Increased expression of SSEA-4 on TKI-resistant non-small cell lung cancer with EGFR-T790M mutation.

Non-small cell lung cancer (NSCLC), a major life-threatening disease accounting for 85% of all lung cancer cases, has been treated with tyrosine kinase inhibitors (TKIs), but often resulted in drug resistance, and approximately 60% of TKI-resistant cases are due to acquired secondary (epithelial growth factor receptor) EGFR-T790M mutation. To identify alternative targets for TKI-resistant NSCLC with EGFR-T790M mutation, we found that the three globo-series glycosphingolipids are increasingly expressed on this type of NSCLC cell lines, and among them, the increase of stage-specific embryonic antigen-4 (SSEA-4) expression is the most significant. Compared to TKI-sensitive cell lines, SSEA-4 and the key enzyme ß3GalT5 responsible for the synthesis of SSEA3 are more expressed in TKI-resistant NSCLC cell lines with EGFR-T790M mutation, and the expression levels strongly correlate with poor survival in patients with EGFR mutation. In addition, we demonstrated that a SSEA-4 targeted monoclonal antibody, especially the homogeneous glycoform with well-defined Fc glycan designed to improve effective functions, is highly effective against this subpopulation of NSCLC in cell-based and animal studies. These findings provide a direction for the prediction of tumor recurrence and treatment of TKI-resistant NSCLC with EGFR-T790M mutation.

Monoclonal enolase-1 blocking antibody ameliorates pulmonary inflammation and fibrosis.

BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is a chronic fatal disease with limited therapeutic options. The infiltration of monocytes and fibroblasts into the injured lungs is implicated in IPF. Enolase-1 (ENO1) is a cytosolic glycolytic enzyme which could translocate onto the cell surface and act as a plasminogen receptor to facilitate cell migration via plasmin activation. Our proprietary ENO1 antibody, HL217, was screened for its specific binding to ENO1 and significant inhibition of cell migration and plasmin activation (patent: US9382331B2). METHODS: In this study, effects of HL217 were evaluated in vivo and in vitro for treating lung fibrosis. RESULTS: Elevated ENO1 expression was found in fibrotic lungs in human and in bleomycin-treated mice. In the mouse model, HL217 reduced bleomycin-induced lung fibrosis, inflammation, body weight loss, lung weight gain, TGF-ß upregulation in bronchial alveolar lavage fluid (BALF), and collagen deposition in lung. Moreover, HL217 reduced the migration of peripheral blood mononuclear cells (PBMC) and the recruitment of myeloid cells into the lungs. In vitro, HL217 significantly reduced cell-associated plasmin activation and cytokines secretion from primary human PBMC and endothelial cells. In primary human lung fibroblasts, HL217 also reduced cell migration and collagen secretion. CONCLUSIONS: These findings suggest multi-faceted roles of cell surface ENO1 and a potential therapeutic approach for pulmonary fibrosis.

Beta-blockers reduce severe exacerbation in patients with mild chronic obstructive pulmonary disease with atrial fibrillation: a population-based cohort study.

BACKGROUND AND OBJECTIVE: Beta-blockers (BBs) decrease mortality and acute exacerbation (AE) rates in patients with chronic obstructive pulmonary disease (COPD) and cardiovascular disease; however, information on their effects in patients with COPD and atrial fibrillation (AF) is limited. We aimed to assess the AE risk in patients with different severities of COPD and AF receiving BBs compared with that in patients receiving calcium channel blockers (CCBs). METHODS: This retrospective cohort study used data from the Taiwan National Health Insurance Database from 2009 to 2018. Outcomes included AE-related emergency room visits and hospitalisation. HRs and 95% CIs were estimated using the Cox proportional hazards model. COPD severity was classified as mild or severe based on exacerbation history. Sensitivity analyses included treatment and subgroup analyses, and competing risk adjustment. RESULTS: After propensity score matching, 4486 pairs of BB and CCB users from 13 462 eligible patients were included. The exacerbation risk for BB users was lower (HR 0.80; 95% CI 0.72 to 0.89) than that of CCB users. After stratification, BB benefits persisted in the mild COPD group (HR 0.75; 95% CI 0.66 to 0.85), unlike the severe COPD group (HR 0.95; 95% CI 0.75 to 1.20). The results of the subgroup analysis showed consistent protective effects even in patients without heart failure or myocardial infarction (adjusted HR 0.82; 95% CI 0.71 to 0.94). CONCLUSION: We found that BB use in patients with mild COPD and AF was associated with a lower exacerbation risk than CCB use, and that close monitoring of BB use in patients with severe COPD and AF is warranted.

