Monomeric pilose antler peptide improves depression-like behavior in mice by inhibiting FGFR3 protein expression.

ETHNOPHARMACOLOGICAL RELEVANCE: It has been found that pilose antler peptide has an antidepressant effect on depression. However, the exact molecular mechanism of its antidepressant effect is still unclear. AIM OF THE STUDY: The study sought to determine the impact of monomeric pilose antler peptide (PAP; sequence LVLVEAELRE) on depression as well as investigate potential molecular mechanisms. MATERIALS AND METHODS: Chronic unexpected mild stress (CUMS) was used to establish the model, and the effect of PAP on CUMS mice was detected by the behavioral test. The influence of PAP on neuronal cells and dendritic spine density was observed by immunofluorescence and Golgi staining. FGFR3 and the CaMKII-associated pathway were identified using quantitative real-time polymerase chain reaction, and Western blot analysis was utilized to measure their proteins and gene expression levels. Molecular docking and microscale thermophoresis were applied to detect the binding of PAP and FGFR3. Finally, the effect of FGFR3's overexpression on PAP treatment of depression was detected. RESULTS: PAP alleviated the changes in depressive behavior induced by CUMS, promoted the growth of nerve cells, and the density of dendritic spines was increased to its original state. PAP therapy successfully downregulated the expression of FGFR3 and ERK1/2 while upregulating the expression of CREB, BDNF, and CaMKII. CONCLUSION: Based on the current research, PAP has a therapeutic effect on depression brought on by CUMS by inhibiting FGFR3 expression and enhancing synaptic plasticity.

Dynamic alterations of spontaneous neural activity in post-stroke aphasia: a resting-state functional magnetic resonance imaging study.

Background and purpose: The dynamic alterations in spontaneous neural activity of the brain during the acute phase of post-stroke aphasia (PSA) remain unclear. Therefore, in this study, dynamic amplitude of low-frequency fluctuation (dALFF) was applied to explore abnormal temporal variability in local functional activity of the brain during acute PSA. Materials and methods: Resting-state functional magnetic resonance imaging (rs-fMRI) data from 26 patients with PSA and 25 healthy controls (HCs) were acquired. The sliding window method was used to assess dALFF, with the k-means clustering method used to identify dALFF states. The two-sample t-test was applied to compare differences in dALFF variability and state metrics between the PSA and HC groups. Results: (1) In the PSA group, greater variance of dALFF in the cerebellar network (CBN) and left fronto-temporo-parietal network (FTPN) was observed. (2) Three dALFF states were identified among all subjects. States 1 and 2 were identified in the PSA patients, and the two dALFF states shared a similar proportion. Moreover, the number of transitions between the two dALFF states was higher in the patients compared with that in HCs. Conclusion: The results of this study provide valuable insights into brain dysfunction that occurs during the acute phase (6.00 ± 3.52 days) of PSA. The observed increase in variability of local functional activities in CBN and left FTPN may be related to the spontaneous functional recovery of language during acute PSA, and it also suggests that cerebellum plays an important role in language.

Neuroinflammation after ischemic stroke involves INPP5D expression mediated by the TMPO-AS1-PU.1 complex.

OBJECTIVES: This study aims to explore the role of lncRNA TMPO-AS1 in ischemic stroke and corresponding mechanism. METHODS: Adult male C57BL/6 J mice were subjected to a middle cerebral artery occlusion (MCAO) model of ischemic stroke, then TMPO-AS1 shRNA lentivirus were injected into ipsilateral striatum of mice. The neurological score and cerebral infarction volume were evaluatedHypoxia/glucose deprivation/reoxygenation (OGD/R)-induced BV2 cells were transfected with TMPO-AS1 shRNA (sh-TMPO-AS1) or together with pcDNA-INPP5D, as well as transfected with sh-PU.1 or together with pcDNA-INPP5D, then TMPO-AS1 level, the expression of PU.1 and INPP5D proteins, the secretion of inflammatory factors (TNF-α, IL-6 and IL-1ß), the levels of iNOS, CD68,Arg1 and CD206 mRNA were detected. RIP and PNA-pull down assays were used to detect the binding of TMPO-AS1 and PU.1, luciferase reporter gene and chromatin immunoprecipitation (ChIP) assays were used to detect the binding activity of PU.1 and INPP5D. RESULTS: TMPO-AS1 level was increased in peripheral blood of ischemic stroke patients , brain tissues of MCAO/R model mice and OGD/R-induced BV2 cells. TMPO-AS1 interference inhibited the inflammation of OGD/R-induced BV2 cells. TMPO-AS1 also enhanced the nuclear accumulation of PU.1 by binding to the transcription factor PU.1, and promoted the transcriptional activation of INPP5D. The anti-inflammatory effects of TMPO-AS1 interference were reversed by INPP5D overexpression. In addition, TMPO-AS1 interference improved the infarct volume of MCAO mice, and improved sensorimotor and cognitive functions. CONCLUSION: INPP5D underexpression mediated by TMPO-AS1-PU.1 complex alleviated neuroinflammation after ischemic stroke.

