Development and validation of a random survival forest model for predicting long-term survival of early-stage young breast cancer patients based on the SEER database and an external validation cohort.

Young breast cancer (YBC) patients often face a poor prognosis, hence it's necessary to construct a model that can accurately predict their long-term survival in early stage. To realize this goal, we utilized data from the Surveillance, Epidemiology, and End Results (SEER) databases between January 2010 and December 2020, and meanwhile, enrolled an independent external cohort from Tianjin Medical University Cancer Institute and Hospital. The study aimed to develop and validate a prediction model constructed using the Random Survival Forest (RSF) machine learning algorithm. By applying the Least Absolute Shrinkage and Selection Operator (LASSO) regression analysis, we pinpointed key prognostic factors for YBC patients, which were used to create a prediction model capable of forecasting the 3-year, 5-year, 7-year, and 10-year survival rates of YBC patients. The RSF model constructed in the study demonstrated exceptional performance, achieving C-index values of 0.920 in the training set, 0.789 in the internal validation set, and 0.701 in the external validation set, outperforming the Cox regression model. The model's calibration was confirmed by Brier scores at various time points, showcasing its excellent accuracy in prediction. Decision curve analysis (DCA) underscored the model's importance in clinical application, and the Shapley Additive Explanations (SHAP) plots highlighted the importance of key variables. The RSF model also proved valuable in risk stratification, which has effectively categorized patients based on their survival risks. In summary, this study has constructed a well-performed prediction model for the evaluation of prognostic factors influencing the long-term survival of early-stage YBC patients, which is significant in risk stratification when physicians handle YBC patients in clinical settings.

Four lathyrane diterpenoids from the seeds of Euphorbia lathyris.

Four new diterpenoids, including three secolathyrane diterpenoids (1-3) and one lathyrane diterpenoid (4), together with seven known diterpenoids, were obtained in the shelled seeds of Euphorbia lathyris. In particular, 1-3 possess a rare split ring structure, and currently only one compound with the same skeleton has been identified in E. lathyris. Compound 4 furnishes an unprecedented oxygen bridge structure. The structures were identified using various spectral techniques, including NMR, HR-ESI-MS, single-crystal X-ray diffraction and calculated electronic circular dichroism (ECD). The biosynthetic pathway of 1-4 was inferred. Furthermore, the cytotoxic activities of all compounds (1-11) were measured on three human tumor cells. New compounds 2 and 3 showed moderate cytotoxic activities against U937 cells with IC50 values of 22.18 and 25.41 µM, respectively.

Mitogenomic phylogenies suggest the resurrection of the subfamily Porrocaecinae and provide insights into the systematics of the superfamily Ascaridoidea (Nematoda: Ascaridomorpha), with the description of a new species of Porrocaecum.

BACKGROUND: The family Toxocaridae is a group of zooparasitic nematodes of veterinary, medical and economic significance. However, the evolutionary relationship of Porrocaecum and Toxocara, both genera currently classified in Toxocaridae, and the monophyly of the Toxocaridae remain under debate. Moreover, the validity of the subgenus Laymanicaecum in the genus Porrocaecum is open to question. Due to the scarcity of an available genetic database, molecular identification of Porrocaecum nematodes is still in its infancy. METHODS: A number of Porrocaecum nematodes collected from the Eurasian marsh harrier Circus aeruginosus (Linnaeus) (Falconiformes: Accipitridae) in the Czech Republic were identified using integrated morphological methods (light and scanning electron microscopy) and molecular techniques (sequencing and analyzing the nuclear 18S, 28S and ITS regions). The complete mitochondrial genomes of the collected nematode specimens and of Porrocaecum (Laymanicaecum) reticulatum (Linstow, 1899) were sequenced and annotated for the first time. Phylogenetic analyses of ascaridoid nematodes based on the amino acid sequences of 12 protein-coding genes of mitochondrial genomes were performed using maximum likelihood and Bayesian inference. RESULTS: A new species of Porrocaecum, named P. moraveci n. sp., is described based on the morphological and genetic evidence. The mitogenomes of P. moraveci n. sp. and P. reticulatum both contain 36 genes and are 14,517 and 14,210 bp in length, respectively. Comparative mitogenomics revealed that P. moraveci n. sp. represents the first known species with three non-coding regions and that P. reticulatum has the lowest overall A + T content in the mitogenomes of ascaridoid nematodes tested to date. Phylogenetic analyses showed the representatives of Toxocara clustered together with species of the family Ascarididae rather than with Porrocaecum and that P. moraveci n. sp. is a sister to P. reticulatum. CONCLUSIONS: The characterization of the complete mitochondrial genomes of P. moraveci n. sp. and P. reticulatum is reported for the first time. Mitogenomic phylogeny analyses indicated that the family Toxocaridae is non-monophyletic and that the genera Porrocaecum and Toxocara do not have an affinity. The validity of the subgenus Laymanicaecum in Porrocaecum was also rejected. Our results suggest that: (i) Toxocaridae should be degraded to a subfamily of the Ascarididae that includes only the genus Toxocara; and (ii) the subfamily Porrocaecinae should be resurrected to include only the genus Porrocaecum. The present study enriches the database of ascaridoid mitogenomes and provides a new insight into the systematics of the superfamily Ascaridoidea.

