Downregulation of RBO-PI4KIIIα Facilitates Aß42 Secretion and Ameliorates Neural Deficits in Aß42-Expressing Drosophila.

Phosphoinositides and their metabolizing enzymes are involved in Aß42 metabolism and Alzheimer's disease pathogenesis. In yeast and mammals, Eighty-five requiring 3 (EFR3), whose Drosophila homolog is Rolling Blackout (RBO), forms a plasma membrane-localized protein complex with phosphatidylinositol-4-kinase Type IIIα (PI4KIIIα) and a scaffold protein to tightly control the level of plasmalemmal phosphatidylinositol-4-phosphate (PI4P). Here, we report that RBO binds to Drosophila PI4KIIIα, and that in an Aß42-expressing Drosophila model, separate genetic reduction of PI4KIIIα and RBO, or pharmacological inhibition of PI4KIIIα ameliorated synaptic transmission deficit, climbing ability decline, premature death, and reduced neuronal accumulation of Aß42 Moreover, we found that RBO-PI4KIIIa downregulation increased neuronal Aß42 release and that PI4P facilitated the assembly or oligomerization of Aß42 in/on liposomes. These results indicate that RBO-PI4KIIIa downregulation facilitates neuronal Aß42 release and consequently reduces neuronal Aß42 accumulation likely via decreasing Aß42 assembly in/on plasma membrane. This study suggests the RBO-PI4KIIIα complex as a potential therapeutic target and PI4KIIIα inhibitors as drug candidates for Alzheimer's disease treatment.SIGNIFICANCE STATEMENT Phosphoinositides and their metabolizing enzymes are involved in Aß42 metabolism and Alzheimer's disease pathogenesis. Here, in an Aß42-expressing Drosophila model, we discovered and studied the beneficial role of downregulating RBO or its interacting protein PI4KIIIα-a protein that tightly controls the plasmalemmal level of PI4P-against the defects caused by Aß42 expression. Mechanistically, RBO-PI4KIIIα downregulation reduced neuronal Aß42 accumulation, and interestingly increased neuronal Aß42 release. This study suggests the RBO-PI4KIIIα complex as a novel therapeutic target, and PI4KIIIα inhibitors as new drug candidates.

Expression of EIF5A2 associates with poor survival of nasopharyngeal carcinoma patients treated with induction chemotherapy.

BACKGROUND: Nasopharyngeal carcinoma (NPC) is a type of head-neck cancer with a distinguishable geographic and racial distribution worldwide. Increasing evidence supports that the accumulation of additional genetic and epigenetic abnormalities is important in driving the NPC tumorigenic process. In this study, we aim to investigate the association between EIF5A2 (Eukaryotic translation initiation factor 5A2) expression status and NPC clinical outcomes. METHODS: The expression status of EIF5A2 was investigated in the NPC tissue microarray. Tissues were from 166 NPC patients staging II-IV, collected between 1999 and 2005. All patients were administered 2-3 cycles of DDP (cisplatin) + 5-Fu (5-fluorouracil) induction therapy and then treated with a uniform conventional two-dimensional radiotherapy. Cell motility assay, tumor growth assay and cytotoxicity assay were performed on the EIF5A2 overexpressed cells and control cells. siRNA was also used in the in vitro studies. RESULTS: Positive staining of EIF5A2 was observed in 85.4 % (105/123) informative tumor cases. Multivariate analyses demonstrated that EIF5A2 was an independent prognostic marker of poor overall survival (OS) (P = 0.041), failure-free survival (FFS) (P = 0.029), and distant failure-free survival (D-FFS) (P = 0.043) in patients with locoregionally advanced NPC patients treated with cisplatin + 5-Fu chemoradiotherapy. The forced expression of EIF5A2 in NPC cells enhanced the cells' motility and growth ability. Knock-down of EIF5A2 in NPC cells decreased the cell's motility and growth ability. Our results also demonstrated that EIF5A2 overexpression induced chemoresistance of NPC cells to 5-Fu. CONCLUSIONS: Our findings suggested that EIF5A2 expression, as examined by immunohistochemistry, could function as an independent prognostic factor of outcomes in NPC patients with cisplatin + 5-Fu chemoradiotherapy. EIF5A2 might be a novel therapeutic target for the inhibition of NPC progress.