Autologous Cytokine-Induced Killer Cell Immunotherapy Enhances Chemotherapy Efficacy against Multidrug-Resistant Tuberculosis.

Objective: Multidrug-resistant tuberculosis (MDR-TB) causes persistent infection and challenges tuberculosis control worldwide. T cell-mediated immunity plays a critical role in controlling Mycobacterium tuberculosis (Mtb) infection, and therefore, enhancing Mtb-specific T cell immune responses represents a promising therapeutic strategy against TB. Cytokine-induced killer (CIK) immunotherapy is based on autologous infusion of in vitro expanded bulk T cells, which include both pathogen-specific and nonspecific T cells from patient peripheral blood mononuclear cells (PBMC) into TB patients. Preclinical mouse studies have shown that the adoptive T cell therapy inhibited Mtb infection. However, the efficacy of CIK immunotherapy in the treatment of MDR-TB infection has not been evaluated in clinical trials. Methods: We performed a retrospective study of MDR-TB patients who received CIK immunotherapy in combination with anti-TB chemotherapy and those who had standard chemotherapy. Results: Our results showed that CIK immunotherapy in combination with anti-TB chemotherapy treatment increased the conversion rate of sputum smear and Mtb culture, alleviated symptoms, improved lesion absorption, and increased recovery. The kinetics of serology and immunology index monitoring data showed good safety profiles for the CIK treatment. Conclusion: Our study has provided strong evidence that CIK immunotherapy in combination with anti-TB chemotherapy is beneficial for MDR-TB patients. A multicenter clinical trial is warranted to evaluate CIK as a new immune therapy for MDR-TB.

Shanzhiside methylester protects against depression by inhibiting inflammation via the miRNA-155-5p/SOCS1 axis.

Inflammation is a key player in the regulation of depression. Shanzhiside methylester (SM) is an iridoid glycoside with strong anti-inflammatory properties. However, the antidepressant effect of SM remains unknown. The present study aimed to investigate whether SM protects against depression by targeting inflammation. A chronic unpredictable mild stress (CUMS)-induced mouse model of depression was established to assess the antidepressant effect of SM in vivo. In addition, an LPS plus ATP-induced cellular model of inflammation was used to explore the related inflammatory mechanism. We found that both SM and miRNA-155-5p sponge markedly remedied CUMS-induced depression-like behaviors in the sucrose preference test (SPT), tail suspension test (TST), and forced swim test (FST), accompanied by decreased Iba1 expression and the production of TNF-α, IL-1ß, and IL-6. Moreover, SM and miRNA-155-5p sponge upregulated the protein levels of SOCS1 and downregulated the protein expression of p-JAK2 and p-STAT3 in the hippocampus of CUMS-exposed mice. miRNA-155-5p expression was also decreased following SM and miRNA-155-5p sponge administration. Furthermore, SM repressed LPS- and ATP-induced inflammatory responses in BV2 cells by regulating the SOCS1/JAK2/STAT3 signaling pathway, which was similar to the anti-inflammatory effects induced by the miRNA-155-5p sponge. Collectively, these findings suggested that SM exerted antidepressant actions by targeting the miRNA-155-5p/SOCS1 axis.

The OX40/OX40L Axis Regulates T Follicular Helper Cell Differentiation: Implications for Autoimmune Diseases.

