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KEY MESSAGE: Unreduced megagametophytes via second-division restitution were confirmed through heterozygosity analysis, and four candidate physical centromeres of rubber were located for the first time. The evaluation of maternal heterozygosity restitution (MHR) is vital in identifying the mechanism of 2n gametogenesis and assessing the utilization value of 2n gametes. In this study, three full-sib triploid populations were employed to evaluate the MHR of 2n female gametes of rubber tree clone GT1 and to confirm their genetic derivation. The 2n female gametes of GT1 were derived from second-division restitution (SDR) and transmitted more than half of the parental heterozygosity. In addition, low recombination frequency markers were developed, and four candidate physical centromeres of rubber tree were located for the first time. The confirmation that 2n female gametes of rubber tree clone GT1 are derived from SDR provides insights into the molecular mechanisms of 2n gametogenesis. In addition, the identified centromere location will aid in the development of centromeric markers for the rapid identification of the 2n gametogenesis mechanism.

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BACKGROUND: Outbreaks of monkeypox have been ongoing in non-endemic countries since May 2022. A thorough assessment of its global zoonotic niche and potential transmission risk is lacking. METHODS: We established an integrated database on global monkeypox virus (MPXV) occurrence during 1958 - 2022. Phylogenetic analysis was performed to examine the evolution of MPXV and effective reproductive number (Rt) was estimated over time to examine the dynamic of MPXV transmissibility. The potential ecological drivers of zoonotic transmission and inter-regional transmission risks of MPXV were examined. RESULTS: As of 24 July 2022, a total of 49 432 human patients with MPXV infections have been reported in 78 countries. Based on 525 whole genome sequences, two main clades of MPXV were formed, of which Congo Basin clade has a higher transmissibility than West African clade before the 2022-monkeypox, estimated by the overall Rt (0.81 vs. 0.56), and the latter significantly increased in the recent decade. Rt of 2022-monkeypox varied from 1.14 to 4.24 among the 15 continuously epidemic countries outside Africa, with the top three as Peru (4.24, 95% CI: 2.89-6.71), Brazil (3.45, 95% CI: 1.62-7.00) and the United States (2.44, 95% CI: 1.62-3.60). The zoonotic niche of MPXV was associated with the distributions of Graphiurus lorraineus and Graphiurus crassicaudatus, the richness of Rodentia, and four ecoclimatic indicators. Besides endemic areas in Africa, more areas of South America, the Caribbean States, and Southeast and South Asia are ecologically suitable for the occurrence of MPXV once the virus has invaded. Most of Western Europe has a high-imported risk of monkeypox from Western Africa, whereas France and the United Kingdom have a potential imported risk of Congo Basin clade MPXV from Central Africa. Eleven of the top 15 countries with a high risk of MPXV importation from the main countries of 2022-monkeypox outbreaks are located at Europe with the highest risk in Italy, Ireland and Poland. CONCLUSIONS: The suitable ecological niche for MPXV is not limited to Africa, and the transmissibility of MPXV was significantly increased during the 2022-monkeypox outbreaks. The imported risk is higher in Europe, both from endemic areas and currently epidemic countries. Future surveillance and targeted intervention programs are needed in its high-risk areas informed by updated prediction.

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INTRODUCTION AND OBJECTIVES: Hepatitis B surface antigen (HBsAg) clearance, indicating functional cure or resolved chronic hepatitis B (CHB), remains difficult to achieve via nucleos(t)ide analogue monotherapy. We investigated whether metformin add-on therapy could help achieve this goal in entecavir-treated patients with hepatitis B e antigen (HBeAg)-negative CHB. PATIENTS AND METHODS: Patients with HBeAg-negative CHB who met eligibility criteria (entecavir treatment for > 12 months, HBsAg < 1000 IU/mL) were randomly assigned (1:1) to receive 24 weeks of either metformin (1000 mg, oral, once a day) or placebo (oral, once a day) add-on therapy. The group allocation was blinded for both patients and investigators. Efficacy and safety analyses were based on the intention-to-treat set. The primary outcome, serum HBsAg level (IU/mL) at weeks 24 and 36, was analysed using mixed models. RESULTS: Sixty eligible patients were randomly assigned to the metformin (n = 29) and placebo (n = 31) groups. There was no substantial between-group difference in the HBsAg level at week 24 (adjusted mean difference 0.05, 95% confidence interval -0.04 to 0.13, p = 0.278) or week 36 (0.06, -0.03 to 0.15, p = 0.187), and no significant effect of group-by-time interaction on the HBsAg level throughout the trial (p = 0.814). The occurrence of total adverse events between the two groups was comparable (9 [31.0%] of 29 vs. 5 [16.1%] of 31, p = 0.227) and no patient experienced serious adverse events during the study. CONCLUSION: Although it was safe, metformin add-on therapy did not accelerate HBsAg clearance in entecavir-treated patients with HBeAg-negative CHB.

