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Profenofos (PFF) as an environmental pollutant seriously harms the health of aquatic animals, and even endangers human safety through the food chain. Albicanol, a sesquiterpenoid extraction from the Dryopteris fragrans, has previously been shown to effectively exhibit anti-aging, anti-oxidant, and antagonize the toxicity of heavy metals. However, the mechanism of hepatocyte toxicity caused by PFF and the role that Albicanol plays in this process are still unclear. In this study, a PFF poisoning model was established by treating grass carp hepatocytes cells with PFF (150μM) for 24 h The results of AO/EB staining, Tunel staining and flow cytometry showed that the proportion of apoptotic liver cells increased significantly after exposure. The results of ROS staining show that compared with the control group, ROS levels and PTEN/PI3K/AKT-related gene expression were up-regulated after PFF exposure. RT-qPCR and Western blotting results showed that the expression of PTEN/PI3K/AKT related genes was up-regulated. These results indicate that PFF can induce oxidative stress in hepatocytes and inhibit the phosphorylation of AKT. We further found that the expressions of Bax, CytC, Caspase-3, Caspase-9, Caspase-8 and TNFR1 after PFF exposure were significantly higher than those of the control group, and Bcl-2/Bax was significantly lower than that of the control group. These results indicate that PFF can induce oxidative stress in hepatocytes and inhibit the phosphorylation of AKT and activate mitochondrial apoptosis. Using Albicanol (5×10−5μg mL−1) can significantly reduce the above-mentioned effects of PFF exposure on grass carp hepatocytes cells. In summary, Albicanol inhibits PFF-induced apoptosis by regulating the ROS/PTEN/PI3K/AKT pathway.

Objective: To investigate the changes of hsMAD2 protein and gene expression levels during chromosome segregation of human embryos. Method: The embryos of spontaneous abortion were collected in our hospital from 2009 to 2013, the chromosomal numbers of the embryonic villi were subsequently detected by fluorescence in situ hybridization (FISH). The patients were then divided into the normal and abnormal groups based on the chromosome number. The hsMAD2 protein and gene expression levels in the villi tissues of the two embryo groups were detected by western blotting and qRT-PCR, respectively. The hsMAD2 protein and gene levels in the embryonic villus tissue of the patient were detected. Results: From 2009 to 2013, we collected 50 embryos from spontaneous abortion patients. The chromosome abnormality and no abnormality were 36 cases (abnormal number of 28 cases (56.0%) and chimerism in 8 cases (16.0%)) and 14 cases (28.0%), respectively. The expression of hsmad2 protein and its gene in the villi of spontaneously aborted embryo with chromosomal abnormality in the abnormal group was significantly higher than that in those without chromosomal abnormalities (0.88 ± 0.20 vs 0.61 ± 0.19, P Conclusion: Abnormal number of chromosomes is closely related to spontaneous abortion Linked, hsMAD2 factor has a card effect on the cell cycle, can block the mitotic process of cells, and play an important role in maintaining the normal separation and stability of chromosomes.

Scientists believed the outbreak of COVID-19 could be linked to the consumption of wild animals, so food safety and hygiene have become the top concerns of the public. An agri-food traceability system becomes very important in this context because it can help the government to trace back the entire production and delivery process in case of food safety concerns. The traceability system is a complicated digitalized system because it integrates information and logistics systems. Previous studies used the technology acceptance model (TAM), information systems (IS) success model, expectation confirmation model (ECM), or extended model to explain the continuance intention of traceability system users. Very little literature can be found integrating two different models to explain user intention, not to mention comparing three models in one research context. This study proposed the technology acceptance model (TAM), technology acceptance model-information systems (TAM-IS) success, and technology acceptance model-expectation confirmation model (TAM-ECM) integrated models to evaluate the most appropriate model to explain agri-food traceability system during the COVID-19 pandemic. A questionnaire was designed based on a literature review, and 197 agri-food traceability system users were sampled. The collected data were analyzed by partial least square (PLS) to understand the explanatory power and the differences between the three models. The results showed that: (1) the TAM model has a fair explanatory power of continuance intention (62.2%), but was recommended for its’ simplicity; (2) the TAM-IS success integrated model had the best predictive power of 78.3%; and (3) the system providers should raise users’ confirmation level, so their continuance intention could be reinforced through mediators, perceived value, and satisfaction. The above findings help to understand agri-food traceability system user intention, and provide theoretical and practical implications for system providers to refine their system design.

Supplemental material, sj-pdf-1-jcb-10.1177_0271678X221111609 for Modification of cerebrovascular morphologies during different stages of life by Boyu Zhang, Zidong Yang, Jing Li, Bei Wang, Huazheng Shi, He Wang and Yuehua Li in Journal of Cerebral Blood Flow & Metabolism

Patients with bladder cancer (BCa) with clinical lymph node (LN) metastasis have an extremely poor prognosis. VEGF-C has been demonstrated to play vital roles in LN metastasis in BCa. However, approximately 20% of BCa with LN metastasis exhibits low VEGF-C expression, suggesting a VEGF-C-independent mechanism for LN metastasis of BCa. Herein, we demonstrate that BCa cell-secreted exosome-mediated lymphangiogenesis promoted LN metastasis in BCa in a VEGF-C-independent manner. We identified an exosomal long noncoding RNA (lncRNA), termed lymph node metastasis-associated transcript 2 (LNMAT2), that stimulated human lymphatic endothelial cell (HLEC) tube formation and migration in vitro and enhanced tumor lymphangiogenesis and LN metastasis in vivo. Mechanistically, LNMAT2 was loaded to BCa cell-secreted exosomes by directly interacting with heterogeneous nuclear ribonucleoprotein A2B1 (hnRNPA2B1). Subsequently, exosomal LNMAT2 was internalized by HLECs and epigenetically upregulated prospero homeobox 1 (PROX1) expression by recruitment of hnRNPA2B1 and increasing the H3K4 trimethylation level in the PROX1 promoter, ultimately resulting in lymphangiogenesis and lymphatic metastasis. Therefore, our findings highlight a VEGF-C-independent mechanism of exosomal lncRNA-mediated LN metastasis and identify LNMAT2 as a therapeutic target for LN metastasis in BCa.