Suboptimal Outcomes and Retreatment Rate of Patients With Crohn's Disease After Forced Discontinuation of Biologics: A Nationwide Population-Based Study.

Taiwan's National Health Insurance (NHI) program forced discontinuation of biologic use in Crohn's disease (CD) after a limited treatment duration, regardless of disease activity. This study investigated the retreatment rate and suboptimal outcomes (i.e., CD-related surgeries, hospitalizations, emergency room visits, and oral steroid flare-ups) after forced discontinuation. This retrospective cohort study was conducted using data from the NHI Database. Patients who received ≥40 weeks of biologic treatment followed by a forced discontinuation were included. The time of biologic retreatment and the cumulative incidence of suboptimal outcomes after the forced discontinuation as well as related risk factors were analyzed. Included were 215 patients (68% male). At the beginning of biologic therapy, the mean age (±SD) was 35.7 (±13.5) years, and the disease duration was 4.46 (±3.52) years. The median (interquartile range) biologic treatment duration was 57.86 (50.3-83.3) weeks. Within the first year after forced discontinuation, 67% of patients (n = 144) were retreated with a second course of biologics, and 53% of patients (n = 114) experienced at least one suboptimal outcome. The independent risk factors associated with the occurrence of suboptimal outcomes were CD-related emergency room visits and hospitalizations during biologic therapy (hazard ratio: 2.49; 95% confidence interval: 1.59-3.89). More than two-thirds of patients with CD required biological retreatment within 1 year after a forced discontinuation. The substantial proportion of patients with poor disease outcomes highlights the need to continue the biologic.

Outcomes of wasp and bee stings in Taiwan.

BACKGROUND: The families of Hymenoptera causing commonly poisonous animal stings in Taiwan include Apoidea (bee) and Vespidae (wasp). This study aimed to investigate the epidemiologic, clinical characteristics, and outcomes of the severity of envenomation following wasp or bee stings in Taiwan. METHODS: We conducted a retrospective study by identifying all wasp and bee sting-related envenomation reported to the Taiwan National Poison Control Center between January 2001 and November 2021. Data were reviewed and abstracted by two independent reviewers. We then used ordinal logistic regression analysis to find potential predictors of severe wasp and bee sting-related envenomation. RESULTS: In Taiwan, bee or wasp stings mainly occur in late summer and autumn. A total of 611 patients were reported to the Taiwan National Poison Control Center with 7.5% resulting in severe or fatal envenomation. Four-hundred and forty-one patients were eligible for the final analysis of the predictors of severity. Logistic regression analysis showed that a greater number of stings, being stung by wasps, older age, and stings over the body were significant predictors for greater severity. The systemic effects following wasp and bee sting included anaphylactic reaction, prolongation of activated partial thromboplastin time, rhabdomyolysis, acute kidney injury, and elevated liver enzymes. CONCLUSIONS: Wasps generally inflicted more severe envenomation than bees. Only 7.5% of patients had severe or fatal outcomes. Patients with older age, multiple stings, and/or multiple sites of stings were more likely to have severe outcomes.

Growth, Physiological, and Photosynthetic Responses of Xanthoceras sorbifolium Bunge Seedlings Under Various Degrees of Salinity.

Xanthoceras sorbifolium Bunge is priced for its medical and energetic values. The species also plays a key role in stabilizing ecologically fragile areas exposed to excess soil salinity. In this study, the effects of salinity on the growth, physiological, and photosynthetic parameters of X. sorbifolium Bunge were investigated. The X. sorbifolium seedlings were subjected to five salt treatments: 0 (control, CK), 70, 140, 210, and 280 mM of sodium chloride (NaCl) solutions. NaCl caused a decrease in plant height, specific leaf area, biomass, and root parameters. Leaf wilting and shedding and changes in root morphology, such as root length, root surface area, and root tips were observed. This study found that X. sorbifolium is tolerant to high salinity. Compared with the CK group, even if the concentration of NaCl was higher than 210 mM, the increase of the relative conductivity was also slow, while intercellular CO2 concentration had a similar trend. Moreover, NaCl stress caused an increase in the malondialdehyde (MDA), soluble proteins, and proline. Among the enzymes in the plant, the catalase (CAT) activity increases first and decreased with the increase in the intensity of NaCl stress, but the salt treatment had no significant effect on superoxide dismutase (SOD) activity. The peroxidase (POD) showed an increasing trend under salt stress. It was found that the photosynthesis of X. sorbifolium was notably impacted by saline stress. NaCl toxicity induced a noticeable influence on leaf net photosynthetic rate (Pn), stomatal conductance (Gs), intercellular CO2 concentration (Ci), transpiration rate (E), and water use efficiency (Wue). As salt concentration increased, the content of chlorophyll decreased. It can be found that a low concentration of NaCl induced the increase of photosynthetic capacity but a high-intensity exposure to stress resulted in the reduction of photosynthetic efficiency and SOD activity, which had a positive correlation. In summary, salt-induced ionic stress primarily controlled root morphology, osmotic adjustment, and enzyme activities of salt-treated X. sorbifolium leaves, whereas the low salt load could, in fact, promote the growth of roots.

