the potential mechanism of latent membrane protein 1 (LMP1)-mediated tumorigenesis and provide a novel therapeutic strategy for targeting the EBV DNA genome. We found that LMP1 upregulated the expression of peroxisome proliferator-activated receptor-γ (PPARγ) coactivator-1β (PGC1β) through activation of nuclear factor-κB (NF-κB). Furthermore, the activated PGC1β upregulated the expression of 8-oxoguanine DNA glycosylase (OGG1) through the coactivation of nuclear respiratory factor 1 (NRF1) and GA-binding protein α (GABPα), preventing reactive oxygen species (ROS)-mediated base incision in the EBV genome and favoring its survival. Interruption of hexokinase domain component 1 (HKDC1) by either shRNA or Tf-D-HKC8 peptide suppressed the interaction of HKDC1 with voltage-dependent anion channel 1 (VDAC1), triggering mitochondrial dysfunction and excessive generation of ROS, thus resulting in EBV suppression through ROS-mediated DNA damage. Suppression of the EBV genome inhibited the expression of the LMP1/PGC1β/HKDC1/OGG1 signaling pathway, forming a positive feed forward loop for the generation of ROS, hence inhibiting the EBV genome and subsequent EBV-associated tumor development. We concluded that LMP1 triggers EBV-associated tumorigenesis through activation of the PGC1β pathway. This study provided a novel therapeutic strategy for the treatment of EBV-associated tumors by targeting HKDC1. Supplementary Information The online version contains supplementary material available at 10.1007/s12015-020-10112-8.

QT prolongation, due to lengthening of the action potential duration in the ventricles, is a major risk factor of lethal ventricular arrhythmias. A widely known consequence of QT prolongation is the genesis of early afterdepolarizations (EADs), which are associated with arrhythmias through the generation of premature ventricular complexes (PVCs). However, the vast majority of the EADs observed experimentally in isolated ventricular myocytes are phase-2 EADs, and whether phase-2 EADs are mechanistically linked to PVCs in cardiac tissue remains an unanswered question. In this study, we investigate the genesis of PVCs using computer simulations with eight different ventricular action potential models of various species. Based on our results, we classify PVCs as arising from two distinct mechanisms: repolarization gradient (RG)-induced PVCs and phase-2 EAD-induced PVCs. The RG-induced PVCs are promoted by increasing RG and L-type calcium current and are insensitive to gap junction coupling. EADs are not required for this PVC mechanism. In a paced beat, a single or multiple PVCs can occur depending on the properties of the RG. In contrast, phase-2 EAD-induced PVCs occur only when the RG is small and are suppressed by increasing RG and more sensitive to gap junction coupling. Unlike with RG-induced PVCs, in each paced beat, only a single EAD-induced PVC can occur no matter how many EADs in an action potential. In the wide parameter ranges we explore, RG-induced PVCs can be observed in all models, but the EAD-induced PVCs can only be observed in five of the eight models. The links between these two distinct PVC mechanisms and arrhythmogenesis in animal experiments and clinical settings are discussed.

Purpose Maximum leaf speed is a configurable parameter of MLC in a treatment‐planning system. This study investigated the influence of MLC on the quality of VMAT plans. Methods Seven MLCs with different maximum leaf speeds (1.0, 1.5, 2.25, 3.5, 5.0, 7.5, and 10 cm/s) were configured for an accelerator in treatment‐planning system. Correspondingly, seven treatment plans, with the identical initial optimization parameter, were designed with the mdaccAutoPlan system. Six nasopharyngeal carcinoma (NPC) patients and nine rectal cancer patients were selected, representing complex and simple clinical circumstances. VMAT plan quality was evaluated with PlanIQTM software. The results were statistically analyzed with a one‐way analysis of variance (ANOVA) and pairwise comparison tests. Results The relative changes of plan scores achieved by the seven configured accelerators, with specific maximum MLC leaf speed (MMLS) for each patient, were studied. Two apparent trends of MMLS influence on VMAT plan scores were observed: Plan scores increased with MMLS; Plan scores increased rapidly when MMLS increased from 1 to 3.5, thus the relative change of plan score decreased in this MMLS range. The stationary point of maximum MLC speed (MMSSP) is defined, for the specific MMLS when the relative changes of plan scores is first

Pancreatic cancer cells are characterized by high cell proliferation and low cell apoptosis, but the factors involved in these processes remain to be further studied. In this study, we report that miR-324-5p regulates the proliferation and apoptosis of pancreatic cancer cells through regulating the expression of Krüppel-like factor 3 (KLF3). In both pancreatic cancer tissues and cell lines, the levels of miR-324-5p are significantly increased. Inhibition of miR-324-5p represses cell proliferation but promotes cell apoptosis, whereas overexpression of miR-324-5p exerts the opposite effect. Furthermore, we identified KLF3, a factor regulating pancreatic cancer cell proliferation and apoptosis, as a new direct downstream target of miR-324-5p. Our results suggest that miR-324-5p plays an important role in pancreatic cancer cell proliferation and apoptosis via downregulating the expression of KLF3., Graphical Abstract, Pancreatic cancer cells are characterized by high cell proliferation and low cell apoptosis, but the factors involved in these processes remain to be further studied. In this study, Zhang and colleagues report that miR-324-5p plays an important role in pancreatic cancer cell proliferation and apoptosis through downregulating the expression of KLF3.