Background Balloon dilation is a commonly used minimally invasive endourological treatment of ureteral stricture, but the postoperative recurrence rate is relatively high. And factors contributing to recurrence after treatment are poorly understood. Herein, we sought to develop a novel clinical nomogram to predict ureteral stricture-free survival in patients suffering from ureter stricture and performed balloon dilation. Methods The nomogram was established based on a retrospective analysis of 321 patients who received endoscopic balloon dilation alone for ureter strictures from January 2016 to January 2020 in Sun Yat-sen Memorial Hospital using the Cox regression model. Perioperative clinical data and disease outcomes were analyzed. The primary endpoint was the onset of ureteral re-stricture after ureter balloon dilation. Discrimination of the nomogram was assessed by the concordance index (C-index) and the calibration curve. The results were internally validated using bootstrap resampling. Results Overall, 321 patients with a median follow-up of 590 days were enrolled in the study, among which 97 patients (30.2%) developed recurrence of ureteral stricture during follow-up. Five variables remained significant predictors of ureteral re-stricture after multivariable analyses: stricture nature (P Conclusions Our study developed the first nomogram to effectively predict stricture-free survival in patients suffering from ureter stricture after balloon dilation. It is helpful to identify the optimal patients with ureter stricture for balloon dilation and improve treatment outcomes. However, further external validation of the nomogram is warranted.

Background Patients with lymph node (LN) metastatic bladder cancer (BCa) present with extremely poor prognosis. BCa‐derived exosomes function as crucial bioactive cargo carriers to mediate the signal transduction in tumor microenvironment triggering tumor metastasis. However, the mechanisms underlying exosome‐mediated LN metastasis in BCa are unclear. Methods We conducted the high‐throughput sequencing to explore the expression profile of long noncoding RNA (lncRNA) in urinary exosomes (urinary‐EXO) from patients with BCa and further evaluated the clinical relevance of exosomal lncRNA BCYRN1 in a larger 210‐case cohort. The functional role of exosomal BCYRN1 was evaluated through the migration and tube formation assays in vitro and the footpad‐popliteal LN metastasis model in vivo. RNA pull‐down assays, luciferase assays, and actinomycin assays were conducted to detect the regulatory mechanism of exosomal BCYRN1. Results LncRNA BCYRN1 was substantially upregulated in urinary‐EXO from patients with BCa, and associated with the LN metastasis of BCa. We demonstrated that exosomal BCYRN1 markedly promoted tube formation and migration of human lymphatic endothelial cells (HLECs) in vitro and lymphangiogenesis and LN metastasis of BCa in vivo. Mechanistically, BCYRN1 epigenetically upregulated WNT5A expression by inducing hnRNPA1‐associated H3K4 trimethylation in WNT5A promoter, which activated Wnt/β‐catenin signaling to facilitate the secretion of VEGF‐C in BCa. Moreover, exosomal BCYRN1 was transmitted to HLECs to stabilize the VEGFR3 mRNA and thus formed an hnRNPA1/WNT5A/VEGFR3 feedforward regulatory loop, ultimately promoting the lymphatic metastasis of BCa. Importantly, blocking VEGFR3 with specific inhibitor, SAR131675 significantly impaired exosomal BCYRN1‐induced the LN metastasis in vivo. Clinically, exosomal BCYRN1 was positively associated with the shorter survival of BCa patients and identified as a poor prognostic factor of patients. Conclusion Our results uncover a novel mechanism by which exosomal BCYRN1 synergistically enhances VEGF‐C/VEGFR3 signaling‐induced lymphatic metastasis of BCa, indicating that BCYRN1 may serve as an encouraging therapeutic target for patients with BCa., lncRNA BCYRN1 is upregulated in urinary exosomes from bladder cancer (BCa) patients and associated with LN metastasis and poor prognosis of patients. BCYRN1 upregulates WNT5A expression to activate WNT/β‐catenin pathway and promotes the secretion of VEGF‐C in BCa. Exosomal BCYRN1 stabilizes VEGFR3 mRNA in HLECs, constituting a feedforward loop to promote lymphangiogenesis and LN metastasis of BCa.

Non-nutritive sweeteners (NNS) have seen increasingly common usage amongst foodstuffs in the westernized diet in the past few decades, including processed foods and flavored beverages. Recently, an increasing body of research has been evaluating the potential of NNS for alternative uses in healthcare and medicine. Some of these sweeteners, such as stevioside, have been implicated as being anti-hypertensive, anti-carcinogenic, and anti-inflammatory in several contexts. To examine the potential of NNS such as stevioside as an anti-carcinogenic agent, cellular behavior of HFF-1 human foreskin fibroblasts (ATCC SCRC-1041) & COLO-829 human melanoma fibroblasts (ATCC CRL-1974) was observed in the presence of sucrose, sucralose, and stevioside on collagen. This study examined the impact of NNS on initial adhesion, cellular proliferation, and integrin expression on healthy and cancerous fibroblast cell lines. The results indicate that 5 mg/mL stevioside caused more than 2-fold decrease in the initial adhesion of healthy cells but caused more than 4-fold increase in the initial adhesion of cancerous cells. In terms of proliferation, stevioside at 5 mg/mL caused a 13-fold decrease in cancer cell proliferation, proportional to concentration, but a 1.5-fold increase in proliferation for healthy cells.

Purpose: Chemoresistance and tumor relapse are the leading cause of deaths in bladder cancer patients. Bladder cancer stem cells (BCSCs) have been reported to contribute to these pathologic properties. However, the molecular mechanisms underlying their self-renewal and chemoresistance remain largely unknown. In the current study, a novel lncRNA termed Low expressed in Bladder Cancer Stem cells (lnc-LBCS) has been identified and explored in BCSCs. Experimental Design: Firstly, we establish BCSCs model and explore the BCSCs-associated lncRNAs by transcriptome microarray. The expression and clinical features of lnc-LBCS are analyzed in three independent large-scale cohorts. The functional role and mechanism of lnc-LBCS are further investigated by gain- and loss-of-function assays in vitro and in vivo. Results: Lnc-LBCS is significantly downregulated in BCSCs and cancer tissues, and correlates with tumor grade, chemotherapy response, and prognosis. Moreover, lnc-LBCS markedly inhibits self-renewal, chemoresistance, and tumor initiation of BCSCs both in vitro and in vivo. Mechanistically, lnc-LBCS directly binds to heterogeneous nuclear ribonucleoprotein K (hnRNPK) and enhancer of zeste homolog 2 (EZH2), and serves as a scaffold to induce the formation of this complex to repress SRY-box 2 (SOX2) transcription via mediating histone H3 lysine 27 tri-methylation. SOX2 is essential for self-renewal and chemoresistance of BCSCs, and correlates with the clinical severity and prognosis of bladder cancer patients. Conclusions: As a novel regulator, lnc-LBCS plays an important tumor-suppressor role in BCSCs’ self-renewal and chemoresistance, contributing to weak tumorigenesis and enhanced chemosensitivity. The lnc-LBCS–hnRNPK–EZH2–SOX2 regulatory axis may represent a therapeutic target for clinical intervention in chemoresistant bladder cancer.