Effect of Premolar Extraction on the Upper Airway in Adult and Adolescent Orthodontic Patients: a Meta-analysis.

OBJECTIVE: To analyse the effects of premolar extraction on the upper airway in adult and adolescent orthodontic patients using CBCT. METHODS: The Embase, Web of Science, Cochrane Library and Medline (via PubMed) databases were searched with no language restrictions. Longitudinal studies in which CBCT was applied to assess the effects of tooth extraction on the upper airway were included in the analysis. Two authors performed the study selection, methodological quality assessment, data extraction and data synthesis independently. RESULTS: A total of 12 studies were included, six of which were eligible for quantitative synthesis. In the adult group, the nasopharynx and oropharynx volume showed no significant change, and the minimum cross-sectional area of the upper airway demonstrated a non-significant decrease compared to the non-extraction group. In the adolescent group, the nasopharynx volume, oropharynx volume and minimum cross-sectional area of the upper airway increased in a non-significant manner. CONCLUSION: The currently available evidence indicates that tooth extraction does not increase the risk of airway collapse in adult and adolescent patients. The present findings should be interpreted with caution and evaluated in further high-quality studies.

Molecular identification of a new species of Rhigonema (Nematoda: Rhigonematidae) and phylogenetic relationships within the infraorder Rhigonematomorpha.

BACKGROUND: The infraorder Rhigonematomorpha comprises a group of obligate parasitic nematodes of millipedes (Arthropoda: Diplopoda). The current species identification of Rhigonematomorpha nematodes remains mainly based on morphological features, with molecular-based identification still in its infancy. Also, current knowledge of the phylogeny of Rhigonematomorpha is far from comprehensive. METHODS: The morphology of Rhigonematomorpha nematodes belonging to the genus Rhigonema, collected from the millipede Spirobolus bungii Brandt (Diplopoda: Spirobolida) in China, was studied in detail using light and scanning electron microscopy. Five different genetic markers, including the nuclear small ribosomal subunit (18S), internal transcribed spacer (ITS) and large ribosomal subunit (28S) regions and the mitochondrial cox1 and cox2 genes of these Rhigonematomorpha nematodes collected from China and Rhigonema naylae collected from Japan were sequenced and analyzed using Bayesian inference (BI) and Assemble Species by Automatic Partitioning (ASAP) methods. Phylogenetic analyses that included the most comprehensive taxa sampling of Rhigonematomorpha to date were also performed based on the 18S + 28S genes using maximum likelihood (ML) and BI methods. RESULTS: The specimens of Rhigonema collected from S. bungii in China were identified as a new species, Rhigonema sinense n. sp. Striking variability in tail morphology was observed among individuals of R. sinense n. sp. ASAP analyses based on the 28S, ITS, cox1 and cox2 sequences supported the species partition of R. sinense n. sp. and R. naylae, but showed no evidence that the different morphotypes of R. sinense n. sp. represent distinct genetic lineages. BI analyses also indicated that R. sinense n. sp. represents a separated species from R. naylae based on the cox1 and cox2 genes, but showed that R. naylae nested in samples of R. sinense n. sp. based on the ITS and 28S data. Phylogenetic results showed that the representatives of Rhigonematomorpha formed two large clades. The monophyly of the families Carnoyidae and Ichthyocephalidae and the genus Rhigonema was rejected. The representatives of the family Ransomnematidae clustered together with the family Hethidae with strong support. CONCLUSIONS: A new species of Rhigonematomorpha, R. sinense n. sp. is described based on morphological and molecular evidence. ASAP analyses using 28S, ITS, cox1 and cox2 data indicate the striking variability in tail morphology of R. sinense n. sp. as intraspecific variation, and also suggest that partial 28S, ITS, cox1 and cox2 markers are effective for molecular identification of Rhigonematomorpha nematodes. The phylogenetic results support the traditional classification of Rhigonematomorpha into the two superfamilies Rhigonematoidea and Ransomnematoidea, and indicate that the families Carnoyidae and Ichthyocephalidae and the genus Rhigonema are non-monophyletic. The present phylogeny strongly supports resurrection of the family Brumptaemiliidae, and also indicates that the family Ransomnematidae is sister to the family Hethidae.