Expression of beta-amyloid induced age-dependent presynaptic and axonal changes in Drosophila.

Alzheimer's disease (AD) is attributable to synapse dysfunction and loss, but the nature and progression of the presynaptic structural and functional changes in AD are essentially unknown. We expressed wild-type or arctic form of beta amyloid(1-42) (Abeta) in a small group of neurons in the adult fly and performed extensive time course analysis of the function and structure of both axon and presynaptic terminals at the identified single-neuron level. Abeta accumulated intracellularly and induced a range of age-dependent changes, including depletion of presynaptic mitochondria, slowdown of bi-directional transports of axonal mitochondria, decreased synaptic vesicles, increased large vacuoles, and elevated synaptic fatigue. These structural and functional synaptic changes correlated with age-dependent deficit in motor behavior. All these alterations were accelerated in flies expressing the arctic form of Abeta. The depletion of presynaptic mitochondria was the earliest detected phenotype and was not caused by the change in axonal transport of mitochondria. Moreover, axonal mitochondria exhibited a dramatic reduction in number but a significant increase in size in aged Abeta-expressing flies, indicating a global depletion of mitochondria in the neuron and an impairment of mitochondria fission. These results suggest that Abeta accumulation depletes presynaptic and axonal mitochondria, leading to other presynaptic deficits.

Effect of sequential high-flow nasal cannula oxygen therapy and non-invasive positive-pressure ventilation in patients with difficult weaning from mechanical ventilation after extubation on respiratory mechanics.

BACKGROUND: Patients with difficult weaning who undergo mechanical ventilation are more likely to be at risk of reintubation and the sequential use of oxygen therapy after extubation is a concern for clinicians. Therefore, the aim of the present study was to compare the effects of transnasal high-flow nasal cannula (HFNC) oxygen therapy and non-invasive positive-pressure ventilation (NIV) on respiratory mechanics in patients with difficult weaning. METHODS: The present study was a single-center, retrospective, observational study. Twenty-nine patients with difficult weaning off invasive mechanical ventilation from the Department of Critical Care Medicine, The First Affiliated Hospital of Guangzhou Medical University, from December 2018 to April 2021, were included. Within 48 h after extubation, alternate respiratory support with HFNC and NIV was provided. Relevant indicators were recorded after each support mode had been maintained for at least 60 min. These included esophageal pressure (Pes), gastric pressure (Pga), transdiaphragmatic pressure (Pdi), pressure-time product of Pes (PTPes), pressure-time product of Pga (PTPga), pressure-time product of Pdi (PTPdi), ratio of the PTPdi to the PTPes (PTPdi/PTPes), and ratio of the Pes to the Pdi (Pes/Pdi), diaphragmatic electromyogram (EMGdi), percentage of esophageal pressure coefficient of variation (CVes%),diaphragmatic electromyogram coefficient of variation (CVEMG),inspiratory time (Ti), expiratory time (Te) and respiratory cycle time (Ttot). RESULTS: Of the 29 patients included, 22 were males and 7 were females [age: 63.97±15.34 years, Acute Physiological and Chronic Health Estimation II (APACHE II) score: 18.00±5.63]. The CVes% and the Pes/Pdi were significantly higher in patients with NIV than HFNC using 40 L/min, CVes%: 9 (-6, 20) vs. -7 (-23, 6) and Pes/Pdi: 0.17 (-0.1, 0.53), vs. -0.12 (-0.43, 0.08) (P<0.05). The remaining indicators were not statistically different. CONCLUSIONS: The sequential NIV and HFNC can be tolerated in patients with such difficult weaning off mechanical ventilation after extubation, and more patients tend to choose HFNC subjectively. Compared with HFNC, NIV reduces the work of adjunctive respiratory muscle, but the patient's Pes dispersion is high when NIV is used, and it is necessary to pay attention to patient-ventilator coordination in clinical practice. We recommend alternating HFNC and NIV during the sequential respiratory therapy after extubation.