Drugs blocking programmed death ligand-1 (PD-L1) have shown unprecedented activity in metastatic and unresectable bladder cancer. The purpose of the present study was to investigate the expression, clinical significance and association of PD-L1 with tumor-infiltrating lymphocytes (TIL) in resectable urothelial cell carcinoma of the bladder (UCB). In this retrospective study, 248 UCB patients who received radical cystectomy or transurethral resection were examined. Immunohistochemistry was used to evaluate PD-L1 expression and stromal CD8+ TIL, Th1 orientation T cell (T-bet+ ) and PD-1+ TIL densities within the intratumoral regions and associated stromal regions. Of the 248 specimens, 23% showed PD-L1 expression in tumor cells and 55% in tumor-infiltrating immune cells. CD8+ TIL, T-bet+ TIL and PD-1+ TIL were distributed throughout the tumor tissues and were more frequently distributed in stromal regions than in intratumoral regions. PD-L1+ tumor cells and PD-L1+ immune cells were positively associated with aggressive clinical features (all P

A novel semirigid ureterorenoscope, named the Sotn ureterorenoscope, was designed with a vacuum suction system. The present study aimed to evaluate the feasibility and safety of using the Sotn ureterorenoscope to manage single proximal ureteral or renal pelvic stones.Data were retrospectively collected from consecutive patients treated with a Sotn ureterorenoscope between February 2010 and August 2015 at Sun Yat-sen Memorial Hospital of Sun Yat-sen University and Jiangmen Wuyi Traditional Chinese Medicine Hospital in China. The primary outcome was the primary stone-free rate (SFR) in 1 month. The secondary outcomes were the final SFR and the perioperative complication rate.A total of 386 patients were evaluated, including 240 males and 146 females. The median (interquartile range [IR]) age was 50 (40-59) years. There were 96 and 290 stones located in the renal pelvis and proximal ureter, respectively. The median (IR) operative time and console time for all patients were 40 (30-70) and 20 (12-38) minutes, respectively. The primary overall SFR was 86.5%, whereas the SFRs for stones with a diameter of ≤1, 1 to 2, and 2 to 3 cm were 95.7%, 86.9%, and 69.0%, respectively. Complications occurred in 90 patients (23.3%); these complications were classified as Clavien-Dindo grades 1 to 2 (minor) in 79 (20.5%) patients, and grades 3 to 4 (major) in 11 (2.8%).The novel semirigid Sotn ureterorenoscope featuring a vacuum suction system is effective and safe for managing proximal ureteral and renal pelvic stones.

Owing to the lack of specific diagnostic methods, Scrub typhus can sometimes be difficult to diagnose in the Asia-Pacific region. Therefore, an efficient and rapid detection method urgently needs to be developed. Based on competitive single-stranded DNA over modified glassy carbon electrode (GCE), an electrochemical biosensor was established to detect the disease. The nano-flower NiFe layered double hydroxide (NiFe-LDH) modified GCE has a large specific surface area, which supported a large amount of gold nanoparticles, so that a wide linear detection range from 25 fM to 0.5 μM was obtained. The beacon DNA (B-DNA) with the same sequence as the Scrub typhus DNA (T-DNA), but labeled with methylene blue, was used to construct a competitive relationship. When T-DNA and B-DNA were present on the sensor simultaneously, they would hybridize with probe DNA in a strong competition, and the corresponding electrochemical response signal would be generated via testing. It contributed to reducing tedious experimental procedures and excessive response time, and achieved fast electrochemical detection of DNA. The strategy provides a worthy avenue and possesses promising applications in disease diagnosis.

Small ubiquitin-like modifier (SUMO) binding (termed SUMOylation) emerged as the inducer for the sorting of bioactive molecules into extracellular vesicles (EVs), triggering lymphangiogenesis and further driving tumor lymph node (LN) metastasis, but the precise mechanisms remain largely unclear. Here, we show that bladder cancer (BCa) cell-secreted EVs mediated intercellular communication with human lymphatic endothelial cells (HLECs) through transmission of the long noncoding RNA ELNAT1 and promoted lymphangiogenesis and LN metastasis in a SUMOylation-dependent manner in both cultured BCa cell lines and mouse models. Mechanistically, ELNAT1 induced UBC9 overexpression to catalyze the SUMOylation of hnRNPA1 at the lysine 113 residue, which mediated recognition of ELNAT1 by the endosomal sorting complex required for transport (ESCRT) and facilitated its packaging into EVs. EV-mediated ELNAT1 was specifically transmitted into HLECs and epigenetically activated SOX18 transcription to induce lymphangiogenesis. Importantly, blocking the SUMOylation of tumor cells by downregulating UBC9 expression markedly reduced lymphatic metastasis in EV-mediated, ELNAT1-treated BCa in vivo. Clinically, EV-mediated ELNAT1 was correlated with LN metastasis and a poor prognosis for patients with BCa. These findings highlight a molecular mechanism whereby the EV-mediated ELNAT1/UBC9/SOX18 regulatory axis promotes lymphangiogenesis and LN metastasis in BCa in a SUMOylation-dependent manner and implicate ELNAT1 as an attractive therapeutic target for LN metastatic BCa.