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SUMMARY: The information technology (IT) based "instant evaluation" is supported by IT, which allows instant evaluation of teaching phenomena based on certain evaluation criteria and provides instant feedback. In anatomy teaching, we explored and practiced the application of instant evaluation based on a platform called "Rain classroom." We found that IT-based instant evaluation had higher practicability and better student satisfaction, which could improve teaching efficiency during class time, help students improve learning methods, and promote knowledge mastery. Additionally, instant evaluation positively impacted teachers' evaluation ability and teaching skills.

RESUMEN: La "evaluación instantánea" basada en la tecnología de la información (TI) está respaldada por ésta y permite la evaluación instantánea de los fenómenos de enseñanza en función de ciertos criterios de evaluación proporcionando retroalimentación instantánea. En la enseñanza de la anatomía, exploramos y practicamos la aplicación de la evaluación instantánea basada en una plataforma llamada "Aula de lluvia" o Rain Classroom. Descubrimos que la evaluación instantánea basada en TI tenía una mayor practicidad y una mejor satisfacción de los estudiantes, lo que podría mejorar la eficiencia de la enseñanza durante el tiempo de clase, ayudar a los estudiantes a mejorar los métodos de aprendizaje y promover el dominio del conocimiento. Además, la evaluación instantánea tuvo un impacto positivo en la capacidad de evaluación y las habilidades de enseñanza de los maestros.

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Abstract Background: Phospholipase C-like 1 (PLCL1), a protein that lacks catalytic activity, has similar structures to the PLC family. The aim of this research was to find the function and underlying mechanisms of PLCL1 in fibroblast-like synoviocyte (FLS) of rheumatoid arthritis (RA). Methods: In this study, we first analyzed the expression of PLCL1 in the synovial tissue of RA patients and K/BxN mice by immunohistochemical staining. Then silencing or overexpressing PLCL1 in FLS before stimulating by TNF-α. The levels of IL-6, IL-1β and CXCL8 in FLS and supernatants were detected by Western Blot (WB), Real-Time Quantitative PCR and Enzyme Linked Immunosorbent Assay. We used INF39 to specifically inhibit the activation of NLRP3 inflammasomes, and detected the expression of NLRP3, Cleaved Caspase-1, IL-6 and IL-1β in FLS by WB. Result: When PLCL1 was silenced, the level of IL-6, IL-1β and CXCL8 were down-regulated. When PLCL1 was overexpressed, the level of IL-6, IL-1β and CXCL8 were unregulated. The previous results demonstrated that the mechanism of PLCL1 regulating inflammation in FLS was related to NLRP3 inflammasomes. INF39 could counteract the release of inflammatory cytokines caused by overexpression of PLCL1. Conclusion: Result showed that the function of PLCL1 in RA FLS might be related to the NLRP3 inflammasomes. We finally confirmed our hypothesis with the NLRP3 inhibitor INF39. Our results suggested that PLCL1 might promote the inflammatory response of RA FLS by regulating the NLRP3 inflammasomes.