Extracting the tongue body accurately from a digital tongue image is a challenge for automated tongue diagnoses, as the blurred edge of the tongue body, interference of pathological details, and the huge difference in the size and shape of the tongue. In this study, an automated tongue image segmentation method using enhanced fully convolutional network with encoder-decoder structure was presented. In the frame of the proposed network, the deep residual network was adopted as an encoder to obtain dense feature maps, and a Receptive Field Block was assembled behind the encoder. Receptive Field Block can capture adequate global contextual prior because of its structure of the multibranch convolution layers with varying kernels. Moreover, the Feature Pyramid Network was used as a decoder to fuse multiscale feature maps for gathering sufficient positional information to recover the clear contour of the tongue body. The quantitative evaluation of the segmentation results of 300 tongue images from the SIPL-tongue dataset showed that the average Hausdorff Distance, average Symmetric Mean Absolute Surface Distance, average Dice Similarity Coefficient, average precision, average sensitivity, and average specificity were 11.2963, 3.4737, 97.26%, 95.66%, 98.97%, and 98.68%, respectively. The proposed method achieved the best performance compared with the other four deep-learning-based segmentation methods (including SegNet, FCN, PSPNet, and DeepLab v3+). There were also similar results on the HIT-tongue dataset. The experimental results demonstrated that the proposed method can achieve accurate tongue image segmentation and meet the practical requirements of automated tongue diagnoses.

Aims/Introduction To investigate the genetic etiology and evaluate the diagnostic application of next‐generation sequencing for diabetes/persistent hyperglycemia in children and adolescents. Materials and Methods Patients with diabetes/persistent hyperglycemia, presenting with at least one other clinical manifestation (other than diabetes) or with a family history of diabetes, were recruited. The clinical and laboratory characteristics of the patients were recorded. Next‐generation sequencing was carried out, and candidate variants were verified by Sanger sequencing. Variant pathogenicity was further evaluated according to the American College of Medical Genetics and Genomics guidelines. Results This study included 101 potential probands, 36 of whom were identified as positive by genetic testing. A further 51.2 and 20.9% of variants were determined to be pathogenic or likely pathogenic, respectively. Variants associated with the disease were primarily identified in 21 genes and three regions of copy number variants. Among the 39 variants in 21 genes, 61.5% (24/39) were novel. The genetic diagnosis of 23 patients was confirmed based on genetic evidence and associated clinical manifestations. We reported GCK variants (21.7%, 5/23) as the most common etiology in our cohort. Different clinical manifestations were observed in one family with WFS1 variants. Conclusions Our findings support the use of next‐generation sequencing as a standard method in patients with diabetes/persistent hyperglycemia and provide insights into the etiologies of these conditions., Few studies have focused on examining the genetic etiology of diabetes in Chinese children and adolescents. A total of 101 potential probands were included in this study to investigate the clinical and genetic features of monogenic diabetes and genetic syndromes associated with it, and evaluate the diagnostic use of next‐generation sequencing for diabetes/persistent hyperglycemia. Our findings expand the gene mutation spectrum and phenotypic spectrum of the rare monogenic diabetes and genetic syndromes associated with diabetes, and provide insights into the current understanding of the underlying etiologies of diabetes/persistent hyperglycemia and support the use of next‐generation sequencing as a diagnostic method in Chinese patients with diabetes/persistent hyperglycemia.

Supplemental Digital Content is available in the text., Rationale: Use of ACEIs (angiotensin-converting enzyme inhibitors) and ARBs (angiotensin II receptor blockers) is a major concern for clinicians treating coronavirus disease 2019 (COVID-19) in patients with hypertension. Objective: To determine the association between in-hospital use of ACEI/ARB and all-cause mortality in patients with hypertension and hospitalized due to COVID-19. Methods and Results: This retrospective, multi-center study included 1128 adult patients with hypertension diagnosed with COVID-19, including 188 taking ACEI/ARB (ACEI/ARB group; median age 64 [interquartile range, 55–68] years; 53.2% men) and 940 without using ACEI/ARB (non-ACEI/ARB group; median age 64 [interquartile range 57–69]; 53.5% men), who were admitted to 9 hospitals in Hubei Province, China from December 31, 2019 to February 20, 2020. In mixed-effect Cox model treating site as a random effect, after adjusting for age, gender, comorbidities, and in-hospital medications, the detected risk for all-cause mortality was lower in the ACEI/ARB group versus the non-ACEI/ARB group (adjusted hazard ratio, 0.42 [95% CI, 0.19–0.92]; P=0.03). In a propensity score-matched analysis followed by adjusting imbalanced variables in mixed-effect Cox model, the results consistently demonstrated lower risk of COVID-19 mortality in patients who received ACEI/ARB versus those who did not receive ACEI/ARB (adjusted hazard ratio, 0.37 [95% CI, 0.15–0.89]; P=0.03). Further subgroup propensity score-matched analysis indicated that, compared with use of other antihypertensive drugs, ACEI/ARB was also associated with decreased mortality (adjusted hazard ratio, 0.30 [95% CI, 0.12–0.70]; P=0.01) in patients with COVID-19 and coexisting hypertension. Conclusions: Among hospitalized patients with COVID-19 and coexisting hypertension, inpatient use of ACEI/ARB was associated with lower risk of all-cause mortality compared with ACEI/ARB nonusers. While study interpretation needs to consider the potential for residual confounders, it is unlikely that in-hospital use of ACEI/ARB was associated with an increased mortality risk.