Sustained-Release and pH-Adjusted Alginate Microspheres-Encapsulated Doxorubicin Inhibit the Viabilities in Hepatocellular Carcinoma-Derived Cells.

The objective of this study aimed to develop biodegradable calcium alginate microspheres carrying doxorubicin (Dox) at the micrometer-scale for sustained release and the capacity of pH regulatory for transarterial chemoembolization. Ultrasonic atomization and CaCl2 cross-linking technologies were used to prepare the microspheres. A 4-by-5 experiment was first designed to identify imperative parameters. The concentration of CaCl2 and the flow rate of the pump were found to be critical to generate microspheres with a constant volume median diameter (~39 µm) across five groups with different alginate: NaHCO3 ratios using each corresponding flow rate. In each group, the encapsulation efficiency was positively correlated to the Dox-loading %. Fourier-transform infrared spectroscopy showed that NaHCO3 and Dox were step-by-step incorporated into the calcium alginate microspheres successfully. Microspheres containing alginate: NaHCO3 = 1 exhibited rough and porous surfaces, high Young's modulus, and hardness. In each group with the same alginate: NaHCO3 ratio, the swelling rates of microspheres were higher in PBS containing 10% FBS compared to those in PBS alone. Microspheres with relatively high NaHCO3 concentrations in PBS containing 10% FBS maintained better physiological pH and higher accumulated Dox release ratios. In two distinct hepatocellular carcinoma-derived cell lines, treatments with microspheres carrying Dox demonstrated that the cell viabilities decreased in groups with relatively high NaHCO3 ratios in time- and dose-dependent manners. Our results suggested that biodegradable alginate microspheres containing relatively high NaHCO3 concentrations improved the cytotoxicity effects in vitro.

High glycosyltransferase 8 domain containing two protein levels contribute to poor prognosis in urothelial carcinoma.

OBJECTIVE: To examine the expression levels of the glycosyltransferase 8 domain containing protein 2 and its clinical implications in urothelial carcinoma patients. METHODS: Data mining, immunohistochemistry together with H-score calculation was carried out to evaluate the glycosyltransferase 8 domain containing protein 2 levels on tissue specimens from urothelial carcinoma patients, retrospectively. Correlations between glycosyltransferase 8 domain containing protein 2 H-score and imperative clinicopathological factors were measured. The indication of glycosyltransferase 8 domain containing protein 2 level on disease-specific and metastasis-free survivals were next analyzed. RESULTS: In upper tract urothelial carcinomas (n = 340) and bladder urothelial carcinomas (n = 295), 170 (50%) and 148 (50%) patients, respectively, were identified to have high glycosyltransferase 8 domain containing protein 2 expression. The glycosyltransferase 8 domain containing protein 2 levels were correlated to several clinicopathological characteristics and patient survival. Upregulation of the glycosyltransferase 8 domain containing protein 2 was correlated to primary tumor (P < 0.001), nodal metastasis (P < 0.001), histological grade (P < 0.001), vascular invasion (P < 0.001), perineural invasion (P < 0.05) and mitotic rate (P < 0.001). High glycosyltransferase 8 domain containing protein 2 levels independently predicted poor disease-specific survival (P = 0.049) and metastasis-free survival (P = 0.008) in upper tract urothelial carcinoma and urinary bladder urothelial carcinoma, respectively. Gene Ontology enrichment analysis additionally showed that multiple biological processes were enriched including "ECM organization" (Gene Ontology:0030198), "extracellular structure organization" (Gene Ontology:0043062), "biological adhesion" (Gene Ontology:0022610), "cell adhesion" (Gene Ontology:0007155), "collagen fibril organization" (Gene Ontology:0030199) and "vasculature development" (Gene Ontology:0001944). CONCLUSIONS: The present findings suggest that upregulation of the glycosyltransferase 8 domain containing protein 2 is an independent and disadvantageous prognosticator in urothelial carcinoma. High glycosyltransferase 8 domain containing protein 2 level might play a crucial role in progression of urothelial carcinoma.