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It has been extensively reported that long noncoding RNAs (lncRNAs) were closely associated with multiple malignancies. The aim of our study was to investigate the effects and mechanism of lncRNA POU6F2-AS1 in lung adenocarcinoma (LADC).The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) datasets provided us the information of LADC clinical samples. High-regulation of POU6F2-AS1 was presented in LADC tissues compared with adjacent normal tissues, which was correlated with poor outcome of LADC patients. Functional experiments in Calu-3 and NCI-H460 cells showed that POU6F2-AS1 significantly promoted LADC cell proliferation, colony formation, invasion and migration. Moreover, through online prediction, luciferase reporter assay and Pearson's correlation analysis, we found that POU6F2-AS1 may act as a competing endogenous RNA (ceRNA) of miR-34c-5p and facilitated the expression of potassium voltage-gated channel subfamily J member 4 (KCNJ4). The promoting effect of cell aggressiveness induced by POU6F2-AS1 was enhanced by KCNJ4, whilst was abrogated due to the overexpression of miR-34c-5p. Collectively, POU6F2-AS1 might function as a ceRNA through sponging miR-34c-5p to high-regulate KCNJ4 in LADC, which indicates that POU6F2-AS1 might be a promising therapeutic target with significant prognostic value for LADC treatment.

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NADPH oxidases are enzymes that have been reported to generate reactive oxygen species (ROS) in animals, plants and many multicellular fungi in response to environmental stresses. Six genes of the NADPH oxidase complex components, including vvnoxa, vvnoxb, vvnoxr, vvbema, vvrac1 and vvcdc24, were identified based on the complete genomic sequence of the edible fungus Volvariella volvacea. The number of vvnoxa, vvrac1, vvbema and vvcdc24 transcripts fluctuated with ageing, and the gene expression patterns of vvnoxa, vvrac1 and vvbema were significantly positively correlated. However, the expression of vvnoxb and vvnoxr showed no significant difference during ageing. In hyphae subjected to mechanical injury stress, both O2- and H2O2 concentrations were increased. The expression of vvnoxa, vvrac1, vvbema and vvcdc24 was substantially upregulated, but vvnoxb and vvnoxr showed no response to mechanical injury stress at the transcriptional level. Additionally, the transcription of vvnoxa, vvrac1, vvbema and vvcdc24 could be repressed when the intracellular ROS were eliminated by diphenyleneiodonium (DPI) chloride and reduced glutathione (GSH) treatments. These results indicated a positive feedback loop involving NADPH oxidase and intracellular ROS, which might be the reason for the oxidative burst during injury stress.

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Abstract The pericarp of Trapa natans L., an annual aquatic floating herb belonging to Lythraceae family, is used as a folk medicine in China. In this study, extracts of Trapa natans pericarp were tested both in vitro and in vivo through a high-fat diet with a single medium dosage streptozotocin injection induced type 2 diabetic mice. Different solvent extracts of Trapa natans pericarp showed α-amylase and α-glucosidase inhibitory activity. After four weeks administration, the ethyl acetate extract of Trapa natans pericarp (50 and 100 mg/kg b.w.) reduced fasting blood glucose level, ameliorated oral glucose tolerance and insulin resistance, improved serum lipids alterations in type 2 diabetic mice as well. Additionally, ethyl acetate extract significantly elevated the insulin receptor substrate 1 and Akt serine/threonine kinase phosphorylation compared to diabetic group. HPLC-MS and HPLC-DAD analysis showed that the ethyl acetate extract was rich in hydrolysable tannins. Results support the notion that Trapa natans pericarp extract has a potential hypoglycemic activity.