Colorectal cancer (CRC) is one of the most common carcinomas with high morbidity and mortality worldwide. However, the underlying molecular mechanisms of CRC are unclear. The aim of the present study was to establish the role that overexpression of LBX2 serves in CRC and to investigate the associated biological pathways. The difference in the expression levels of LBX2 between CRC tissues and adjacent normal colorectal tissues was assessed using the Oncomine database and Tumor Immune Estimation Resource. The expression levels of LBX2 and its prognostic significance in CRC were analyzed using a t-test and χ2 test using data from The Cancer Genome Atlas database. The Kaplan-Meier method and Cox regression analysis were used to estimate the prognostic value of LBX2 in CRC. Furthermore, the potential mechanisms of LBX2 dysregulation and its underlying molecular mechanisms in CRC were investigated using Gene Set Enrichment Analysis (GSEA). LBX2 expression levels were significantly upregulated in CRC samples compared with corresponding normal colorectal tissues (P

Background Pancreatic cancer (PC) is a highly invasive tumor with a poor prognosis, short overall survival rate and few chemotherapeutic choices. Despite the importance of finding ways to treat pancreatic cancer, the mechanisms of tumor progression have not been fully elucidated. microRNA-455-3p (miR-455-3p) has been reported to play an important role in several cancers, but its function in pancreatic cancer remains unclear. Methods To investigate the biological functions, miRNAs mimics or inhibitors were transfected into pancreatic cancer cells. Flow cytometry was used to detect cell apoptosis. Wound healing and Transwell assays were employed to observe cell invasion and migration abilities. The expression of Bcl-2, Bax, caspase-3, E-cadherin, N-cadherin, Snail, β-Catenin, c-Myc and Cyclin D1 were evaluated by qPCR and Western blot. Results We confirmed that inhibition of miR-455-3p decreases cell apoptosis and increases cell migration, invasion and EMT of pancreatic cancer, whereas forced overexpression of miR-455-3p has the opposite effect. Furthermore, we demonstrated that the tumor suppression effects of miR-455-3p were partially reversed by TAZ overexpression. In addition, miR-455-3p led to inactivation of Wnt/β-catenin signaling in pancreatic cancer cells, and TAZ overexpression restored the inhibition of Wnt/β-catenin signaling. Conclusion Taken together, our data demonstrated that miR-455-3p functions as an important tumor suppressor that suppresses the Wnt/β-catenin signaling pathway via TAZ to inhibit tumor progression in pancreatic cancer. We conclude that the miR-455-3p/TAZ/Wnt axis may be a potential therapeutic target for pancreatic cancer.

Organophosphate pesticides (OPs) are often used as insecticides and acaricides in agriculture, thus improving yields. OP residues may pose a serious threat, due to inhibition of the enzyme acetylcholinesterase (AChE). Therefore, a competitive bio-barcode immunoassay was designed for simultaneous quantification of organophosphate pesticide residues using AuNP signal amplification technology and Au@Pt catalysis. The AuNP probes were labelled with antibodies and corresponding bio-barcodes (ssDNAs), MNP probes coated with ovalbumin pesticide haptens and Au@Pt probes functionalized with the complementary ssDNAs were then prepared. Subsequently, pesticides competed with MNP probes to bind the AuNP probes. The recoveries of the developed assay were ranged from 71.26 to 117.47% with RSDs from 2.52 to 14.52%. The LODs were 9.88, 3.91, and 1.47 ng·kg−1, for parathion, triazophos, and chlorpyrifos, respectively. The assay was closely correlated with the data obtained from LC-MS/MS. Therefore, the developed method has the potential to be used as an alternative approach for detection of multiple pesticides.

Background Australia is theoretically at risk of epidemic chikungunya virus (CHIKV) activity as the principal vectors are present on the mainland Aedes aegypti) and some islands of the Torres Strait (Ae. aegypti and Ae. albopictus). Both vectors are highly invasive and adapted to urban environments with a capacity to expand their distributions into south-east Queensland and other states in Australia. We sought to estimate the epidemic potential of CHIKV, which is not currently endemic in Australia, by considering exclusively transmission by the established vector in Australia, Ae. aegypti, due to the historical relevance and anthropophilic nature of the vector. Methodology/Principal findings We estimated the historical (1995–2019) epidemic potential of CHIKV in eleven Australian locations, including the Torres Strait, using a basic reproduction number equation. We found that the main urban centres of Northern Australia could sustain an epidemic of CHIKV. We then estimated future trends in epidemic potential for the main centres for the years 2020 to 2029. We also conducted uncertainty and sensitivity analyses on the variables comprising the basic reproduction number and found high sensitivity to mosquito population size, human population size, impact of vector control and human infectious period. Conclusions/Significance By estimating the epidemic potential for CHIKV transmission on mainland Australia and the Torres Strait, we identified key areas of focus for controlling vector populations and reducing human exposure. As the epidemic potential of the virus is estimated to rise towards 2029, a greater focus on control and prevention measures should be implemented in at-risk locations., Author summary Chikungunya virus (CHIKV) is transmitted primarily by Aedes aegypti and Aedes albopictus mosquitoes and causes a potentially debilitating febrile and arthralgic disease. The virus is a threat to public health in regions where the primary vectors are established, as evidenced by past epidemics in the Indian Ocean Islands, South America and the Caribbean. In Australia, there are established populations of Ae. aegypti both on the mainland and in the Torres Strait, and of Ae. albopictus in the Torres Strait. This provides a theoretical potential for CHIKV transmission, as seen historically with dengue virus (DENV). It is therefore important to understand the epidemic potential of CHIKV in Australia. We estimated the basic reproduction number (R0) of CHIKV during the years 1995–2019 for 11 Urban Centres and Localities (UCLs) in Australia, and found that Brisbane, Cairns, Darwin, Rockhampton, Thursday Island, and Townsville were all susceptible to CHIKV epidemics. We then forecasted epidemic potential from 2020–2029 and found an increase in R0 across the six main UCLs. By highlighting factors that significantly influence the epidemic potential of CHIKV in Australia, our study supports evidence-based decision making for vector control and public health programs.