T Follicular helper (Tfh) cells, a unique subset of CD4+ T cells, play an essential role in B cell development and the formation of germinal centers (GCs). Tfh differentiation depends on various factors including cytokines, transcription factors and multiple costimulatory molecules. Given that OX40 signaling is critical for costimulating T cell activation and function, its roles in regulating Tfh cells have attracted widespread attention. Recent data have shown that OX40/OX40L signaling can not only promote Tfh cell differentiation and maintain cell survival, but also enhance the helper function of Tfh for B cells. Moreover, upregulated OX40 signaling is related to abnormal Tfh activity that causes autoimmune diseases. This review describes the roles of OX40/OX40L in Tfh biology, including the mechanisms by which OX40 signaling regulates Tfh cell differentiation and functions, and their close relationship with autoimmune diseases.

Commensal microbiota contributes to predicting the response to immune checkpoint inhibitors in non-small-cell lung cancer patients.

Immunotherapy against cancer, through immune checkpoint inhibitors targeting the programmed cell death-1/programmed cell death-ligand 1 axis, is particularly successful in tumors by relieving the immune escape. However, interindividual responses to immunotherapy are often heterogeneous. Therefore, it is essential to screen out predictive tumor biomarkers. In this study, we analyzed the commensal microbiota in stool samples and paired sputum samples from 75 metastatic non-small-cell lung cancer (NSCLC) patients at baseline and during treatment with immune checkpoint inhibitors. Results showed distinct microbes' signatures between the gut microbiota and paired respiratory microbiota. The alpha diversity between the gut and respiratory microbiota was uncorrelated, and only the gut microbiota alpha diversity was associated with anti-programmed cell death-1 response. Higher gut microbiota alpha diversity indicated better response and more prolonged progression-free survival. Comparison of bacterial communities between responders and nonresponders showed some favorable/unfavorable microbes enriched in responders/nonresponders, indicating that commensal microbiota had potential predictive value for the response to immune checkpoint inhibitors. Generally, some rare low abundance gut microbes and high abundance respiratory microbes lead to discrepancies in microbial composition between responders and nonresponders. A significant positive correlation was observed between the abundance of Streptococcus and CD8+ T cells. These results highlighted the intimate relationship between commensal microbiota and the response to immunotherapy in NSCLC patients. Gut microbiota and respiratory microbiota are promising biomarkers to screen suitable candidates who are likely to benefit from immune checkpoint inhibitor-based immunotherapy.

The Inhibitory Effect of 6-Gingerol on Ubiquitin-Specific Peptidase 14 Enhances Autophagy-Dependent Ferroptosis and Anti-Tumor in vivo and in vitro.

Lung cancer is the most common malignant tumor and is the leading cause of cancer-related deaths worldwide. Extraction of bioactive substances from herbs is considered as an alternative method to traditional treatment. 6-Gingerol is a naturally occurring phenol found in ginger that can be used to treat tumors and suppress inflammation. To determine whether 6-Gingerol can be used as a therapeutic agent for tumors. In this study, tumor-bearing mice were used as an animal model and A549 as a cell model. Western blot was used to detect the expression of autophagy related proteins ubiquitin-specific peptidase 14 (USP14), Beclin1, microtubule-associated protein light chain 3 (LC3) and ferroptosis related proteins nuclear receptor coactivator 4 (NCOA4), ferritin heavy chain 1 (FTH1), transferrin receptor 1 (TfR1), glutathione peroxidase 4 (GPX4), activating transcription factor4 (ATF4) in vivo and in vitro. MTT and EdU were used to detect the viability of A549 cells. H&E and immunofluorescence were used to localize and detect the expression of proteins. The detection of reactive oxygen species was performed using fluorescence probes. It was found that the administration of 6-Gingerol decreased the expression of USP14, greatly increased the number of autophagosomes, reactive oxygen species (ROS) and iron concentration, decreased the survival and proliferation rate of A549 cells, and significantly decreased tumor volume and weight. The results indicate that 6-Gingerol inhibits lung cancer cell growth via suppression of USP14 expression and its downstream regulation of autophagy-dependent ferroptosis, revealing the function and efficacy of 6-Gingerol as a therapeutic compound in A549 and its possible mechanism of action.

Penisarins A and B, Sesquiterpene Coumarins Isolated from an Endophytic Penicillium sp.