Supplemental material, sj-pdf-1-jcb-10.1177_0271678X211023660 for Deep learning-based identification of acute ischemic core and deficit from non-contrast CT and CTA by Chengyan Wang, Zhang Shi, Ming Yang, Lixiang Huang, Wenxing Fang, Li Jiang, Jing Ding and He Wang in Journal of Cerebral Blood Flow & Metabolism

Psocids are a new risk for global food security and safety because they are significant worldwide pests of stored products. Among these psocids, Liposcelis bostrychophila has developed high levels of resistance or tolerance to heat treatment in grain storage systems, and thus has led to investigation of molecular mechanisms underlying heat tolerance in this pest. In this study, the time-related effects of thermal stress treatments at relatively high temperatures on the activities of antioxidant enzymes, including superoxide dismutase (SOD), catalase (CAT), peroxidases (POD), glutathione-S-transferases (GST) and malondialdehyde (MDA), of L. bostrychophila were determined. Thermal stress resulted that L. bostrychophila had a significantly higher MDA concentration at 42.5 &deg, C, which indicated that the heat stress increased lipid peroxidation (LPO) contents and oxidative stress in this psocid pest. Heat stress also resulted in significant elevation of SOD, CAT and GST activities but decreased POD activity. Our data indicates that different antioxidant enzymes contribute to defense mechanisms, counteracting oxidative damage in varying levels. POD play minor roles in scavenging deleterious LPO, while enhanced SOD, CAT and GST activities in response to thermal stress likely play a more important role against oxidative damage. Here, we firstly identified five LbHsps (four LbHsp70s and one LbHsp110) from psocids, and most of these LbHsps (except LbHsp70-1) are highly expressed at fourth instar nymph and adults, and LbHsp70-1 likely presents as a cognate form of HSP due to its non-significant changes of expression. Most LbHsp70s (except LbHsp70-4) are significantly induced at moderate high temperatures (&lt, 40 &deg, C) and decreased at extreme high temperatures (40&ndash, 45 &deg, C), but LbHsp110-1 can be significantly induced at all high temperatures. Results of this study suggest that the LbHsp70s and LbHsp110 genes are involved in tolerance to thermal stress in L. bostrychophila, and antioxidant enzymes and heat shock proteins may be coordinately involved in the tolerance to thermal stress in psocids.

Purpose: The aim of this study was to review the clinicopathologic characteristics, treatments, and outcomes of patients with primary large cell neuroendocrine carcinoma of the bladder (LCNEC). Patients and Methods: We report one patient diagnosed with primary pure LCNEC of the bladder in Sun Yat-sen Memorial Hospital. In addition, we performed a systematic literature review, in April 2020, on case report and case series of LCNEC of the bladder. The clinicopathologic characteristics, treatments and outcomes of this rare disease were analyzed. Results: A total of 39 patients were included in our analysis (1 case from our institution and 38 cases from the literature). Most patients (79.5%) were male. The average age at the surgery for the patients is 61.5 years (range 19-85 years). The most common symptom was hematuria (n = 20, 76.9%). Almost all patients (38, 97.4%) underwent surgery, with 26 (66.7%) receiving multimodality therapy. Out of 24 patients with available data, regional or distant recurrences developed in 14 patients (58.3%). The median overall survival of the patients was 11.5 months, with 1- and 3-year survival rates of 54.0 and 21.4%, respectively. In the survival analysis, theT1-2 tumors (P = 0.025), no distant metastases at diagnosis (P = 0.001), and multimodality therapy (P = 0.017) were associated with better overall survival (OS). Conclusions: LCNEC of the bladder is an extremely rare neoplasm. The available data suggest that the disease has an aggressive natural history with poor prognosis. Early pathologic stage and multimodality treatment may be important factors in determining prognosis.

Purpose: We assessed the performance of metagenomic next-generation sequencing (mNGS) in the diagnosis of infectious encephalitis and meningitis. Methods: This was a prospective multicenter study. Cerebrospinal fluid samples from patients with viral encephalitis and/or meningitis, tuberculous meningitis, bacterial meningitis, fungal meningitis, and non-central nervous system (CNS) infections were subjected to mNGS. Results: In total, 213 patients with infectious and non-infectious CNS diseases were finally enrolled from November 2016 to May 2019; the mNGS-positive detection rate of definite CNS infections was 57.0%. At a species-specific read number (SSRN) ≥2, mNGS performance in the diagnosis of definite viral encephalitis and/or meningitis was optimal (area under the curve [AUC] = 0.659, 95% confidence interval [CI] = 0.566-0.751); the positivity rate was 42.6%. At a genus-specific read number ≥1, mNGS performance in the diagnosis of tuberculous meningitis (definite or probable) was optimal (AUC=0.619, 95% CI=0.516-0.721); the positivity rate was 27.3%. At SSRNs ≥5 or 10, the diagnostic performance was optimal for definite bacterial meningitis (AUC=0.846, 95% CI = 0.711-0.981); the sensitivity was 73.3%. The sensitivities of mNGS (at SSRN ≥2) in the diagnosis of cryptococcal meningitis and cerebral aspergillosis were 76.92 and 80%, respectively. Conclusion: mNGS of cerebrospinal fluid effectively identifies pathogens causing infectious CNS diseases. mNGS should be used in conjunction with conventional microbiological testing. Trial Registration: Chinese Clinical Trial Registry, ChiCTR1800020442.

Long noncoding RNA FOXF1 adjacent non-coding developmental regulatory RNA (FENDRR) dysregulation associates with multiple types of human cancer. However, the biological functions of FENDRR in renal cell carcinoma are unresolved. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was performed to determine the expression level of FENDRR in renal cell carcinoma tissues. An RNA interference assay and ectopic expression experiments were conducted to evaluate the effects of FENDRR on cell proliferation, migration, invasion and colony formation in vitro. RNA immunoprecipitation was conducted to identify proteins associated with FENDRR. It was observed that FENDRR is frequently downregulated in renal cell carcinoma and overexpression of FENDRR attenuated proliferation, migration, invasion and colony growth of renal carcinoma cells. Conversely, knockdown of FENDRR promotes proliferation and invasiveness of renal carcinoma cells. Downregulation of FENDRR associates with poor prognosis of renal cell carcinoma. Mechanistically, it was identified that FENDRR may bind to Polycomb Repressive Complex 2 and lysin methyltransferase 2A histone modifying complexes. In summary, FENDRR acts as an tumor suppressor in renal cell carcinoma and may serve as a candidate target for gene therapy.