Collision tumor of squamous cell carcinoma and neuroendocrine carcinoma in the head and neck: A case report.

BACKGROUND: There are many disputes about the definition, diagnosis, therapy, and prognosis of collision tumors. CASE SUMMARY: We describe a rare patient with a collision tumor consisting of neuroendocrine carcinoma (NEC) and squamous cell carcinoma (SCC) in the nasal cavity and paranasal sinus. She received operation, concurrent chemoradiotherapy, and then two cycles of palliative chemotherapy. Follow-up at 12 mo after diagnosis showed that this patient experienced a complete response with no signs of recurrence or metastasis. A literature review of previous 26 cases diagnosed with collision tumor of NEC and SCC in the head and neck was also undertaken. CONCLUSION: It is challenging to manage collision tumors because there are two morphologically and etiologically distinct tumors. Well-designed multimodality therapy including surgery and chemoradiotherapy might lead to a long survival in these patients.

LINC01554-Mediated Glucose Metabolism Reprogramming Suppresses Tumorigenicity in Hepatocellular Carcinoma via Downregulating PKM2 Expression and Inhibiting Akt/mTOR Signaling Pathway.

Background and Aims: Cancer cells prefer aerobic glycolysis to maintain growth advantages, but the role of long non-coding RNAs (lncRNAs) in glycometabolism still remains unclear. Here we identified one cytoplasmic lncRNA LINC01554 as a significantly downregulated lncRNA in hepatocellular carcinoma (HCC) and aimed to investigate its role in cellular glucose metabolism in the development and progression of HCC. Methods: Quantitative real-time PCR was used to determine the expression level of LINC01554. Downregulation of LINC01554 by miR-365a at transcriptional level was assessed by luciferase reporter assay. Subcellular fractionation assay and RNA fluorescence in situ hybridization were performed to detect the subcellular localization of LINC01554. RNA pull-down assay, mass spectrometry, and RNA immunoprecipitation assay were used to identify the underlying molecular mechanisms. The tumor-suppressive function of LINC01554 was determined by both in vitro assay and nude mice xenograft model. Results: LINC01554 was frequently downregulated in HCC, which was significantly associated with tumor invasion (P = 0.005), tumor size (P = 0.041), tumor staging (P = 0.023) and shorter survival (P = 0.035) of HCC patients. Luciferase reporter assay unraveled that LINC01554 was negatively regulated by miR-365a. Subcellular fractionation assay and RNA FISH revealed the cytoplasmic predominance of LINC01554 in MIHA cells and HCC clinical samples. Ectopic expression of LINC01554 inhibited HCC cell growth, colony formation in soft agar, foci formation, and tumor formation in nude mice. LINC01554 promoted the ubiquitin-mediated degradation of PKM2 and inhibited Akt/mTOR signaling pathway to abolish aerobic glycolysis in HCC cells. Further study found that LINC01554-knockout could effectively reverse the tumor-suppressive effect of LINC01554. Conclusions: Our results identify LINC01554 as a novel tumor suppressor in HCC and unravel its underlying molecular mechanism in reprogramming cellular glucose metabolism. LINC01554 could possibly serve as a novel prognostic biomarker and provide the rationale for HCC therapy.

RHCG Suppresses Tumorigenicity and Metastasis in Esophageal Squamous Cell Carcinoma via Inhibiting NF-κB Signaling and MMP1 Expression.