High G protein subunit beta 4 protein level is correlated to poor prognosis of urothelial carcinoma.

Data mining on a public domain detected eight potential transcripts which were upregulated in advanced UBUCs, suggesting that they may take part in UC development or/and progression. Retrospectively, immunohistochemistry along with H-score recording was carried out to evaluate the GNB4 protein levels on tissues from UC patients. Correlations between GNB4 H-score and imperative clinicopathological factors, as well as the implication of GNB4 protein level on disease-specific and metastasis-free survivals were assessed. In UTUCs (n = 340) and UBUCs (n = 295), 170 (50.0%) and 148 (50.0%) cases, respectively, were identified to be of high GNB4 expression. The GNB4 protein levels were correlated to numerous clinicopathological features and patients' survivals. Upregulation of the GNB4 protein was significantly associated with primary tumor, nodal metastasis, histological grade, vascular invasion and mitotic rate. High GNB4 protein levels independently and significantly predicted poor disease-specific and metastasis-free in UTUC and UBUC, respectively. Ingenuity pathway analysis furthermore showed that multiple signaling pathways were enriched including 'Communication between Innate and Adaptive Immune Cells' and 'NFκB Signaling'. Our findings demonstrated that the upregulation of the GNB4 protein is an independent unfavorable prognosticator in UC. High GNB4 gene expression plays an important role in UC progression.

Safety and tolerability of prescription omega-3 fatty acids: A systematic review and meta-analysis of randomized controlled trials.

BACKGROUND: Omega-3 fatty acids [eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA)] are widely recommended for health promotion. Over the last decade, prescription omega-3 fatty acid products (RxOME3FAs) have been approved for medical indications. Nonetheless, there is no comprehensive analysis of safety and tolerability of RxOME3FAs so far. METHODS: A systematic review of randomized controlled trials (RCTs) was carried out based on searches in six electronic databases. The studies involving marketed RxOME3FA products were included, and adverse-effect data were extracted for meta-analysis. Subgroup analysis and meta-regression were conducted to explore the sources of potential heterogeneity. RESULTS: Among the 21 included RCTs (total 24,460 participants; 12,750 from RxOME3FA treatment cohort and 11,710 from control cohort), there was no definite evidence of any RxOME3FA-emerging serious adverse event. Compared with the control group, RxOME3FAs were associated with more treatment-related dysgeusia (fishy taste; p = 0.011) and skin abnormalities (eruption, itching, exanthema, or eczema; p < 0.001). Besides, RxOME3FAs had mild adverse effects upon some non-lipid laboratory measurements [elevated fasting blood sugar (p = 0.005); elevated alanine transaminase (p = 0.022); elevated blood urea nitrogen (p = 0.047); decreased hemoglobin (p = 0.002); decreased hematocrit (p = 0.009)]. Subgroup analysis revealed that EPA/DHA combination products were associated with more treatment-related gastrointestinal adverse events [eructation (belching; p = 0.010); nausea (p = 0.044)] and low-density lipoprotein cholesterol elevation (p = 0.009; difference in means = 4.106mg/dL). CONCLUSION: RxOME3FAs are generally safe and well tolerated but not free of adverse effects. Post-marketing surveillance and observational studies are still necessary to identify long-term adverse effects and to confirm the safety and tolerability profiles of RxOME3FAs.

Toll-Like Receptor 9 Alternatively Spliced Isoform Negatively Regulates TLR9 Signaling in Teleost Fish.