Hui Huang,1– 3 Xiaodong Huang,3 Kaojuan Zeng,3 Fan Deng,3 Changqing Lin,3 Wenjie Huang1,2 1The First School of Clinical Medicine, Southern Medical University, Guangzhou, 510515, People’s Republic of China; 2Department of Respiratory and Critical Care Medicine, General Hospital of Southern Theatre Command, Guangzhou, 510010, People’s Republic of China; 3Department of Respiratory and Critical Care Medicine, Huizhou Municipal Central Hospital, Huizhou, 516001, People’s Republic of ChinaCorrespondence: Wenjie HuangThe First School of Clinical Medicine, Southern Medical University, No. 1023-1063, Shatai South Road, Guangzhou, 510010, People’s Republic of ChinaTel +86 13600466358Fax +8620-88686500Email huangyelu1029@vip.163.comPurpose: Persistent chronic inflammation of chronic obstructive pulmonary disease (COPD) is associated with poor outcomes and frequently results in acute exacerbation. Predicting the number of exacerbations is important. Because interleukin 6 (IL-6) plays an important role in inducing and maintaining chronic inflammation, we sought to observe whether IL-6 measurement can predict the frequency of acute exacerbation of COPD.Methods: We reviewed serum IL-6 concentrations of stable COPD patients from January 2016 to December 2017 and statistically analyzed them to determine the optimal threshold value to predict the frequency of COPD acute exacerbations. Outpatients with stable COPD were then recruited between January 2018 and December 2019 and grouped into a low IL-6 group and a high IL-6 group according to this threshold value. We then compared the number of exacerbations of COPD in 1 year between the two groups.Results: We reviewed data from 95 COPD patients, who had a median of 1.00 exacerbations in preceding year; 35 of these patients had no fewer than two. The median IL-6 concentration was 8.80 pg/mL. IL-6 and hs-CRP were positively correlated with frequency of acute exacerbation in the preceding year, COPD assessment test (CAT) score and British medical research council (mMRC) score, and negatively correlated with forced expiratory volume in one second as percentage of predicted value (FEV1%pred) and FEV1/FVC% (forced vital capacity). IL-6 was the risk factor of COPD patients with two or more exacerbations in 1 year. Finally, we enrolled 65 COPD patients and divided into low IL-6 group and high IL-6 group; the high IL-6 group experienced more frequent exacerbations than did the low IL-6 group.Conclusion: An IL-6 measurement of 14.030 pg/mL or more is a risk factor for ≥ 2 acute exacerbations of COPD in the following year.Keywords: hs-CRP, IL-6, risk factor, prediction, stable COPD, biomarkers

Objective:To compare the long-term oncological outcomes of endoscopic resection versus open surgery in combination with radiotherapy for locally advanced sinonasal malignancies.Methods:Data for continuous patients with sinonasal epithelial tumors treated in our center between Jan 1999 and Dec 2016 were retrospectively reviewed. Those who received surgery (endoscopic or open surgery) combined with radiotherapy were identified, and 1:1 matching with propensity scores was performed. The primary endpoints of overall survival (OS) and progression-free survival (PFS) were evaluated using the Kaplan-Meier method and Cox proportional hazards modeling. The local recurrence rate (LRR) was assessed by competing risk analysis.Results:We identified 267 eligible patients, 90 of whom were included after matching: 45 patients in the endoscopy group and 45 in the open group. The median follow-up time was 87 months. In the endoscopic group, 84.4% of patients received intensity-modulated radiotherapy (IMRT), with a mean gross tumor volume (GTV) dose of 68.28 Gy; in the open surgery group, 64.4% of patients received IMRT, with a mean GTV dose of 64 Gy. The 5-year OS, PFS and LRR were 69.9%, 58.6%, and 24.5% in the endoscopic group and 64.6%, 54.4%, and 31.8% in the open surgery group, respectively. Multivariable regression analysis revealed that surgical approach was not associated with lower OS, PFS or LRR. Age, histopathology and stage were independent risk factors for OS.Conclusion:For patients with locally advanced sinonasal carcinoma, minimally invasive endoscopic resection, in combination with a higher radiation dose and new radiation techniques such as IMRT, yields survival outcomes similar to those of open surgery in combination with radiotherapy.