The protective effort of GPCR kinase 2-interacting protein-1 in neurons via promoting Beclin1-Parkin induced mitophagy at the early stage of spinal cord ischemia-reperfusion injury.

In spinal cord ischemia-reperfusion (I/R) injury, large amounts of reactive oxygen species can cause mitochondrial damage. Therefore, mitophagy acts as the main mechanism for removing damaged mitochondria and protects nerve cells. This study aimed to illustrate the important role of GPCR kinase 2-interacting protein-1 (GIT1) in mitophagy in vivo and in vitro. The level of mitophagy in the neurons of Git1 knockout mice was significantly reduced after ischemia-reperfusion. However, the overexpression of adeno-associated virus with Git1 promoted mitophagy and inhibited the apoptosis of neurons. GIT1 regulated the phosphorylation of Beclin-1 in Thr119, which could promote the translocation of Parkin to the mitochondrial outer membrane. This process was independent of PTEN-induced kinase 1 (PINK1), but it could not rescue the role in the absence of PINK1. Overall, GIT1 enhanced mitophagy and protected neurons against ischemia-reperfusion injury and, hence, might serve as a new research site for the protection of ischemia-reperfusion injury.

Morphological and Genetic Characterization of the Poorly Known Species Subulura chinensis Schwartz, 1926 (Nematoda: Ascaridida) from Athene noctua (Scopoli) (Strigiformes: Strigidae).

INTRODUCTION: Subulura chinensis Schwartz, 1926 is a hitherto poorly known nematode species. The morphology of S. chinensis has not been sufficiently well described. In addition, the molecular data from species of the Subuluroidea are extremely limited. METHODS: The detailed morphology of S. chinensis was studied using light microscopy and, for the first time, scanning electron microscopy, based on newly collected specimens from the little owl Athene noctua (Scopoli) (Strigiformes: Strigidae) in China. The internal transcribed spacer (ITS) of ribosomal DNA and mitochondrial cytochrome c oxidase subunit 1 (cox1) target regions of S. chinensis were first amplified by polymerase chain reaction (PCR), then sequenced and analysed for the molecular identification of this species. RESULTS: Our SEM observations showed for the fist time the detailed morphology of the cephalic extremity, precloacal pseudo-sucker, caudal papillae, gubernaculum, phasmids and vulva of S. chinensis, and also determined the presence of a small, single medio-ventral precloacal papilla in the male. Moreover, we detected the presence of 0.08-0.40% and 0-1.30% nucleotide divergence among different individuals of S. chinensis in the ITS and cox1 regions, respectively. The supplementary morphological characters and genetic data will be very useful for the diagnosis of this poorly known species.

GIT1 contributes to autophagy in osteoclast through disruption of the binding of Beclin1 and Bcl2 under starvation condition.