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BACKGROUND: This study aims to investigate the expression of Id-1 in human colorectal adenocarcinoma tissues and explore its correlation with the clinical pathological parameters of colorectal cancer. METHODS: The Id-1 mRNA and protein expression levels of 50 specimens of normal colorectal tissues and 50 specimens of colorectal adenocarcinoma tissues were detected using reverse-transcription polymerase chain reaction and western blot. Furthermore, Id-1 protein was detected using immunohistochemistry. The correlation between the expression of Id-1 and clinicopathologic features was analyzed. RESULTS: The mRNA expression level of Id-1 in colorectal adenocarcinoma tissues and normal colorectal tissues was 0.96 ± 0.03 vs. 0.20 ± 0.04, respectively; and the difference was statistically significant (P=0.011). Furthermore, Id-1 protein expression was higher in colorectal adenocarcinoma tissues than in normal colorectal tissues (0.82 ± 0.04 vs. 0.31 ± 0.02, P=0.020). In addition, the positive protein expression rate of Id-1 was higher in colorectal adenocarcinoma tissues than in normal colorectal tissues (72.00% vs. 24.00%, X2=23.431, P=0.000). The expression of Id-1 was correlated with the depth of tumor invasion, TNM stage, lymph node metastasis, vessel invasion, and liver metastasis (P<0.01). However, this expression was not correlated with tumor size and differentiation degrees (P>0.05). CONCLUSIONS: The high Id-1 expression in colorectal adenocarcinoma tissues play an important role in the process of cancer, and is expected to become a new tumor monitoring indicator for clinical diagnosis, treatment, and prognosis judgment.

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SUMMARY BACKGROUND: This study aims to investigate the expression of Id-1 in human colorectal adenocarcinoma tissues and explore its correlation with the clinical pathological parameters of colorectal cancer. METHODS: The Id-1 mRNA and protein expression levels of 50 specimens of normal colorectal tissues and 50 specimens of colorectal adenocarcinoma tissues were detected using reverse-transcription polymerase chain reaction and western blot. Furthermore, Id-1 protein was detected using immunohistochemistry. The correlation between the expression of Id-1 and clinicopathologic features was analyzed. RESULTS: The mRNA expression level of Id-1 in colorectal adenocarcinoma tissues and normal colorectal tissues was 0.96 ± 0.03 vs. 0.20 ± 0.04, respectively; and the difference was statistically significant (P=0.011). Furthermore, Id-1 protein expression was higher in colorectal adenocarcinoma tissues than in normal colorectal tissues (0.82 ± 0.04 vs. 0.31 ± 0.02, P=0.020). In addition, the positive protein expression rate of Id-1 was higher in colorectal adenocarcinoma tissues than in normal colorectal tissues (72.00% vs. 24.00%, X2=23.431, P=0.000). The expression of Id-1 was correlated with the depth of tumor invasion, TNM stage, lymph node metastasis, vessel invasion, and liver metastasis (P<0.01). However, this expression was not correlated with tumor size and differentiation degrees (P>0.05). CONCLUSIONS: The high Id-1 expression in colorectal adenocarcinoma tissues play an important role in the process of cancer, and is expected to become a new tumor monitoring indicator for clinical diagnosis, treatment, and prognosis judgment.

RESUMO OBJETIVO: O objetivo deste estudo é investigar a expressão de Id-1 em tecidos de adenocarcinoma colorretal em humanos e investigar sua correlação com os parâmetros patológicos clínicos de câncer colorretal. MÉTODOS: Os níveis de expressão de proteína e mRNA Id-1 em 50 amostras de tecido colorretal normal e 50 amostras de tecido de adenocarcinoma colorretal foram detectados através de reação em cadeia de polimerase precedida de transcrição reversa e western blot. Além disso, a proteína Id-1 foi detectada através de imuno-histoquímica. A correlação entre a expressão de Id-1 e características clínico-patológicas foi analisada. RESULTADOS: O nível de expressão de mRNA Id-1 em tecidos de adenocarcinoma colorretal e tecidos colorretais normais foi de 0,96 ± 0,03 versus 0,20 ± 0,04, respectivamente; a diferença foi estatisticamente significativa (P= 0,011). Além disso, a expressão da proteína Id-1 foi maior em tecidos de adenocarcinoma colorretal do que em tecidos colorretais normais (0,82 ± 0,04 versus 0,31 ± 0,02, P= 0,020). Além disso, a taxa de expressão positiva de proteínas Id-1 foi maior em tecidos de adenocarcinoma colorretal do que em tecidos colorretais normais (72,00% vs. 24,00%, X2=23,431, p=0,000). A expressão de Id-1 foi correlacionada com a profundidade da invasão tumoral, estágio TNM, metástases linfonodais, invasão vascular e metástase hepática (P<0,01). Todavia, essa expressão não se correlacionou com o tamanho do tumor e graus de diferenciação (P>0,05). CONCLUSÃO: A alta expressão de Id-1 em tecidos de adenocarcinoma colorretal desempenham um importante papel no processo do câncer, e é esperado que se torne um novo indicador de monitoramento de tumores para o diagnóstico clínico, tratamento e estimativa de prognóstico.