Purpose: To define the clinical characteristics of irradiation-induced nasopharyngeal necrosis (INN) after intensity-modulated radiotherapy (IMRT) and identify the influence of treatment strategies on INN in primary nasopharyngeal carcinoma (NPC) patients.Patients and methods: From 2008 to 2019, NPC patients pathologically diagnosed with INN after primary IMRT were reviewed. Those patients were matched with propensity scores for patients without INN in our center. The impact of treatment strategies on INN occurrence was assessed using univariate and multivariate logistic regression analysis. Results: The incidence rate of INN was 1.9% among the primary NPC population, and 53 patients with INN were enrolled. Headache and foul odor were the main symptoms, and 71.7% of cases had pseudomembrane during or at the end of radiotherapy. All patients were in early or middle stage INN, and no one presented with skull-based osteoradionecrosis. Then 212 non-INN patients were included based on propensity scores match. Overall survival (p=0.248) and progression-free survival (p=0.266) curves were similar between the INN and non-INN groups. Treatment strategies including combining chemotherapy or molecular targeted therapy with radiotherapy were not associated with INN occurrence, while boost dose (OR 7.360; 95% CI 2.301-23.547; P = 0.001) was a predictor factor for it. However, the optimal threshold for an accumulated dose to predict INN's occurrence was failed to determine.Conclusion: In the IMRT era, the severity of INN in primary NPC patients is lessened. This study showed that treatment strategies contributed little to develop INN, while the accumulated dose of radiation may relate to its occurrence.

PurposeThe high intracranial efficacy of targeted therapeutic agents poses a challenge in determining the optimal sequence of local radiation therapy (RT) and systemic treatment with tyrosine kinase inhibitors (TKIs) in non-small cell lung cancer (NSCLC) patients with brain metastasis (BM). Therefore, we conducted a cohort study to elucidate the appropriate treatment strategy, either upfront RT or deferred RT including a toxicity assessment, in these patients.Patients and MethodsWe retrospectively evaluated patients with gene-driven BMs from a single institution and divided them into deferred and upfront RT groups. Survival was estimated using a log-rank test. Intracranial progression was estimated using Fine-Gray competing risks model. Cox proportional hazards regression was performed for multivariable analysis in the entire group and subgroups.ResultsAmong the 198 eligible patients, 94 and 104 patients received deferred and upfront RT, respectively. The upfront RT group showed a lower intracranial progression risk with an adjusted sub-distribution hazard ratios of 0.41 (95% CI, 0.30–0.57) than did the deferred RT group (median intracranial progression-free survival [iPFS], 19.9 months vs. 11.1 months; p < 0.001). The median overall survival (OS; 43.2 months vs. 49.1 months, p = 0.377) and BM-specific survival (92.1 months vs. 82.9 months, p = 0.810) after salvage therapy were not significantly different between the upfront and deferred groups. Among patients with progressed extracranial disease, the deferred RT group showed significantly better OS than did the upfront RT group (44.0 vs. 28.1 months, p = 0.022). Grade 3–4 treatment-related adverse events were rare, and similar toxicities were observed between the two groups.ConclusionCompared to the deferred RT group, the upfront RT group achieved longer iPFS and similar survival outcomes in most patients with gene-driven NSCLC BM, although patients with progression of extracranial disease might benefit from deferred RT. Both groups showed well-tolerated toxicities.Trial registration IDNCT04832672.

AEBP1 is differentially expressed in various tumors. However, the correlation between AEBP1 and immune cell infiltration in gastric cancer remains unclear. Kaplan-Meier survival curves were used to evaluate the relationship between AEBP1 expression and overall survival and progression-free survival. The relationship between AEBP1 expression and infiltrating immune cells, and their corresponding gene marker sets was examined using the TIMER database.The expression of AEBP1 was lower in gastric cancer (GC) tumor tissues than in normal tissues (P

Background. Chaiqin Qingning Capsule (CQ-C) is a traditional Chinese medicine (TCM) formula commonly used to treat respiratory infectious diseases in China. The aim of this study was to detect the effect and mechanism of CQ-C treated with influenza virus in vitro and vivo. Methods. The cytotoxicity and antiviral activity of CQ-C in vitro was determined by methyl thiazolyl tetrazolium (MTT) assay. The regulation of CQ-C on cytokine/chemokine expression was evaluated using RT-qPCR. In addition, the effect of CQ-C on the pathway protein, NF-κB, and its phosphorylation level was verified by western blotting. After virus inoculation, BALB/c mice were administered with CQ-C of different concentrations for 7 days. Body weight, viral titer, lung pathology, and mortality of the mice were measured, and the level of inflammatory cytokines was also examined using real-time RT-qPCR. Results. CQ-C inhibited the proliferation of influenza virus of various strains in vitro, with the 50% inhibitory concentration (IC50) ranging from 49 to 59 µg/mL. CQ-C downregulated virus-induced gene expression of IL-6, TNF-α, CXCL8, CXCL10, CCL5, and COX-2 in a dose-dependent manner in A549 cells. Also, CQ-C inhibited the expression of NF-κB protein of the signaling pathway. Moreover, a decrease of the lung index and mortality of mice was observed in the CQ-C (1 g/kg/d) group. The related cytokine/chemokine expression was also decreased in the early stages of infection in the mRNA level. Conclusion. As a clinically applied Chinese prescription, our study shows that CQ-C has a wide range of effects on several influenza viruses. Moreover, CQ-C could play an important role in anti-influenza activity and anti-inflammation in vitro and in vivo. Thus, CQ-C may be a promising treatment option for influenza.