Approximately 10-15% of all bone fractures do not heal properly, causing patient morbidity and additional medical care expenses. Therefore, better mechanism-based fracture repair approaches are needed. In this study, a reduced number of osteoclasts (OCs) and autophagosomes/autolysosomes in OC can be observed in GPCR kinase 2-interacting protein 1 (GIT1) knockout (KO) mice on days 21 and 28 post-fracture, compared with GIT1 wild-type (GIT1 WT) mice. Furthermore, in vitro experiments revealed that GIT1 contributes to OC autophagy under starvation conditions. Mechanistically, GIT1 interacted with Beclin1 and promoted Beclin1 phosphorylation at Thr119, which induced the disruption of Beclin1 and Bcl2 binding under starvation conditions, thereby, positively regulating autophagy. Taken together, the findings suggest a previously unappreciated role of GIT1 in autophagy of OCs during fracture repair. Targeting GIT1 may be a potential therapeutic approach for bone fractures.

GPCR kinase 2-interacting protein-1 protects against ischemia-reperfusion injury of the spinal cord by modulating ASK1/JNK/p38 signaling.

GPCR kinase 2-interacting protein-1 (GIT1) is a scaffold protein that plays an important role in cell adaptation, proliferation, migration, and differentiation; however, the role of GIT1 in the regulation of neuronal death after spinal cord injury remains obscure. Here, we demonstrate that GIT1 deficiency remarkably increased neuronal apoptosis and enhanced JNK/p38 signaling, which resulted in stronger motor deficits by ischemia-reperfusion in vivo, consistent with the finding of oxygen-glucose deprivation/reoxygenation-induced neuronal injury in vitro. After treatment with JNK and p38 inhibitors, abnormally necroptotic cell death caused by GIT1 knockdown could be partially rescued, with the recovery of neuronal viability, which was still poorer than that in control neurons. Meanwhile, overactivation of JNK/p38 after GIT1 depletion was concomitant with excessive activity of apoptosis signal-regulating kinase-1 (ASK1) that could be abolished by ASK1 silencing in HEK293T cells. Finally, GIT1 could disrupt the oligomerization of ASK1 via interaction between the synaptic localization domain that contains the coiled-coil (CC)-2 domain of GIT1 and the C-terminal CC domain of ASK1. It suppressed the autophosphorylation of ASK1 and led to decreasing activity of the ASK1/JNK/p38 pathway. These data reveal a protective role for GIT1 in neuronal damage by modulating ASK1/JNK/p38 signaling.-Chen, J., Wang, Q., Zhou, W., Zhou, Z., Tang, P.-Y., Xu, T., Liu, W., Li, L.-W., Cheng, L., Zhou, Z.-M., Fan, J., Yin, G.-Y. GPCR kinase 2-interacting protein-1 protects against ischemia-reperfusion injury of the spinal cord by modulating ASK1/JNK/p38 signaling.

[Efficacy of regular or intermittent inhalation of corticosteroids in treatment of asthma and its effects on growth and development in children].

OBJECTIVE: To observe the efficacy of regular or intermittent inhalation of salmeterol/fluticasone propionate (SM/FP) in the treatment of bronchial asthma and its effects on growth and development in children. METHODS: A total of 112 children diagnosed with bronchial asthma between September 2012 and October 2013 were assigned to standardized treatment (standard group, n=56) and non-standardized treatment (intermittent group, n=56). Comparisons of clinical symptom scores and main pulmonary function indicators between the two groups were carried out before treatment and at 6 and 12 months after treatment. The growth velocity and changes in body mass index (BMI) were observed in the two groups. RESULTS: At 6 and 12 months after the treatment, the standard group had significantly reduced clinical symptom scores and significantly increased pulmonary function indicators (percentage of predicted peak expiratory flow, PEF%; percentage of forced expiratory volume in 1 second, FEV1%) (P<0.05); the intermittent group had significantly reduced clinical symptom scores and significantly increased FEV1% (P<0.05), but PEF% was significantly increased only at 6 months after treatment (P<0.05). At 12 months after treatment, the standard group had significantly lower clinical symptom scores and significantly higher PEF% and FEV1% when compared with the intermittent group (P<0.05). The growth velocity and BMI showed no significant differences between the two groups at 6 and 12 months after treatment (P>0.05). CONCLUSIONS: Compared with intermittent inhalation, long-term regular inhalation of SM/FP performs better in controlling clinical symptoms and enhancing pulmonary function in children with asthma. Inhalation of SM/FP for one year reveals no apparent effect on the growth and development of these children.