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ABSTRACT Folium Eriobotryae, the dried leaves of loquat (Eriobotrya japonica, (Thunb.) Lindl., Rosaceae), is a traditional Chinese medicine used to treat cough with phlegm in China. Fallen and growing loquat leaves were tested for their effect on coughing and expectoration in mice. HPLC-ELSD and HPLC-MS analyses of aqueous and ethanol extracts of fallen or growing leaves were used to identify the chemical components responsible for this effect. Both the aqueous and ethanol extracts of growing and fallen leaves of loquat contained antitussive and expectorant activities. Moreover, an aqueous extract of growing loquat leaves with a higher flavonoid content displayed a stronger expectorant activity while the ethanol extract of fallen loquat leaves that contained a higher content of triterpenoid acids induced a stronger antitussive activity.

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Background: D-Hydroxyphenylglycine is considered to be an important chiral molecular building-block of antibiotic reagents such as pesticides, and β-lactam antibiotics. The process of its production is catalyzed by D-hydantoinase and D-carbamoylase in a two-step enzyme reaction. How to enhance the catalytic potential of the two enzymes is valuable for industrial application. In this investigation, an Escherichia coli strain genetically engineered with D-hydantoinase was immobilized by calcium alginate with certain adjuncts to evaluate the optimal condition for the biosynthesis of D-carbamoyl-p-hydroxyphenylglycine (D-CpHPG), the compound further be converted to D-hydroxyphenylglycine (D-HPG) by carbamoylase. Results: The optimal medium to produce D-CpHPG by whole-cell immobilization was a modified Luria-Bertani (LB) added with 3.0% (W/V) alginate, 1.5% (W/V) diatomite, 0.05% (W/V) CaCl2 and 1.00 mM MnCl2.The optimized diameter of immobilized beads for the whole-cell biosynthesis here was 2.60 mm. The maximized production rates of D-CpHPG were up to 76%, and the immobilized beads could be reused for 12 batches. Conclusions: This investigation not only provides an effective procedure for biological production of D-CpHPG, but gives an insight into the whole-cell immobilization technology.

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UNLABELLED:  Background. Acute-on-chronic liver failure has high mortality. Currently, robust models for predicting the outcome of hepatitis B virus (HBV)-associated ACLF are lacking. AIM: To assess and compare the performance of six prevalent models for short- and longterm prognosis in patients with HBV-ACLF. MATERIAL AND METHODS: The model for end-stage liver disease (MELD), MELD sodium (MELD-Na), MELD to sodium ratio (MESO), integrated MELD, Child-Turcotte-Pugh (CTP), and modified CTP (mCTP) were validated in a prospective cohort of 232 HBV-ACLF patients. The six models were evaluated by determining discrimination, calibration and overall performance at 3 months and 5 years. RESULTS: According to the Hosmer-Lemeshow tests and calibration plots, all models could adequately describe the data except CTP at 3 months. Discrimination analysis showed that the iMELD score had the highest AUC of 0.76 with sensitivity of 62.6% and specificity of 80.2% for an optimal cut-off value of 52 at 3 months. It also had the highest AUC of 0.80 with sensitivity of 89.9% and specificity of 48.2% for an optimal cut-off value of 43 at 5 years. The overall performance of iMELD, assessed with Nagelkerke's R2 and the Brier score, was also the best among the six models. CONCLUSION: Integrated MELD may be the best model to predict short- and long-term prognosis in patients with HBV-ACLF.