Tumor-associated macrophages (TAMs) are the most abundant inflammatory infiltrates in the tumor microenvironment and contribute to lymph node (LN) metastasis. However, the precise mechanisms of TAMs-induced LN metastasis remain largely unknown. Herein, we identify a long noncoding RNA, termed Lymph Node Metastasis Associated Transcript 1 (LNMAT1), which is upregulated in LN-positive bladder cancer and associated with LN metastasis and prognosis. Through gain and loss of function approaches, we find that LNMAT1 promotes bladder cancer-associated lymphangiogenesis and lymphatic metastasis. Mechanistically, LNMAT1 epigenetically activates CCL2 expression by recruiting hnRNPL to CCL2 promoter, which leads to increased H3K4 tri-methylation that ensures hnRNPL binding and enhances transcription. Furthermore, LNMAT1-induced upregulation of CCL2 recruits macrophages into the tumor, which promotes lymphatic metastasis via VEGF-C excretion. These findings provide a plausible mechanism for LNMAT1-modulated tumor microenvironment in lymphatic metastasis and suggest that LNMAT1 may represent a potential therapeutic target for clinical intervention in LN-metastatic bladder cancer.

Chronic hepatitis is a general designation class of diseases, which results in different degrees of liver necrosis and inflammatory reaction, followed by liver fibrosis, may eventually develop into cirrhosis. However, the molecular pathogenesis of chronic hepatitis is too complex to elucidate. Herbal medicines, featured with multiple targets and compounds, have long displayed therapeutic effect in treating chronic hepatitis, though their molecular mechanisms of contribution remain indistinct. This research utilized the network pharmacology to confirm the molecular pathogenesis of chronic hepatitis through providing a comprehensive analysis of active chemicals, drug targets and pathways' interaction of Sinisan formula for treating chronic hepatitis. The outcomes showed that 80 active ingredients of Sinisan formula interacting with 91 therapeutic proteins were authenticated. Sinisan formula potentially participates in immune modulation, anti-inflammatory and antiviral activities, even has regulating effects on lipid metabolism. These mechanisms directly or indirectly are involved in curing chronic hepatitis by an interaction way. The network pharmacology based analysis demonstrated that Sinisan has multi-scale curative activity in regulating chronic hepatitis related biological processes, which provides a new potential way for modern medicine in the treatment of chronic diseases.

Autism spectrum disorder (ASD) is a genetically heterogeneous neurodevelopmental disorder characterized by impairments in social interaction and communication, and by restricted and repetitive behaviors. The genetic architecture of ASD has been elucidated, including chromosomal rearrangements, de novo or inherited rare variants, and copy number variants. However, the genetic mechanism of Chinese families with ASD children is explored rarely. To identify genetic pathogenesis, we performed massively parallel sequencing on 13 Chinese ASD trio families, and found two de novo variations. The novel de novo splice alteration c.664 + 2T > G in the DEAF1 gene and the novel de novo missense mutation c.95 C > T in the AADAT gene associated with ASD may be important clues for exploring the etiology of this disorder.

Long non-coding RNAs (lncRNAs) have been identified as significant regulators in cancer progression. Positive feedback loops between lncRNAs and transcription factors have attracted increasing attention. Akt pathway plays a crucial role in bladder cancer growth and recurrence. In the present study, we demonstrate a novel regulatory pattern involving FOXD2-AS1, Akt, and E2F1. FOXD2-AS1 is highly expressed in bladder cancer and is associated with tumor stage, recurrence, and poor prognosis. Further experiments showed that FOXD2-AS1 promotes bladder cancer cell proliferation, migration, and invasion in vitro and in vivo. Microarray analysis demonstrated that FOXD2-AS1 negatively regulates the expression of Tribbles pseudokinase 3 (TRIB3), a negative regulator of Akt. Mechanistically, FOXD2-AS1 forms an RNA-DNA complex with the promoter of TRIB3, the transcriptional activity of which is subsequently repressed, and leads to the activation of Akt, which further increases the expression of E2F1, a vital transcription factor involved in the G/S transition. Interestingly, E2F1 could bind to the FOXD2-AS1 promoter region and subsequently enhance its transcriptional activity, indicating that FOXD2-AS1/Akt/E2F1 forms a feedback loop. In summary, this regulatory pattern of positive feedback may be a novel target for the treatment of bladder cancer and FOXD2-AS1 has the potential to be a new recurrence predictor.

The prognosis for bladder cancer patients with lymph node (LN) metastasis is dismal and only minimally improved by current treatment modalities. Elucidation of the molecular mechanisms that underlie LN metastasis may provide clinical therapeutic strategies for LN-metastatic bladder cancer. Here, we report that a long noncoding RNA LINC00958, which we have termed bladder cancer-associated transcript 2 (BLACAT2), was markedly upregulated in LN-metastatic bladder cancer and correlated with LN metastasis. Overexpression of BLACAT2 promoted bladder cancer-associated lymphangiogenesis and lymphatic metastasis in both cultured bladder cancer cell lines and mouse models. Furthermore, we demonstrate that BLACAT2 epigenetically upregulated VEGF-C expression by directly associating with WDR5, a core subunit of human H3K4 methyltransferase complexes. Importantly, administration of an anti-VEGF-C antibody inhibited LN metastasis in BLACAT2-overexpressing bladder cancer. Taken together, these findings uncover a molecular mechanism in the lymphatic metastasis of bladder cancer and indicate that BLACAT2 may represent a target for clinical intervention in LN-metastatic bladder cancer.

Background Gastrointestinal stromal tumors (GISTs) are the most common mesenchymal tumors of the gastrointestinal tract. Rectal locations are very rare, and minimally invasive surgery is a good choice for the treatment of rectal GISTs. Case presentation Two women each had a mass located on the lower vaginal-rectal space as determined by transvaginal ultrasound (TV-US), pelvis MR imaging, and colonoscopy. The patients successfully underwent transvaginal excision. The spindle-shaped cells were found in pathological test. The immunohistochemical analysis showed that CD117 and Dog-1 were stained positively. These results confirmed the masses as GISTs. The postoperative period was uneventful without anal dysfunction. Two patients were received adjuvant treatment with imatinib after surgery. Conclusion Transvaginal excision could be a minimally invasive and safe alternative treatment in the management of rectal GISTs in lower locations.