Penisarins A (1) and B (2), sesquiterpene coumarins with an unusual tricyclic sesquiterpene system, were isolated from endophytic Penicillium sp. KMU18029. Their structures were elucidated on the basis of spectroscopic methods, single-crystal X-ray diffraction, and electronic circular dichroism calculations. Compound 2 showed significant cytotoxicities against two human cancer cell lines, HL-60 and SMMC-7721, with IC50 values of 3.6 ± 0.2 and 3.7 ± 0.2 µM, respectively.

Risk Factor Analysis and Risk Prediction Model Construction of Pressure Injury in Critically Ill Patients with Cancer: A Retrospective Cohort Study in China.

BACKGROUND The aim of this study was to analyze the risk factors of pressure injury (PI) in critically ill patients with cancer to build a risk prediction model for PI. MATERIAL AND METHODS Between January 2018 and December 2019, a total of 486 critically ill patients with cancer were enrolled in the study. Univariate analysis and binary logistic regression analysis were used to explore risk factors. Then, a risk prediction equation was constructed and a receiver operator characteristic (ROC) curve analysis model was used for prediction. RESULTS Of the 486 critically ill patients with cancer, 15 patients developed PI. Risk factors found to have a significant impact on PI in critically ill patients with cancer included the APACHE II score (P<0.001), semi-reclining position (P=0.006), humid environment/moist skin (P<0.001), and edema (P<0.001). These 4 independent risk factors were used in the regression equation, and the risk prediction equation was constructed as Z=0.112×APACHE II score +2.549×semi-reclining position +2.757×moist skin +1.795×edema-9.086. From the ROC curve analysis, the area under the curve (AUC) was 0.938, sensitivity was 100.00%, specificity was 83.40%, and Youden index was 0.834. CONCLUSIONS The PI risk prediction model developed in this study has a high predictive value and provides a basis for PI prevention and treatment measures for critically ill patients with cancer.

Virtual Screening for Type II B Inhibitors of B-RafV600E Kinase.

BACKGROUND: B-RafV600E kinase was identified as an important target in current cancer treatment, and the type II B inhibitors show good qualities in preclinical studies. Therefore, it is very important to discover novel II B inhibitors of B-RafV600E kinase. METHODS: In order to discover novel II B inhibitors of B-RafV600E kinase, virtual screening against ZINC database was performed by using a combination of pharmacophore modelling, molecular docking, 3DQSAR model and binding free energy (ΔGbind) calculation studies. The inhibitory activities against A375 cell lines of the hit compounds were tested by using MTT assay. RESULTS: Five promising hit compounds were obtained after screening, and all the five hit compounds showed good inhibitory rates against A375 cell lines. CONCLUSION: The combined approach of the virtual screening in our work is effective, which can be used to discover novel inhibitors with a new skeleton. In addition, the five compounds obtained from the screening showed good inhibitory rates against A375 cell lines, which can be considered to develop new II B inhibitors of B-RafV600E kinase.

New azaphilones from Penicillium variabile, a fungal endophyte from roots of Aconitum vilmorinianum.

Two new azaphilone derivatives, comazaphilones G and H (1 and 2), together with eight known analogues (3-10), were isolated from an endophytic fungus Penicillium variabile. Their structures were established on the basis of extensive spectroscopic analysis. Compounds 1, 2 and 4-10 were tested their nitric oxide inhibitory activities in lipopolysaccharide-activated RAW 264.7 macrophage cells. Compounds 1, 2 and 4-9 showed significant nitric oxide inhibitory activities with IC50 values ranged from 4.35 ± 0.05 to 40.52 ± 0.47 µM.

IL-36ß Promotes CD8+ T Cell Activation and Antitumor Immune Responses by Activating mTORC1.