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OBJECTIVE: To investigate the association between bone morphogenetic protein 4 (BMP4) rs17563 polymorphism and nonsyndromic cleft lip with or without palate (NSCL/P) risk. METHODS: Four online databases were researched and the related publications were collected. Odds ratio (OR) with 95% confidence interval (CI) was applied to assess the relationship; publication bias, metaregression, and sensitivity analysis were conducted to guarantee the strength of results. RESULTS: Six published case-control studies were collected. Overall, no significant association between BMP4 rs17563 polymorphism and NSCL/P risk was found. It was notable that significant susceptibility on different ethnicity was observed in the stratified analysis. For Chinese population, the BMP4 rs17563 polymorphism was a significantly increased risk for NSCL/P (C versus T: OR = 1.52, 95% CI = 1.28-1.82, P < 0.01, I (2) = 0%; CC versus TT: OR = 2.58, 95% CI = 1.74-3.82, P < 0.01, I (2) = 0%; TC + CC versus TT: OR = 1.45, 95% CI = 1.14-1.84, P < 0.01, I (2) = 0%; CC versus TT + TC: OR=2.46, 95% CI = 1.46-4.14, P < 0.01, I(2) = 47.0%). On the contrary, significantly protective effects were found in Brazilian population (C versus T: OR = 0.69, 95% CI = 0.50-0.96, P = 0.03, I(2) = 68.5%; TC versus TT: OR = 0.52, 95% CI = 0.40-0.68, P < 0.01, I(2) = 0%; TC + CC versus TT: OR = 0.52, 95% CI = 0.35-0.78, P < 0.010, I(2) = 54.4%). CONCLUSION: This meta-analysis indicated that BMP4 rs17563 polymorphism could play a different role during the development of NSCL/P based on ethnicity diversity.

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BACKGROUND: International surveys have demonstrated that use of traditional and complementary/alternative medicine (TCAM) is highly prevalent among children with cancer; however, little is known about its use among children with cancer in Latin America. As part of a regional initiative, we present the results of the first survey exploring use of TCAM among children with cancer residing in Latin America. PROCEDURE: A cross-sectional sample of 100 parents whose children received treatment in Guatemala City were interviewed from May 2008 to February 2010. Upon consent, an in-person interview was performed during a routine clinical visit. Information on the form, duration, cost, and satisfaction of TCAM was collected. Approval from the institutional review board was obtained. RESULTS: The median age of patients was 9.2 years with 63% undergoing treatment. Use of TCAM was reported by 90% of parents. Most often, more than one type of therapy was utilized. Most patients (67%) relied on TCAM for supportive care indications; however, a significant percentage (34%) used TCAM for curative purposes. The most commonly reported form was the use of oral supplements with the majority perceiving TCAM as effective for the intended purpose. CONCLUSIONS: Use of TCAM was highly prevalent among children with cancer residing in Guatemala. Most importantly, TCAM was used alongside conventional therapy for supportive care indications. These findings underscoring the need for open lines of communication between clinicians and families. Future research may consider exploring the role of TCAM within the scope of supportive care and its effect on existing supportive care interventions.

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Heliconema hainanensis sp. nov. collected from Uroconger lepturus (Richardson) (Anguilliformes: Congridae), Muraenesox cinereus (Forsskål) and Congresox talabonoides (Bleeker) (Anguilliformes: Muraenesocidae) in the South China Sea was described using light and scanning electron microscopy. The new species differs from its congeners by the following morphology: pseudolabia, the number and arrangement of caudal papillae (4 pairs of pedunculate precloacal papillae arranged in 2 groups of 2 and 2 pairs and 6 pairs of pedunculate postcloacal papillae arranged in 4 groups of 1, 2, 1 and 2 pairs), the length of spicules [left spicule 0.51-0.69 mm, right spicule 0.20-0.27 mm, spicule (right:left) ratio 1:2.20-2.69] and the morphology of the female tail tip. In addition, specimens of the new species collected from the three different hosts and specimens of an unidentified species of Heliconema collected from U. lepturus were characterised using molecular methods by sequencing the internal transcribed spacer (ITS) of ribosomal DNA. Analyses and comparison of the ITS sequence of H. hainanensis sp. nov. with Heliconema sp. support the validity of the new species based on morphological observations. An identification key to the species of Heliconema is also provided.