BACKGROUND Severe acute pancreatitis (SAP) is a severe form of inflammatory disease with a high mortality rate. Ulinastatin, a urinary trypsin inhibitor, has anti-inflammatory properties and may be beneficial to critically ill patients with SAP. Nevertheless, there is currently insufficient evidence to conclude whether there is a dose-effect relationship between ulinastatin and SAP treatment outcomes. The present study examined the efficacy of ulinastatin at different doses in the treatment of SAP. METHODS A retrospective study was conducted examining the clinical outcomes of 130 SAP patients. Patients were categorized into a control group and three groups receiving different daily doses of ulinastatin (200,000; 400,000; and 600,000 IU). The study compared the 1-week mortality rate; the Acute Physiology and Chronic Health Evaluation II (APACHE-II) score; abdominal pain relief time; time to recover a normal heart and respiratory rate; blood amylase, glucose, C-reactive protein, and procalcitonin levels; and white blood cell (WBC) count among the different groups. RESULTS The 400,000 and 600,000 IU groups had significantly lower mortality rates and WBC count compared to the 200,000 IU group (P

Purpose: The overall biological roles and clinical significance of most long noncoding RNAs (lncRNA) in chemosensitivity are not fully understood. We investigated the biological function, mechanism, and clinical significance of lncRNA NR_034085, which we termed miRNA processing–related lncRNA (MPRL), in tongue squamous cell carcinoma (TSCC). Experimental Design: LncRNA expression in TSCC cell lines with cisplatin treatment was measured by lncRNA microarray and confirmed in TSCC tissues. The functional roles of MPRL were demonstrated by a series of in vitro and in vivo experiments. The miRNA profiles, RNA pull-down, RNA immunoprecipitation, serial deletion analysis, and luciferase analyses were used to investigate the potential mechanisms of MPRL. Results: We found that MPRL expression was significantly upregulated in TSCC cell lines treated with cisplatin and transactivated by E2F1. MPRL controlled mitochondrial fission and cisplatin sensitivity through miR–483-5p. In exploring the underlying interaction between MPRL and miR–483-5p, we identified that cytoplasmic MPRL directly binds to pre–miR–483 within the loop region and blocks pre–miR–483 recognition and cleavage by TRBP–DICER-complex, thereby inhibiting miR–483-5p generation and upregulating miR–483-5p downstream target-FIS1 expression. Furthermore, overexpression or knockdown MPRL altered tumor apoptosis and growth in mouse xenografts. Importantly, we found that high expression of MPRL and pre–miR–483, and low expression of miR–483-5p were significantly associated with neoadjuvant chemosensitivity and better TSCC patients’ prognosis. Conclusions: We propose a model in which lncRNAs impair microprocessor recognition and are efficient of pre-miRNA cropping. In addition, our study reveals a novel regulatory network for mitochondrial fission and chemosensitivity and new biomarkers for prediction of neoadjuvant chemosensitivity in TSCC. These findings uncover a novel mechanism by which lncRNA determines mitochondrial fission and cisplatin chemosensitivity by inhibition of pre-miRNA processing and provide for the first time the rationale for lncRNA and miRNA biogenesis for predicting chemosensitivity and patient clinical prognosis.

Malignant brain tumor domain containing protein 1 (MBTD1) is a member of the polycomb group protein family that is associated with tumorigenesis. The present study investigated the role of MBTD1 within defined clinicopathological parameters and the prognosis of patients with prostate cancer (PCa). A human tissue microarray containing samples from 71 patients with PCa and seven healthy donors was employed for immunohistochemistry (IHC). The clinicopathological characteristics and prognostic value of MBTD1 were investigated using a dataset of 499 patients from The Cancer Genome Atlas (TCGA). IHC illustrated that the levels of MBTD1 protein were enhanced and markedly associated with aggressive clinical stage and advanced tumor invasion, distant metastasis and lymph node metastasis in patients with PCa. In the TCGA data set, the level of MBTD1 was found to positively correlate with the prostate-specific antigen level, Gleason score and distant metastasis. The multivariate analysis of Cox regression revealed that the levels of MBTD1 may act as an independent prognostic factor for low non-biochemical, recurrence-free survival. In conclusion, MBTD1 was overexpressed in PCa tissues and is associated with aggressive clinicopathological characteristics. It may therefore act as a novel prognostic factor and diagnostic marker in PCa.

Virus-like particles (VLPs) vaccines combine many of the advantages of whole-virus vaccines and recombinant subunit vaccines, integrating key features that underlay their immunogenicity, safety and protective potential. We have hypothesized here the effective insertion of the VP1 epitopes (three amino acid residues 21-40, 141-160 and 200-213 in VP1, designated VPe) of foot-and-mouth disease (FMDV) within the external loops of PPV VP2 could be carried out without altering assembly based on structural and antigenic data. To investigate the possibility, development of two recombinant adenovirus rAd-PPV:VP2-FMDV:VPe a or rAd-PPV:VP2-FMDV:VPe b were expressed in HEK-293 cells. Out of the two insertion strategies tested, one of them tolerated an insert of 57 amino acids in one of the four external loops without disrupting the VLPs assembly. Mice were inoculated with the two recombinant adenoviruses, and an immunogenicity study showed that the highest levels of FMDV-specific humoral responses and T cell proliferation could be induced by rAd-PPV:VP2-FMDV:VPe b expressing hybrid PPV:VLPs (FMDV) in the absence of an adjuvant. Then, the protective efficacy of inoculating swine with rAd-PPV:VP2-FMDV:VPe b was tested. All pigs inoculated with rAd-PPV:VP2-FMDV:VPe b were protected from viral challenge, meanwhile the neutralizing antibody titers were significantly higher than those in the group inoculated with swine FMD type O synthetic peptide vaccine. Our results clearly demonstrate the potential usefulness of adenovirus-derived PPV VLPs as a vaccine strategy in prevention of FMDV.

Ubiquitin-specific peptidase 39 (USP39) has been reported to participate in the mitotic spindle checkpoint and the process of cytokinesis. and has been identified as a therapeutic target for various types of cancer. However, the effect of USP39 in colorectal cancer (CRC) has not been investigated. To explore the functional role of USP39 in CRC cell growth, lentivirus-mediated RNA interference was applied to inhibit USP39 expression in SW1116 and HCT116 cells. The relative USP39 mRNA and protein expression levels were significantly reduced in the USP39 knockdown cells, as verified by reverse transcription-quantitative polymerase chain reaction and western blot analysis. USP39 knockdown significantly reduced the proliferation and colony formation abilities of CRC cells, and induced apoptosis and cell cycle arrest in the G2/M phases, as determined by an MTT assay, a colony formation assay and flow cytometry analysis. Furthermore, western blot analysis demonstrated that USP39 knockdown may have induced apoptosis through the upregulation of p53, p-p53, PARP and caspase-3 expression in SW1116 cells. In conclusion, USP39 may be a novel biological marker for targeted therapy against CRC, and requires further investigation.