Background and Aims: Esophageal squamous cell carcinoma (ESCC), a major histologic subtype of esophageal cancer, is increasing in incidence, but the genetic underpinnings of this disease remain unexplored. The aim of this study is to identify the recurrent genetic changes, elucidate their roles and discover new biomarkers for improving clinical management of ESCC. Methods: Western blotting and immunohistochemistry were performed to detect the expression level of RHCG. Bisulfite genomic sequencing (BGS) and methylation-specific PCR (MSP) were used to study the methylation status in the promoter region of RHCG. The tumor-suppressive effect of RHCG was determined by both in-vitro and in-vivo assays. Affymetrix cDNA microarray was used to identify the underlying molecular mechanism. Results:RHCG was frequently downregulated in ESCCs, which was significantly correlated with poor differentiation (P = 0.001), invasion (P = 0.003), lymph node metastasis (P = 0.038) and poorer prognosis (P < 0.001). Demethylation treatment and bisulfite genomic sequencing analyses revealed that the downregulation of RHCG in both ESCC cell lines and clinical samples was associated with its promoter hypermethylation. Functional assays demonstrated that RHCG could inhibit clonogenicity, cell motility, tumor formation and metastasis in mice. Further study revealed that RHCG could stabilize IκB by decreasing its phosphorylation, and subsequently inhibit NF-κB/p65 activation by blocking the nuclear translocation of p65, where it acted as a transcription regulator for the upregulation of MMP1 expression. Conclusions: Our results support the notion that RHCG is a novel tumor suppressor gene that plays an important role in the development and progression of ESCC.

Overexpression of HN1L promotes cell malignant proliferation in non-small cell lung cancer.

Non-small cell lung cancer (NSCLC) is a progressive malignant disease, involving the activation of oncogenes and inactivation of tumor suppressors. In this study, we identified and characterized a novel oncogene hematopoietic- and neurologic-expressed sequence 1-like (HN1L) in human NSCLC. Overexpression of HN1L was frequently detected in primary NSCLC compared with their non-tumor counterparts (P < 0.001), which was significantly associated with tumor size (P = 0.022). In addition, Kaplan-Meier analysis showed that upregulation of HN1L correlated with worse overall survival (P = 0.029) and disease-free survival (P = 0.011) for NSCLC patients. Both in vitro and in vivo studies demonstrated that inhibition of HN1L expression with shRNA dramatically inhibited cell growth, adherent and non-adherent colony formation, and tumorigenicity in nude mice. The positive correlation of HN1L expression and Ki67 level in a large NSCLC samples further suggested the key role of HN1L in the regulation of cell growth. Further study showed that knockdown of HN1L resulted in dramatic cell cycle arrest by interfering with MAPK pathway via interacting with RASA4 protein. In conclusion, HN1L plays a crucial role in the progression of NSCLC by contributing to malignant proliferation, with possible use as a new intervention point for therapeutic strategies.

CD68 and interleukin 13, prospective immune markers for esophageal squamous cell carcinoma prognosis prediction.

PURPOSE: Oncology immunity was reported to play a key role in cancer development and progression, so we investigated the prediction role of several immune markers in esophageal squamous cell carcinoma (ESCC) patients after operation in this study. PATIENTS AND METHODS: 66 primary ESCC tumor tissues and four sets of tissue microarrays including 705 primary ESCC tumor tissues from four centers were collected and analyzed. Expressions of several immune markers in ESCC tumor tissue were detected with immunohistochemistry staining. Their distribution densities were analyzed with InForm™ 2.0.1 software. All statistic analyses were performed with SPSS16.0 and Stata version 10.0. RESULTS: Survival analyses assessed by Kaplan-Meier plots and log-rank tests demonstrated that densities of CD68 and interleukin 13 (IL-13) in tumor stroma were positively correlated with the overall survival of ESCC patients after operation (p < 0.01 for CD68, p < 0.001 for IL-13). Further, a model based on tumor stroma densities of CD68 and IL-13 was constructed and it could significantly classify patients with poor or good prognosis. This model could further identify high-risk group and low-risk group at the same Tumor lymph Nodes Metastases (TNM) stage. Lastly, a more accuracy model based on TNM stage, densities of CD68 and IL-13 was constructed to predict the prognosis of ESCC patient after operation. CONCLUSION: Combining the TNM staging system and densities of CD68 and IL-13 could substantially improve the prognosis prediction accuracy of ESCC patient after operation, which might be an excellent tool for selecting patients for individualized therapy in future.