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Heliconema hainanensis sp. nov. collected from Uroconger lepturus (Richardson) (Anguilliformes: Congridae), Muraenesox cinereus (Forsskål) and Congresox talabonoides (Bleeker) (Anguilliformes: Muraenesocidae) in the South China Sea was described using light and scanning electron microscopy. The new species differs from its congeners by the following morphology: pseudolabia, the number and arrangement of caudal papillae (4 pairs of pedunculate precloacal papillae arranged in 2 groups of 2 and 2 pairs and 6 pairs of pedunculate postcloacal papillae arranged in 4 groups of 1, 2, 1 and 2 pairs), the length of spicules [left spicule 0.51-0.69 mm, right spicule 0.20-0.27 mm, spicule (right:left) ratio 1:2.20-2.69] and the morphology of the female tail tip. In addition, specimens of the new species collected from the three different hosts and specimens of an unidentified species of Heliconema collected from U. lepturus were characterised using molecular methods by sequencing the internal transcribed spacer (ITS) of ribosomal DNA. Analyses and comparison of the ITS sequence of H. hainanensis sp. nov. with Heliconema sp. support the validity of the new species based on morphological observations. An identification key to the species of Heliconema is also provided.

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Our group established a method to culture spheres under serum-free culture condition. However, the biological characteristics and the tumorigenicity of spheres are unknown. Here, we demonstrate that sphere cells expressed high levels of the putative colorectal cancer stem cell markers CD133 and CD44. The CD133-positive rates were 13.27 ± 5.62, 52.71 ± 16.97 and 16.47 ± 2.45 percent in sphere cells, regular Colo205 cells and differentiated sphere cells, respectively, while the CD44-positive rates were 62.92 ± 8.38, 79.06 ± 12.10 and 47.80 ± 2.5 percent, respectively, and the CD133/CD44-double-positive rates were 10.77 ± 4.96, 46.89 ± 19.17 and 12.41 ± 2.27 percent, respectively (P < 0.05). Cancer sphere cells formed crypt-like structures in 3-D culture. Moreover, cells from cancer spheres exhibited more tumorigenicity than regular Colo205 cells in a xenograft assay. The cancer sphere cells displayed much higher oncogenicity than regular Colo205 cells to initiate neoplasms, as assayed by H&E staining, Musashi-1 staining and electron microscopy. Our findings indicated that the sphere cells were enriched with cancer stem cells (CSCs), and exhibited more proliferation capacity, more differentiation potential and especially more tumorigenicity than regular Colo205 cells in vitro and in vivo. Further isolation and characterization of these CSCs may provide new insights for novel therapeutic targets and prognostic markers.

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Our group established a method to culture spheres under serum-free culture condition. However, the biological characteristics and the tumorigenicity of spheres are unknown. Here, we demonstrate that sphere cells expressed high levels of the putative colorectal cancer stem cell markers CD133 and CD44. The CD133-positive rates were 13.27 ± 5.62, 52.71 ± 16.97 and 16.47 ± 2.45% in sphere cells, regular Colo205 cells and differentiated sphere cells, respectively, while the CD44-positive rates were 62.92 ± 8.38, 79.06 ± 12.10 and 47.80 ± 2.5%, respectively, and the CD133/CD44-double-positive rates were 10.77 ± 4.96, 46.89 ± 19.17 and 12.41 ± 2.27%, respectively (P < 0.05). Cancer sphere cells formed crypt-like structures in 3-D culture. Moreover, cells from cancer spheres exhibited more tumorigenicity than regular Colo205 cells in a xenograft assay. The cancer sphere cells displayed much higher oncogenicity than regular Colo205 cells to initiate neoplasms, as assayed by H&E staining, Musashi-1 staining and electron microscopy. Our findings indicated that the sphere cells were enriched with cancer stem cells (CSCs), and exhibited more proliferation capacity, more differentiation potential and especially more tumorigenicity than regular Colo205 cells in vitro and in vivo. Further isolation and characterization of these CSCs may provide new insights for novel therapeutic targets and prognostic markers.

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