Background Pathogenic variants in POC1A led to SOFT syndrome and variant POC1A-related (vPOC1A) syndrome. SOFT syndrome is a rare primordial dwarfism condition characterized by short stature, onychodysplasia, facial dysmorphism and hypotrichosis.The main clinical differences between SOFT and vPOC1A syndrome include dyslipidemia with insulin resistance and acanthosis nigricans. To our knowledge, this is the first report of a SOFT syndrome patient diagnosed with a homozygous splicing variant, which could help to extend our understanding of the genotypic and phenotypic information of the disease. Case presentation We reported a seven-year-old boy with SOFT syndrome. The patient presented symmetrical short stature and facial features, including prominent forehead, inverted triangular face, epicanthal fold, small teeth and enlarged ears. Laboratory tests displayed mild insulin resistance. Whole-exome sequencing (WES) led to the identification of a homozygous splicing variant (c.981+1G>A) in POC1A gene of the patient, which was inherited from his heterozygous parents confirmed by Sanger sequencing. Further transcriptional experiments of the splicing variant revealed aberrant percentage of exon 9 skipping transcripts. Conclusions This is the firstly reported case of a SOFT syndrome patient with a novel homozygous splicing variant and detailed delineation of the aberrant transcript in proband and carrier of the variant in Chinese. Our study enriched mutational spectrum of POC1A which could help in further genetic diagnosis and counselling of SOFT syndrome patients.

Purpose To provide a direct evidence for the current practice of prescribing antiemetic with aprepitant, dexamethasone and ondansetron in patients with locally advanced head and neck squamous cell carcinoma (LA-HNSCC) receiving concurrent chemoradiotherapy (CCRT). Methods Treatment-naïve LA-HNSCC patients received intensity-modulated radiotherapy with concomitant cisplatin 100 mg/m² every 3 weeks for two cycles. All patients were given orally aprepitant 125 mg once on d1, then 80mg once on d2-5; ondansetron 8 mg once on d1; and dexamethasone 12 mg once on d1, then 8mg on d2-5. The primary endpoint was complete response (CR). Pursuant to δ=0.2 and α=0.05, the expected CR rate was 80%. Results A total of 43 patients with LA-HNSCC were enrolled. The median age was 53 years old, and 86.0% were male. All patients received radiotherapy and 86.0% of patients completed both cycles as planned. The overall CR rate was 86.0% (95% CI: 72.1-94.7). The CR rates for cycles 1 and 2 were 88.4% (95% CI: 74.9-96.1) and 89.2% (95% CI: 74.6-97.0). The complete protection rate in the overall phase was 72.1% (95% CI: 56.3-84.7). The emesis-free response and nausea-free response in overall phase were 88.4% (95% CI: 74.9-96.1) and 60.5% (95% CI: 44.4-75.0), respectively. The adverse events related to antiemetics were constipation (65.1%) and hiccups (16.3%), but both were grade 1-2. There was no grade 4 or 5 treatment-related adverse event with antiemetic usage. Conclusion The addition of aprepitant into ondansetron and dexamethasone provided effective protection from nausea and emesis in patients with LA-HNSCC receiving CCRT. Trial registration ID: NCT03572829.

Both the distribution and orientation of anisotropic materials have great effects on the performance of civil and mechanical structures. Taking the maximum structural stiffness as the design object...

n-Pentanol exhibits some advantageous fuel properties, such as a higher energy density, lubricity, viscosity, and low hygroscopicity, when compared to short-chain alcohols. It is miscible with pure diesel in large proportions and burns directly on diesel engines. Therefore, n-pentanol is considered to be a promising diesel fuel additive. However, the differences between the chemical and physical properties of n-pentanol and commercial diesel affect the spray, combustion, and emissions performance of commercial diesel engines. In this study, visualization and engine experimental test methods were used to study the spray, combustion, and emissions performance of diesel/n-pentanol mixtures. The results showed that the atomization characteristics of diesel/n-pentanol mixtures were better than those of diesel. As the engine load increased, the maximum in-cylinder pressure and heat release rate increased. The brake specific fuel consumption (BSFC), nitrogen oxide (NOX), hydrocarbons (HC), and carbon monoxide (CO) emissions decreased, but soot emissions increased. Compared to diesel fuel, adding n-pentanol to pure diesel resulted in a decrease in soot emissions, while the BSFC, HC, and NOX emissions increased. Upon adding 50% n-pentanol to pure diesel (P50), the soot emissions decreased by up to 77.15% and the brake thermal efficiency (BTE) decreased by 1.86%. In summary, P50 could be burned directly on the diesel engine without any modifications, which can significantly reduce soot emissions; in this case, the BTE experienced only a slight decrease.

Objective: Restless legs syndrome (RLS) is one of the most common non-motor symptoms of Parkinson’s disease (PD), but its pathogenesis in a PD background is unclear. Abnormal iron metabolism may be...