A novel tumor suppressor gene ECRG4 interacts directly with TMPRSS11A (ECRG1) to inhibit cancer cell growth in esophageal carcinoma.

BACKGROUND: The esophageal carcinoma related gene 4 (ECRG4) was initially identified and cloned from human normal esophageal epithelium in our laboratory (GenBank accession no.AF325503). ECRG4 has been described as a novel tumor suppressor gene associated with prognosis in esophageal squamous cell carcinoma (ESCC). METHODS: In this study, binding affinity assay in vitro and co-immunoprecipitation experiment in vivo were utilized to verify the physical interaction between ECRG4 and transmembrane protease, serine 11A (TMPRSS11A, also known as ECRG1, GenBank accession no. AF 071882). Then, p21 protein expression, cell cycle and cell proliferation regulations were examined after ECRG4 and ECRG1 co-transfection in ESCC cells. RESULTS: We revealed for the first time that ECRG4 interacted directly with ECRG1 to inhibit cancer cell proliferation and induce cell cycle G1 phase block in ESCC. Binding affinity and co-immunoprecipitation assays demonstrated that ECRG4 interacted directly with ECRG1 in ESCC cells. Furthermore, the ECRG4 and ECRG1 co-expression remarkably upregulatd p21 protein level by Western blot (P < 0.001), induced cell cycle G1 phase block by flow cytometric analysis (P < 0.001) and suppressed cell proliferation by MTT and BrdU assay (both P < 0.01) in ESCC cells. CONCLUSIONS: ECRG4 interacts directly with ECRG1 to upregulate p21 protein expression, induce cell cycle G1 phase block and inhibit cancer cells proliferation in ESCC.

[Mechanism of loss of human esophageal cancer-related gene 4 (ECRG4) gene expression in esophageal squamous cell carcinoma cell line EC9706].

OBJECTIVE: To investigate the mechanism of loss of human esophageal cancer-related gene 4 (ECRG4) expression in esophageal squamous cell carcinoma (ESCC.) METHODS: PCR-SSCP and DNA sequencing analysis were used to detect the mutation of ECRG4 exons in esophageal cancer and matched adjacent normal tissues of 80 patients. DNA bisulfite-modifying ssPCR sequencing assay was used to examine the methylation status of ECRG4 promoter in human esophageal squamous cell carcinoma EC9706 cells. The re-expression of ECRG4 mRNA was examined by RT-PCR in EC9706 cells, after treatment with either demethylation drug 5-aza-2'-deoxycytidine or arsenic trioxide. RESULTS: No mutation in the four ECRG4 exons was found in all the ESCC and matched normal adjacent tissues. RT-PCR showed that 11 of 16 CpG islands of ECRG4 promoter were hypermethylated, while ECRG4 mRNA expression level was undetectable in the EC9706 cells. The ECRG4 mRNA was re-expressed after treatment with either demethylation drug 5-aza-2'-deoxycytidine or arsenic trioxide. CONCLUSION: The epigenetic mechanism of methylation is a reason of loss of ECRG4 gene expression in the ESCC cell line EC9706.

[Tumor-suppressing function of human esophageal cancer related gene 4 in esophageal squamous cell carcinoma].