Purpose To investigate the topologic architecture of white matter connectivity networks in preschool-aged children with a diagnosis of autism spectrum disorder (ASD) versus typical development (TD). Materials and Methods Forty-two participants were enrolled, including 21 preschool children with ASD (14 male children and seven female children; mean age, 4.56 years ± 0.97 [standard deviation]) and 21 children with TD (11 males and 10 females; mean age, 5.13 years ± 0.82). The diagnosis of ASD was determined according to the Diagnostic and Statistical Manual of Mental Disorders Global Assessment of Functioning scores (mean score, 8.00 ± 0.50). All participants underwent diffusion-tensor imaging (DTI) and T2-weighted imaging on a 3-T magnetic resonance system. A graph theoretical analysis was applied to investigate the topologic organization of the brain network including global and local topologic parameters. Statistical analysis was then performed for the comparison between the groups. Results Compared with the TD group, children with ASD demonstrated shortened characteristic path length (t

The L-plastin gene is involved in the invasion and metastasis of prostate cancer. However, the molecular mechanisms underlying L-plastin transcription are unclear. We hypothesize that the occurrence of polymorphic genetic variations in the L-plastin promoter might affect an individual's susceptibility to prostate cancer. In this study, we identified a single nucleotide polymorphism (SNP) at position -1,687 in the L-plastin promoter by genotype sequencing. The SNP -1,687 showed different transcriptional activity in the luciferase assay in vitro. The TRANSFAC software was applied to predict the multiple cis-elements, and luciferase assay was used to further identify the L-plastin regulatory region. We performed EMSAs, supershift assays and ChIP-qPCR demonstrated that the transcriptional suppressor NKX3.1 binds to the SNP site of the L-plastin promoter. SNP -1,687 (T/T) led to an increase in the affinity of NKX3.1 for L-plastin promoter, resulted in lower levels of L-plastin RNA and protein expression. Furthermore, we collected and sequenced samples from 640 individuals (372 prostate cancer patients and 268 healthy controls) from 2000 to 2013. The results showed that SNP -1,687 (T/T) occurred more frequently in the healthy individuals than that in the prostate cancer patients compared to SNP -1,687 (C/C). Similarly, SNP -1,687 (T/T) genotype occurred more frequently compared to SNP -1,687 (C/C) genotype in the patients with low and moderately differentiated tumors. In conclusion, SNP -1,687, located in the NKX3.1 binding site within the L-plastin promoter, might reduce the expression of L-plastin and potentially decrease the tumorigenesis and progression of prostate cancer. This SNP could be a potential prognostic factor for prostate cancer.

Air pollution has been classified as Group 1 carcinogenic to humans, but the underlying tumorigenesis remains unclear. In Xuanwei City of Yunnan Province, the lung cancer incidence is among the highest in China attributed to severe air pollution generated by combustion of smoky coal, providing a unique opportunity to dissect lung carcinogenesis of air pollution. Here we analyzed the somatic mutations of 164 non-small cell lung cancers (NSCLCs) from Xuanwei and control regions (CR) where smoky coal was not used. Whole genome sequencing revealed a mean of 289 somatic exonic mutations per tumor and the frequent C:G → A:T nucleotide substitutions in Xuanwei NSCLCs. Exome sequencing of 2010 genes showed that Xuanwei and CR NSCLCs had a mean of 68 and 22 mutated genes per tumor, respectively (p, Highlights • Somatic genomic mutations of air pollution-related lung cancer were characterized. • Mutations in air pollution-related lung cancers were 3 times as high as lung cancers from control regions. • 167 of the 2010 genes had higher mutation rates in patients from highly polluted region than those from control regions. • The mutation rates of 70 genes were associated with patients' lifetime benzo(a)pyrene exposure.

// Bo Wang 1, 2, * , Hao Liu 2, * , Xiaoliang Dong 1, * , Shaoxu Wu 2 , Hong Zeng 3 , Zhuowei Liu 4 , Di Wan 2 , Wen Dong 2 , Wang He 2 , Xu Chen 2 , Limin Zheng 5 , Jian Huang 2 , Tianxin Lin 1, 2 1 Guangdong Provincial Key Laboratory of Malignant Tumor Epigenetics and Gene Regulation, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, Guangzhou, PR China 2 Department of Urology, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen (Zhosngshan) University, Guangzhou, PR China 3 Department of Pathology, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen (Zhongshan) University, Guangzhou, PR China 4 Department of Urology, Cancer Center, Sun Yat-Sen (Zhongshan) University, Guangzhou, PR China 5 State Key Laboratory of Oncology in South China, Cancer Center, Sun Yat-Sen (Zhongshan) University, Guangzhou, PR China * These authors have contributed equally to this work Correspondence to: Tianxin Lin, e-mail: tianxinl@sina.com Jian Huang, e-mail: urolhj@sina.com Keywords: tumor-infiltrating macrophages, CD204, CD169, urothelial cell carcinoma of the bladder (UCB) Received: December 03, 2014 Accepted: April 25, 2015 Published: May 07, 2015 ABSTRACT Macrophages (Mφs) are a major cell type that can infiltrate solid tumors and exhibit distinct phenotypes in different tumor microenvironments. This study attempted to investigate the prognostic values of various tumor-infiltrating Mφ phenotypes in patients with urothelial cell carcinoma of the bladder (UCB), with a focus on Mφ tissue microlocalization. Mφs were assessed by immunohistochemistry in tissues from 302 UCB patients using CD68 as a pan-Mφ marker, and CD204 and CD169 as robust pro- and anti-tumoral Mφ phenotype markers, respectively. Our data showed that these Mφ phenotypes were predominately distributed in stromal (ST) rather than in intratumoral (INT) regions (all P < 0.0001). Surprisingly, CD204 and CD169 can be co-expressed by the same CD68 + Mφs. Kaplan-Meier analysis revealed that all INT- and ST-infiltrating CD204 + or CD169 + Mφ densities were inversely associated with overall survival (all P < 0.01). By multivariate analysis, ST-infiltrating CD204 + Mφ density emerged as an independent prognostic factor for overall survival (HR, 1.981; P = 0.022). Moreover, the density of ST-infiltrating CD204 + Mφs was positively associated with the tumor size ( P = 0.001), tumor stage ( P < 0.0001), nodal metastasis ( P < 0.0001), and histological grade ( P < 0.0001). Our findings suggest that CD204 + Mφs might play detrimental protumoral roles and represent the predominant Mφ phenotype in human bladder cancer.