A magnetic graphene/polydopamine (MG/PDA) nanocomposite has been prepared and used as sorbent for magnetic dispersive solid-phase extraction (MDSPE) of four benzoylurea insecticides in environmental water samples. The obtained nanocomposites were characterized by transmission electron microscopy, scanning electron microscopy, vibrating sample magnetometry, powder X-ray diffraction, fourier transform infrared spectroscopy, surface area and porosity analysis and thermogravimetric analysis. To investigate the adsorption performance of MG/PDA for target analytes, various parameters affecting the MG/PDA-based MDSPE procedure were optimized. Under the optimal conditions, the established method exhibits good linearity (R2 ≥ 0.9988) in the concentration range 2.5–500 µg L−1. A low limit of detection (0.75 µg L−1, signal/noise = 3:1), a low limit of quantification (2.50 µg L−1, signal/noise = 10:1), and good precision (intraday relative standard deviation ≤3.6%, interday relative standard deviation ≤4.5%) are also achieved. Finally, the simple, fast, and sensitive sample preparation technique was successfully used to determine benzoylurea insecticides in environmental water samples.

The viscoelasticity of constituent materials has a significant effect on the dispersion relation of waves in viscoelastic phononic crystals (PCs). This paper extends the bi-directional evolutionary structure optimization (BESO) method to the design of viscoelastic PCs with the maximum attenuation and stiffness. The attenuation factor is calculated by the k(ω)-method, and the effective elasticity matrix of composite PCs is extracted by the homogenization theory. The inverse design of viscoelastic PCs is formulated with a topology optimization problem, which is then solved by the proposed BESO method. Generally, BESO re-distributes the material phases of viscoelastic PCs within the primitive unit cell step by step based on sensitivity analysis. The optimization process is stopped until the optimized viscoelastic PC with the maximum attenuation factor and the desirable bulk modulus is achieved. Numerical examples are systematically presented for the propagation of out-of-plane or in-plane waves, and combined out-of-plane and in-plane waves at various frequencies. Novel topological patterns of the optimized viscoelastic PCs are obtained and discussed.

Discography can increase disc degeneration, but the influence of different discography variables on the degeneration of discs has not been reported. The aim of this study was to investigate the effects of discography variables of needle diameter, type of contrast agent and volume of contrast agent on disc degeneration. Three separate experiments examined needle diameter, and type and volume of contrast agent. Coccygeal discs (Co7-10) adult male rats were used. X-rays were used to detect the disc height degeneration index at 1, 2 and 4 weeks after the procedure. MRI was used to study the changes in the disc structure and the signal intensity of IVD 2 and 4 weeks after the procedure. Disc water content and histology were measured at 4 weeks after the procedure. A 21-g needle significantly increased disc degeneration when compared with the 30-g needle as detected by X-ray, MRI, disc water content and histology (P

Coronavirus disease (COVID-19) is a new infectious disease associated with high mortality, and traditional Chinese medicine decoctions (TCMDs) have been widely used for the treatment of patients with COVID-19 in China; however, the impact of these decoctions on severe and critical COVID-19-related mortality has not been evaluated. Therefore, we aimed to address this gap. In this retrospective cohort study, we included inpatients diagnosed with severe/critical COVID-19 at the Tongren Hospital of Wuhan University and grouped them depending on the recipience of TCMDs (TCMD and non-TCMD groups). We conducted a propensity score-matched analysis to adjust the imbalanced variables and treatments and used logistic regression methods to explore the risk factors associated with in-hospital death. Among 282 patients with COVID-19 who were discharged or died, 186 patients (66.0%) received TCMD treatment (TCMD cohort) and 96 (34.0%) did not (non-TCMD cohort). After propensity score matching at a 1:1 ratio, 94 TCMD users were matched to 94 non-users, and there were no significant differences in baseline clinical variables between the two groups of patients. The all-cause mortality was significantly lower in the TCMD group than in the non-TCMD group, and this trend remained valid even after matching (21.3% [20/94] vs. 39.4% [37/94]). Multivariable logistic regression model showed that disease severity (odds ratio: 0.010; 95% CI: 0.003, 0.037; [Formula: see text]¡ 0.001) was associated with increased odds of death and that TCMD treatment significantly decreased the odds of in-hospital death (odds ratio: 0.115; 95% CI: 0.035, 0.383; [Formula: see text]¡ 0.001), which was related to the duration of TCMD treatment. Our findings show that TCMD treatment may reduce the mortality in patients with severe/critical COVID-19.

BACKGROUND The increasingly younger age of stroke onset has posed a serious threat to the physical and mental health of young and middle-aged adults. Young and middle-aged stroke patients often suffer from varying degrees of motor, cognitive, and language disabilities, which can trigger considerable psychological trauma. However, these patients may have certain posttraumatic growth (PTG). As a positive psychological experience, PTG provides a new entry point for clinical psychological interventions. Here, we explored the trajectories of PTG in young and middle-aged stroke patients and their predictive effects on mental health. METHODS A total of in 165 patients who were treated at our center from January to December 2019 were enrolled with randomized sampling. The survey was conducted at 5 times follow up through use of the Posttraumatic Growth Inventory (PTGI), Social Support Rating Scale (SSRS), and Symptom Checklist-90 (SCL-90). Data were analyzed by using the latent growth mixture modeling (LGMM). RESULTS The PTG levels in young and middle-aged stroke patients showed a gradual increase (F=9.927; P