Toll-like receptor 9 (TLR9) recognizes and binds unmethylated CpG motifs in DNA, which are found in the genomes of bacteria and DNA viruses. In fish, Tlr9 is highly diverse, with the number of introns ranging from 0 to 4. A fish Tlr9 gene containing two introns has been reported to express two alternatively spliced isoforms, namely gTLR9A (full-length) and gTLR9B (with a truncated C'-terminal signal transducing domain), whose regulation and function remain unclear. Here, we report a unique regulatory mechanism of gTLR9 signaling in orange-spotted grouper (Epinephelus coioides), whose gTlr9 sequence also contains two introns. We demonstrated that the grouper gTlr9 gene indeed has the capacity to produce two gTLR9 isoforms via alternative RNA splicing. We found that gTLR9B could function as a negative regulator to suppress gTLR9 signaling as demonstrated by the suppression of downstream gene expression. Following stimulation with CpG oligodeoxynucleotide (ODN), gTLR9A and gTLR9B were observed to translocate into endosomes and co-localize with ODN and the adaptor protein gMyD88. Both gTLR9A and gTLR9B could interact with gMyD88; however, gTLR9B could not interact with downstream IRAK4 and TRAF6. Further analysis of the expression profile of gTlr9A and gTlr9B upon immune-stimulation revealed that the two isoforms were differentially regulated in a time-dependent manner. Overall, these data suggest that fish TLR9B functions as a negative regulator, and that its temporal expression is mediated by alternative RNA splicing. This has not been observed in mammalian TLR9s and might have been acquired relatively recently in the evolution of fish.

Effect of sialylation on EGFR phosphorylation and resistance to tyrosine kinase inhibition.

Epidermal growth factor receptor (EGFR) is a heavily glycosylated transmembrane receptor tyrosine kinase. Upon EGF-binding, EGFR undergoes conformational changes to dimerize, resulting in kinase activation and autophosphorylation and downstream signaling. Tyrosine kinase inhibitors (TKIs) have been used to treat lung cancer by inhibiting EGFR phosphorylation. Previously, we demonstrated that EGFR sialylation suppresses its dimerization and phosphorylation. In this report, we further investigated the effect of sialylation on the phosphorylation profile of EGFR in TKI-sensitive and TKI-resistant cells. Sialylation was induced in cancer progression to inhibit the association of EGFR with EGF and the subsequent autophosphorylation. In the absence of EGF the TKI-resistant EGFR mutant (L858R/T790M) had a higher degree of sialylation and phosphorylation at Y1068, Y1086, and Y1173 than the TKI-sensitive EGFR. In addition, although sialylation in the TKI-resistant mutants suppresses EGFR tyrosine phosphorylation, with the most significant effect on the Y1173 site, the sialylation effect is not strong enough to stop cancer progression by inhibiting the phosphorylation of these three sites. These findings were supported further by the observation that the L858R/T790M EGFR mutant, when treated with sialidase or sialyltransferase inhibitor, showed an increase in tyrosine phosphorylation, and the sensitivity of the corresponding resistant lung cancer cells to gefitinib was reduced by desialylation and was enhanced by sialylation.

Transcriptome characterization and gene expression of Epinephelus spp in endoplasmic reticulum stress-related pathway during betanodavirus infection in vitro.

BACKGROUND: Grouper (Epinephelus spp) is an economically important fish species worldwide. However, viral pathogens such as nervous necrosis virus (NNV) have been causing severe infections in the fish, resulting in great loss in the grouper aquaculture industry. Yet, the understanding of the molecular mechanisms underlying the pathogenicity of NNV is still inadequate, mainly due to insufficient genomic information of the host. RESULTS: De novo assembly of grouper transcriptome in the grouper kidney (GK) cells was conducted by using short read sequencing technology of Solexa/Illumina. A sum of 66,582 unigenes with mean length of 603 bp were obtained, and were annotated according to Gene Ontology (GO) and Clusters of Orthologous Groups (COG). In addition, the tag-based digital gene expression (DGE) system was used to investigate the gene expression and pathways associated with NNV infection in GK cells. The analysis revealed endoplasmic reticulum (ER) stress response was prominently affected in NNV-infected GK cells. A further analysis revealed an interaction between the NNV capsid protein and the ER chaperone immunoglobulin heavy-chain binding protein (BiP). Furthermore, exogenous expression of NNV capsid protein was able to induce XBP-1 mRNA splicing in vivo, suggesting a role of the capsid protein in the NNV-induced ER stress. CONCLUSIONS: Our data presents valuable genetic information for Epinephelus spp., which will benefit future study in this non-model but economically important species. The DGE profile of ER stress response in NNV-infected cells provides information of many important components associated with the protein processing in ER. Specifically, we showed that the viral capsid protein might play an important role in the ER stress response.