Cytokine-amplified functional CD8+ T cells ensure effective eradication of tumors. Interleukin 36α (IL-36α), IL-36ß, and IL-36γ share the same receptor complex, composed of the IL-36 receptor (IL-36R), and IL-1RAcP. Recently, we revealed that IL-36γ greatly promoted CD8+ T cell activation, contributing to antitumor immune responses. However, the underlying mechanism of IL-36-mediated CD8+ T cell activation remains understood. In the current study, we proved that IL-36ß had the same effect on CD8+ T cell as IL-36γ, and uncovered that IL-36ß significantly activated mammalian target of rapamycin complex 1 (mTORC1) of CD8+ T cells. When mTORC1 was inhibited by rapamycin, IL-36ß-stimulated CD8+ T cell activation and expansion was drastically downregulated. Further, we elucidated that IL-36ß-mediated mTORC1 activation was dependent on the pathway of phosphatidylinositol 3 kinase (PI3K)/Akt, IκB kinase (IKK) and myeloid differentiation factor 88 (MyD88). Inhibition of PI3K or IKK by inhibitor, or deficiency of MyD88, respectively, suppressed mTORC1 signal, causing arrest of CD8+ T cell activation. Additionally, it was validated that IL-36ß significantly promoted mTORC1 activation and antitumor function of CD8+ tumor-infiltrating lymphocytes (TILs) in vivo, resulting in inhibition of tumor growth and prolongation of survival of tumor-bearing mice. Taken together, we substantiated that IL-36ß could promote CD8+ T cell activation through activating mTORC1 dependent on PI3K/Akt, IKK and MyD88 pathways, leading to enhancement of antitumor immune responses, which laid the foundations for applying IL-36ß into tumor immunotherapy.

B7-H3 modulates endothelial cell angiogenesis through the VEGF cytokine.

B7-H3 is a cell surface molecule in the immunoglobulin superfamily that has been shown to perform both immunological and non-immunological functions. It has also been found that vascular endothelial growth factor (VEGF) is an important molecule in the modulation of endothelial cell behavior. In this study, we analyzed the serum expression of B7-H3 in 113 rheumatoid arthritis and systemic lupus erythematous patients using the ELISA and found a positive correlation between B7-H3 and VEGF. Next, we investigated the involvement of B7-H3 in angiogenesis using human umbilical vein endothelial cells (HUVECs) with transient knockdown of B7-H3 and an in vivo Matrigel model. Data from the in vitro experiments showed that B7-H3 increased cell proliferation, migration, and tube formation, and correlated with the expression of VEGF. Furthermore, B7-H3 affected the formation of functional vascular networks in Matrigel plugs, which were dissected from mice injected with different HUVECs. Our data suggest that B7-H3 promotes angiogenesis through the enhancement of VEGF secretion. This is the first study proposing a significant role for B7-H3 in the promotion of angiogenesis and may provide further understanding of this gene's biological function.

Filamin A inhibits tumor progression through regulating BRCA1 expression in human breast cancer.

Filamin A (FlnA) is an actin cross-linking protein. Previous studies have demonstrated its role in tumor progression in a wide range of cancer types. It has been reported that FlnA interacts with the DNA damage response protein, breast cancer gene 1 (BRCA1), which is a tumor suppressor gene. However, to the best of our knowledge, there are no studies evaluating the association of these genes in human carcinomas. In the present study, the immunohistochemistry of a tissue microarray was used to investigate the clinical significance of FlnA and BRCA1 expression in pathological specimens collected from 424 patients treated for breast cancer. In addition, FlnA and BRCA1 expression was downregulated in the breast cancer cell line, MCF-7, through FlnA RNA interference. FlnA expression was exhibited by cancer tissues collected from 137 patients with breast cancer, which also exhibited high expression of BRCA1 and were associated with a relatively long survival time. A significant association was identified between FlnA protein expression and tumor size, and between FlnA protein expression and progesterone receptor expression. These results suggest that BRCA1 expression could be regulated by FlnA in the breast cancer cell line, MCF-7. Overall, the present study demonstrates that FlnA expression was associated with BRAC1 expression and tumor size in breast cancer, which provides important implications for future study of FlnA in the progression of human breast cancer.

Clinicopathological analysis of PD-L2 expression in colorectal cancer.

BACKGROUND: (PD-L2), a ligand of programmed cell death protein 1 (PD-1), is an inhibitory receptor of T cells and activated B cells. Many studies have focused on PD-L1, another ligand of PD-1, and the prognostic significance of PD-L1 has been reported in many tumors. However, the expression of PD-L2 in relation to clinical outcomes has not been fully investigated in cancer patients. PATIENTS AND METHODS: In this study, we investigated the expression of PD-L2 via immunohistochemistry (IHC) in the pathological specimens of 348 patients treated for colorectal cancer (CRC). RESULTS: Strong PD-L2 expression was found in the cancer tissues from 41% of the CRC patients who also had a high TNM stage and carcinoembryonic antigen (CEA) concentration. We also carried out functional studies in vitro, which showed that PD-L2 did not influence the growth of the CRC cell line HCT116, but increased cell invasion. CONCLUSION: Collectively, these findings suggest that PD-L2 may be a potential therapeutic target for CRC.