Impaired wound healing is one of a variety of severe diabetic complications and involves many factors, including consistent oxidative stress, prolonged inflammation, impaired angiogenesis, and delayed re-epithelialization. Despite the severe negative impacts that impaired wound healing has on patients’ lives, detailed mechanisms and effective therapies are still not fully developed. In this study, we aim to investigate the potential effects and mechanisms of topical administration of pterostilbene and resveratrol on burn wound healing in diabetes. Our in vitro experiments in human umbilical vein endothelial cells showed that long term exposure of hyperglycemia induces oxidative stress and suppression of hypoxia inducible factor1α (HIF1α) signaling pathway, and pterostilbene treatment completely, while resveratrol treatment partly, reversed this effect. Further in vivo experiments in diabetic rats showed that topical administration of pterostilbene exhibited stronger efficacy than resveratrol in normalizing oxidative stress, HIF1α activity, and accelerating burn wound healing in diabetes. We conclude that topical administration of pterostilbene accelerates burn wound healing in diabetes through activation of the HIF1α signaling pathway; thus, pterostilbene may be a potential candidate for clinical treatment of burn wound healing in diabetes.

To test the pharmacokinetics and toxicology of whole organs and tissues after intradiscal injection of simvastatin in rabbits. To provide the information needed to support human clinical trials. Twelve male and twelve female rabbits were randomly divided into four groups: control group (0 mg/ml), low dose group (0.1 mg/ml), medium dose group (1 mg/ml) and high dose group (10 mg/ml). Simvastatin at different concentrations of 10 μl was injected into L3/4, L4/5 and L5/6 intervertebral discs in each group. Poly (ethylene glycol) -poly (lactic-co-glycolic acid) -poly (ethylene glycol) (PEG-PLGA-PEG) polymer as the drug carrier. The pharmacokinetics of blood samples were measured by LC-MS/MS. Cerebrospinal fluid was obtained and the drug concentration was measured. Blood routine, blood biochemistry and urine of all animals were analyzed and evaluated. The heart, kidney, liver and spleen of each animal were observed and weighed. The intervertebral disc tissues were stained with hematoxylin and hematoxylin (H&E), and then qualitatively analyzed by optical microscopy. 28 days after intradiscal injection of simvastatin, 28 days after simvastatin intradiscal injection, there was no significant difference between the weight, food residue, blood routine, blood biochemistry, urine routine results and the weight of each organ in the four groups (p > 0.05). The serum concentration of simvastatin is lower than the lowest measurable concentration. The histological score of the intervertebral disc in the high-dose group was significantly higher than that in the other three groups at 28 days (p < 0.05). Three doses of simvastatin were injected into male and female animals respectively, showing no toxic effects. Microscopic histological evaluation of the intervertebral disc showed that the high dose group (10 mg/ml) had damage to the intervertebral disc tissue.

The present study aimed to investigate the association between the serum SIRT1 protein and the severity of spinal cord injury (SCI) as well as the neurological recovery in mice. In this study, the wild-type (WT), Mx1-Cre+ SIRT1loxP/loxP (Mx1), and LCK-Cre+SIRT1loxP/loxP (LCK) mice were subjected to sham surgery, mild, moderate, or severe SCI, respectively. The serum was collected at intervals of 12 h, 1 day (d), 3 d, 5 d, 7 d, 10 d, 14 d, and 21 d after the injury. The locomotor function of all the animals was assessed using the Basso mouse scale (BMS) and the serum SIRT1 proteins were analyzed using enzyme-linked immunosorbent assay (ELISA). The results demonstrated that about 7–10 d after SCI, the levels of SIRT1 protein in the serum correlated significantly with the severity of the injury and at 28 d post-injury, there was a distant neurological recovery (BMS score). The serum SIRT1 concentration in both the Mx1 and LCK mice in the sham group was significantly reduced compared to that in the WT mice, and there was a delayed increase in the serum SIRT1 levels after injury. These findings indicate that the SIRT1 concentrations in the serum of the SCI mice closely correlated with the acute severity and neurological outcome.

To study the effect of intradiscal injection of simvastatin on discogenic pain in rats and its possible mechanism, 30 adult female rats were used in this experiment. Twenty rats were randomly divided into sham operation group (Control group), intervertebral disk degeneration group (DDD group), intervertebral disk degeneration + hydrogel group (DDD + GEL group), and intervertebral disk degeneration + simvastatin group (DDD + SIM group). The mechanical pain threshold and cold sensation in rats were measured. The contents of NF-kappa B1, RelA, GAP43, SP, CGRP, TRPM 8, IL-1β, and TNF-α in the intervertebral disk (IVD), the corresponding contents of dorsal root ganglion (DRG) and plantar skin GAP43 and TRPM 8 were quantitatively detected by PCR. The corresponding IVDs were stained to detect their degeneration. There was no significant difference in the mechanical pain threshold between the groups at each time point. From the first day to the 8th week after surgery, the cold-sensing response of the DDD group was significantly higher than that of the Control group (P < 0.05). At 7 and 8 weeks postoperatively, the cold-sensing response of the DDD + SIM group was significantly lower than that of the DDD + GEL group (P < 0.05). The levels of NF-κB1, RelA, GAP43, SP, CGRP, TRPM8, IL-1β, and TNF-α in the IVD of DDD + SIM group were significantly lower than those in DDD group (P < 0.05). The content of GAP43 and TRPM8 in rat plantar skin decreased significantly and TRPM8 in DRG decreased significantly (P < 0.05).