Stemness and chemotherapeutic drug resistance induced by EIF5A2 overexpression in esophageal squamous cell carcinoma.

Esophageal squamous cell carcinoma (ESCC) is one of the most lethal malignancies of the digestive tract in East Asian countries. Multimodal therapies, including adjuvant chemotherapy and neo-adjuvant chemotherapy, have become more often used for patients with advanced ESCC. However, the chemotherapy effect is often limited by patients' drug resistance. This study demonstrated that EIF5A2 (eukaryotic translation initiation factor 5A2) overexpression induced stemness and chemoresistance in ESCC cells. We showed that EIF5A2 overexpression in ESCC cells resulted in increased chemoresistance to 5-fluorouracil (5-FU), docetaxel and taxol. In contrast, shRNAs suppressing eIF5A2 increased tumor sensitivity to these chemotherapeutic drugs. In addition, EIF5A2 overexpression was correlated with a poorer overall survival in patients with ESCC who underwent taxane-based chemotherapy after esophagectomy (P < 0.05). Based on these results, we suggest that EIF5A2 could be a predictive biomarker for selecting appropriate chemo-treatment for ESCC patients and EIF5A2 inhibitors might be considered as combination therapy to enhance chemosensitivity in patients with ESCC.

Proteomic analysis of a nasopharyngeal carcinoma cell line and a nasopharyngeal epithelial cell line.

BACKGROUND: Nasopharyngeal carcinoma (NPC) is an epithelial malignancy exhibiting a strong geographic preference and a close association with Epstein-Barr virus (EBV). The aim of this study was to investigate the precise mechanism of nasopharyngeal epithelial-to-NPC tumorigenesis and to identify possible biomarkers and targets for therapy. METHODS: Proteomic analysis was performed on the immortalized nasopharyngeal epithelial cell line NP69 and the NPC cell line C666. RESULTS: A comparative analysis of total lysates from the cell lines using 2-D gel electrophoresis-mass spectrometry resulted in the identification of 87 different protein spots. The different proteins were grouped into 5 main categories: (i) energy production and general metabolism, (ii) adaptation/stress tolerance, (iii) cell proliferation, (iv) cell structure and (v) epithelial-mesenchymal transition. The detection of metabolism-related proteins indicated that the NPC cells relied on aerobic glycolysis, with reduced use of the citric acid cycle. Glucose uptake and lactate secretion increased in the medium of C666 compared with NP69. CONCLUSIONS: The present study revealed that glycolysis was up-regulated in the NPC cell lines compared with nasopharyngeal epithelial cells. A number of molecules involved in metabolism were identified, and further investigations will be needed to validate these potential biomarkers or targets.

[Enhancing hGH expression level in insect cells by shortening the 5'-UTR of hGH cDNA].

The regulation of foreign gene expression in Insect-Baculovirus Expression System is very complex. In this report, the effect of 5'-UTR in the expression of hGH gene in cultured Sf9 cells was examined. A 18 bp length in the end of 5'-UTR of hGH (human Growth Hormone, hGH) cDNA including a stem-loop structure was deleted by PCR. The truncated hGH cDNA, delta 1hGH was cloned in pFastBac1, named pFast-Bac-delta 1hGH. After transforming into E. coli. DH10Bac, which have a shuttle vetor-Bacmid, the delta 1hGH was integrated into Bacmid by site-specific transposition, and an expression vector, rBacmid-delta 1hGH DNA was acquired. By transfecting the cultured Sf9 cells with the recombinant expression vector DNA, pure recombinant virus, rAcV-Bac-delta 1hGH was obtained, and hGH gene was expressed. Immuno-blot and Chemiluminescent assay revealed that the expressed hGH had normal immunological activity, the amount of hGH expression level in Sf9 cell supernatant infected with rAcV-Bac-delta 1hGH containing the truncated 5'UTR was four to five times higher than that infected with rAcV-Bac-hGH.