[Expression and purification of FLT3 and FLT3-ITD mutated proteins in HEK293T cells].

Objective To construct the eukaryotic expression vectors of human fms related tyrosine kinase 3 (FLT3) gene and FLT3-internal tandem duplication (FLT3-ITD) mutants and purify the native proteins through immunoprecipitation from HEK293T cell lysates. Methods The cDNA fragments of FLT3wt and FLT3-ITD were amplified from bone marrow cells of healthy individuals and FLT3-ITD-mutated acute myeloid leukemia (AML) patients with specific primers, and the PCR products were cloned in CD530A-T2A-GFP expression vectors. FLT3wt and FLT3-ITD plasmids were transfected in HEK293T cells by Polyjet reagent, and the recombinant proteins were purified by immunoprecipitation and competing elution methods. Results FLT3wt and FLT3-ITD-mutated DNA sequences were successfully cloned in CD530A-T2A-GFP expression vectors. FLT3wt and FLT3-ITD mutated proteins were successfully expressed and purified in HEK293T cells as verified by Western blotting and sliver staining. Conclusion FLT3wt and FLT3-ITD expression vectors were successfully constructed, and purified proteins were successfully obtained from HEK293T cells.

Reduced sB7-H3 Expression in the Peripheral Blood of Systemic Lupus Erythematosus Patients.

Both membrane-bound and soluble forms of costimulatory molecules play important roles in immune-regulatory networks. B7-H3, a member of the B7 family, has been found with aberrant expression in tumors and infectious disease. However, the significance of sB7-H3 expression in systemic lupus erythematosus (SLE) has not been investigated. Using the peripheral blood of 78 SLE patients, we established a comprehensive database containing clinical data and relevant laboratory tests. We found that sB7-H3 expression in SLE patients was significantly lower compared with the healthy individuals. In addition, sB7-H3 levels in the patients were positively correlated with the disease activity as indicated by SLE disease activity index score, rashes, fever, and inflammatory cytokines. Moreover, sB7-H3 was associated with the counts of red blood cells and hemoglobin. Our findings suggest that sB7-H3 might counteract the aberrant immune response and potentially serve as a monitoring indicator of disease progression and therapeutic target in SLE treatment.

Post-treatment serum lactic dehydrogenase as a predictive indicator for distant metastasis and survival of patients with nasopharyngeal carcinoma.

PURPOSE: To examine the function of serum lactic dehydrogenase (SLDH) level after intensity-modulated radiotherapy (IMRT) as a predictive factor for and loco-regional relapse free survival (LRFS), distant metastasis-free survival (DMFS), disease free survival (DFS), and overall survival(OS) among patients with in-situ nasopharyngeal carcinoma (NPC). RESULTS: Compared with the normal pt-SLDH group, elevated pt-SLDH demonstrated significant lower DMFS (46 versus 66 months, hazard ratio (HR) 4.07, 95% CI 2.43-6.80, p < 0.001), DFS (46 versus 63 months, HR 2.78, 95% CI 1.70-4.53, p < 0.001), and OS (54 versus 66 months, HR 2.93, 95% CI 1.65-5.23, p < 0.001). Distant metastasis were observed in 32.8% (20/61) patients with elevated pt-SLDH, and 8% (54/678) in normal SLDH (odds ratio (OR) 6.13, 95% CI 3.35-11.18, p < 0.001). COX regression showed that pt-SLDH was an independent prognostic factors for OS (HR 2.91, 95% CI 1.57-5.41, p < 0.001), DMFS (HR 4.21, 95% CI 2.51-7.07, p < 0.001), LRFS (HR 2.53, 95% CI 1.22-5.24, p < 0.001), and DFS (HR 2.81, 95% CI 1.72-4.59, p < 0.001). MATERIALS AND METHODS: The records of 739 in-situ NPC patients admitted to Zhejiang Cancer Hospital between January 2007 and May 2012 were retrospectively reviewed. The relationships between post-treatment SLDH (pt-SLDH) and LRFS, DMFS, DFS, and OS were analyzed. CONCLUSIONS: Our finding indicated that elevated pt-SLDH could be a simple available prognostic indicator for distant metastasis and survival for in-situ NPC patients.