Growing evidence indicates that dys-regulation of PBRM1 contributes to tumorigenesis. However, little is known about the biological function of PBRM1 in the development or progression of bladder cancer. In this study, we aimed to elucidate the pathophysiological role of PBRM1 in bladder cancer. We assessed the expression of PBRM1 in 64 bladder cancer tissue samples with matching normal tissues. We explored the biological functions of PBRM1 both in vitro and in vivo. Mutational status of PBRM1 was analyzed. Effect of PBRM1 on cell cycle was evaluated. qRT-PCR and Western blot were carried out to evaluate the expression of cyclins affected by PBRM1. Our results showed that PBRM1 expression was significantly reduced in bladder cancer cells and tissues compared to their normal counterparts. The reduced expression of PBRM1 was associated with advanced tumor stage, low differentiation grade and worse patient outcome. Further functional analysis demonstrated that PBRM1 suppressed bladder cancer cell proliferation, migration, colony formation in vitro and tumorigenicity in vivo. Genetic alteration analysis showed no amino-acid sequence altering mutations. We found that PBRM1 could block the G2/M transition by repressing cyclin B1. Our data indicated that PBRM1 functions as a tumor suppressor in bladder cancer by repressing cyclin B1 expression.

In the past few years, metabolic disorders, such as type 2 diabetes and metabolic syndrome, have reached global prevalence. Lycopene is one of the major carotenoids in tomatoes, watermelons, red grapefruits, and guava. In the current study, using high fat diet (HFD)-fed mice, we investigated the effect of Lycopene on insulin resistance. We showed that diet containing Lycopene significantly prevented HFD-induced increase of fasting blood glucose and insulin level, glucose and insulin intolerance, and decrease of hepatic glycogen content. We found that Lycopene notably prevented the increase of IL-1β, TNFα and CRP levels in mice fed HFD. We showed that Lycopene improved the lipid profiles in HFD-fed mice, as evidenced by decrease of systemic and hepatic TC, TG and LDL, and increase of HDL. Lycopene suppressed the increase of the expression of Srebp-1c, FAS and ACC-1 in mice fed HFD. The administration of Lycopene notably prevented the expression and phosphorylation of STAT3 in livers of mice induced by HFD. The treatment of adenovirus carrying STAT3 significantly suppressed the decrease of Srebp-1c expression induced by Lycopene. Furthermore, enhancement of STAT3 signaling by adenovirus markedly blocked the reduction of fasting blood glucose and insulin level. In conclusion, in the current study, we found that Lycopene prevented STAT3 signaling and inhibited Srebp-1c and downstream gene expression, resulting in inhibition of lipid accumulation, inflammation, insulin resistance and metabolic dysfunction. Overall, the data in the study provide better understanding of the beneficial effects of Lycopene against insulin resistance and metabolic disorder.

// Fengjin Zhao 1,* , Tianxin Lin 1,2,* , Wang He 1 , Jinli Han 1 , Dingjun Zhu 1 , Kaishun Hu 3 , Weicong Li 1 , Zaosong Zheng 1 , Jian Huang 1 and Wenlian Xie 1 1 Department of Urology, Sun Yat-sen Memorial Hospital, Sun Yat-sen University, Guangzhou, China 2 Lin Bai-xin Research Center, Sun Yat-sen Memorial Hospital, Sun Yat-sen University, Guangzhou, China 3 Guangdong Provincial Key Laboratory of Malignant Tumor Epigenetics and Gene Regulation, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, Guangzhou, China * These Authors contributed equally to this work Correspondence: Wenlian Xie, email: // Keywords : lincRNA, bladder cancer, proliferation, apoptosis Received : September 29, 2014 Accepted : November 24, 2014 Published : November 25, 2014 Abstract Long intergenic noncoding RNAs (lincRNAs) play important roles in regulating various biological processes in cancer, including proliferation and apoptosis. However, the roles of lincRNAs in bladder cancer remain elusive. In this study, we identified a novel lincRNA, which we termed AATBC. We found that AATBC was overexpressed in bladder cancer patient tissues and positively correlated with tumor grade and pT stage. We also found that inhibition of AATBC resulted in cell proliferation arrest through G1 cell cycle mediated by cyclin D1, CDK4, p18 and phosphorylated Rb. In addition, inhibition of AATBC induced cell apoptosis through the intrinsic apoptosis signaling pathway, as evidenced by the activation of caspase-9 and caspase-3. The investigation for the signaling pathway revealed that the apoptosis following AATBC knockdown was mediated by activation of phosphorylated JNK and suppression of NRF2. Furthermore, JNK inhibitor SP600125 could attenuate the apoptotic effect achieved by AATBC knockdown, confirming the involvement of JNK signaling in the induced apoptosis. Moreover, mouse xenograft model revealed that knockdown of AATBC led to suppress tumorigenesis in vivo . Taken together, our study indicated that AATBC might play a critical role in pro-proliferation and anti-apoptosis in bladder cancer by regulating cell cycle, intrinsic apoptosis signaling, JNK signaling and NRF2. AATBC could be a potential therapeutic target and molecular biomarker for bladder cancer.

Background The purpose of this analysis was to create a prognostic score model for newly diagnosed nasopharyngeal carcinoma (NPC) which has not been created before. Methods We retrospectively reviewed the medical records of 419 patients with newly diagnosed, nondisseminated, and biopsy proven NPC. Results Independent prognostic factors included age >45, stage III/IV, antienzyme rate of Epstein–Barr virus DNase-specific neutralizing antibody (AER) >58%, and absolute neutrophil count (ANC) >4.7 × 109/L. Four prognostic groups based on prognostic score model were obtained: low-risk group (total score = 0–2); low-intermediate-risk group (total score = 3–4); high-intermediate-risk group (total score = 5–6); and high-risk group (total score = 7). Conclusion Our proposed prognostic score model was a useful tool for improving the prognostic assessment of patients with NPC, stratifying patients into different risk groups that call for different treatments, and comparing clinical trials. © 2014 Wiley Periodicals, Inc. Head Neck 37: 624–629, 2015