OBJECTIVE: To investigate the tumor-suppressing function of human esophageal cancer related gene 4 (ECRG4) in esophageal squamous cell carcinoma (ESCC). METHODS: The recombinant plasmid pcDNA3.1-ECRG4 with ECRG4 open reading frame was constructed. The EC9706 cell was transfected with either pcDNA3.1 or pcDNA3.1-ECRG4. And the effects of ECRG4 on tumor cell were examined by in vivo assays of cell proliferation, adhesion, migration, invasion and tumor growth. The expression levels of P53 and P21 proteins were detected in EC9706 cell with transfection of ECRG4 gene by Western blotting. RESULTS: The final tumor volume and weight in ECRG4 transfection group were (264±43) mm3 and (0.39±0.09) g, versus (464±128) mm3 and (0.76±0.13) g in control group (both P<0.05). And the capacities of tumor cells adhesion, migration and invasion decreased in ECRG4 transfection group versus that in control group (all P>0.05). Furthermore, the expression levels of P53 and P21 proteins were higher in ECRG4 transfection group than those in control group (100.00±3.87, 35.71±2.36 vs 16.6±1.92, 1.09±0.11, both P<0.05). CONCLUSION: As a novel candidate tumor suppressor in ESCC, ECRG4 may induce the up-regulation of p21 protein through p53 pathway to inhibit tumor growth in ESCC.

Expression of esophageal cancer related gene 4 (ECRG4), a novel tumor suppressor gene, in esophageal cancer and its inhibitory effect on the tumor growth in vitro and in vivo.

The ECRG4 gene was initially identified and cloned in our laboratory from human normal esophageal epithelium (GenBank accession no. AF325503). We revealed the expression of ECRG4 protein was downregulated in 68.5% (89/130) ESCC samples using tissue microarray. The low ECRG4 protein expression was significantly associated with regional lymph node metastasis, primary tumor size, and tumor stage in ESCC (p < 0.05). ECRG4 mRNA expression was downregulated in ESCC due to the hypermethylation in the gene promoter. The treatment with 5-aza-2'-deoxycytidine, which is a DNA methyltransferase inhibitor restored ECRG4 mRNA expression in ESCC cells. The result indicated that promoter hypermethylation may be 1 main mechanism leading to the silencing of ECRG4. The high expression of ECRG4 in patients with ESCC was associated with longer survival compared with those with low ECRG4 expression by Kaplan-Meier survival analysis (p < 0.05). ECRG4 protein was an independent prognostic factor for ESCC by multivariable Cox proportional hazards regression analysis (p < 0.05). The restoration of ECRG4 expression in ESCC cells inhibited cell proliferation, colony formation, anchorage-independent growth, cell cycle progression and tumor growth in vivo (p < 0.05). The transfection of ECRG4 gene in ESCC cells inhibited the expression of NF-kappaB and nuclear translocation, in addition to the expression of COX-2, a NF-kappaB target gene, was attenuated. Taken together, ECRG4 is a novel candidate tumor suppressor gene in ESCC, and ECRG4 protein is a candidate prognostic marker for ESCC.

[Demethylation of estrogen receptor gene and its re-expression in estrogen receptor-negative breast].

OBJECTIVE: To investigate the correlation between the lack of estrogen receptor (ER) gene expression and hypermethylation of ER gene, and detect whether re-expressed ER protein is activated. METHODS: The methylation status of ER gene promoter in the ER-negative breast cancer cells was evaluated by methylation specific PCR (MSP) and genomic sequencing. The expression of ER and progesterone receptor (PR) mRNA as well as the production of ER protein were detected by RT-PCR and Western blot method, respectively. MTI assay was used to examine the function of re-expressed ER protein. RESULTS: The ER gene promoter was highly methylated, while ER mRNA and ER protein were not expressed in the ER-negative breast cell line MDA-MB-231. The ER-negative breast cells treated with demethylating agent 5 -aza-2'-deoxycytidine (5-AZA-2'-deoxyC) restored the expression of ER mRNA and ER protein. Expression of the endogenous ER-responsive PR gene was activated and the methylation of ER gene was simultaneously decreased. After MDA-MB-231 was treated with 5-AZA-2'-deoxyC, the protein of ER was re-expressed and the growth of cells treated with tamoxifen were inhibited significantly (P < 0.05). CONCLUSION: inactivation of ER gene has a close relationship with the abnormal methylation of ER gene promoter. 5-AZA-2'-deoxyC may effectively cause demethylation and restore functional expression of ER silenced by aberrant hypermethylation. The result may offer a new measure and theory for breast cancer patients with ER-negative expression to receive endocrine therapies.