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ObjectiveGastric cancer (GC) comprises multiple molecular subtypes. Recent studies have highlighted mesenchymal-subtype GC (Mes-GC) as a clinically aggressive subtype with few treatment options. Combining multiple studies, we derived and applied a consensus Mes-GC classifier to define the Mes-GC enhancer landscape revealing disease vulnerabilities.DesignTranscriptomic profiles of ~1000 primary GCs and cell lines were analysed to derive a consensus Mes-GC classifier. Clinical and genomic associations were performed across >1200 patients with GC. Genome-wide epigenomic profiles (H3K27ac, H3K4me1 and assay for transposase-accessible chromatin with sequencing (ATAC-seq)) of 49 primary GCs and GC cell lines were generated to identify Mes-GC-specific enhancer landscapes. Upstream regulators and downstream targets of Mes-GC enhancers were interrogated using chromatin immunoprecipitation followed by sequencing (ChIP-seq), RNA sequencing, CRISPR/Cas9 editing, functional assays and pharmacological inhibition.ResultsWe identified and validated a 993-gene cancer-cell intrinsic Mes-GC classifier applicable to retrospective cohorts or prospective single samples. Multicohort analysis of Mes-GCs confirmed associations with poor patient survival, therapy resistance and few targetable genomic alterations. Analysis of enhancer profiles revealed a distinctive Mes-GC epigenomic landscape, withTEAD1as a master regulator of Mes-GC enhancers and Mes-GCs exhibiting preferential sensitivity to TEAD1 pharmacological inhibition. Analysis of Mes-GC super-enhancers also highlightedNUAK1kinase as a downstream target, with synergistic effects observed between NUAK1 inhibition and cisplatin treatment.ConclusionOur results establish a consensus Mes-GC classifier applicable to multiple transcriptomic scenarios. Mes-GCs exhibit a distinct epigenomic landscape, and TEAD1 inhibition and combinatorial NUAK1 inhibition/cisplatin may represent potential targetable options.

3G study protocol

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Gastric cancer cases are often diagnosed at an advanced stage with poor prognosis. Platinum-based chemotherapy has been internationally accepted as first-line therapy for inoperable or metastatic gastric cancer. To achieve greater benefits, selection of patients eligible for this treatment is critical. Although gene expression profiling has been widely used as a genomic classifier to identify molecular subtypes of gastric cancer and to stratify patients for different chemotherapy regimens, its prediction accuracy can be improved. Adenosine-to-inosine (A-to-I) RNA editing has emerged as a new player contributing to gastric cancer development and progression, offering potential clinical utility for diagnosis and treatment. Using a systematic computational approach followed by both in vitro validations and in silico validations in The Cancer Genome Atlas (TCGA), we conducted a transcriptome-wide RNA editing analysis of a cohort of 104 patients with advanced gastric cancer and identified an RNA editing (GCRE) signature to guide gastric cancer chemotherapy. RNA editing events stood as a prognostic and predictive biomarker in advanced gastric cancer. A GCRE score based on the GCRE signature consisted of 50 editing sites associated with 29 genes, predicting response to chemotherapy with a high accuracy (84%). Of note, patients demonstrating higher editing levels of this panel of sites presented a better overall response. Consistently, gastric cancer cell lines with higher editing levels showed higher chemosensitivity. Applying the GCRE score on TCGA dataset confirmed that responders had significantly higher levels of editing in advanced gastric cancer. Overall, this newly defined GCRE signature reliably stratifies patients with advanced gastric cancer and predicts response from chemotherapy.Significance:This study describes a novel A-to-I RNA editing signature as a prognostic and predictive biomarker in advanced gastric cancer, providing a new tool to improve patient stratification and response to therapy.

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Supplementary Information, Figures 1-10 from Inhibition of Gastric Cancer Invasion and Metastasis by PLA2G2A, a Novel β-Catenin/TCF Target Gene

Elevated expression of the PLA2G2A phospholipase in gastric cancer (GC) is associated with improved patient survival. To elucidate function and regulation of PLA2G2A in GC, we analyzed a panel of GC cell lines. PLA2G2A was specifically expressed in lines with constitutive Wnt activity, implicating β-catenin–dependent Wnt signaling as a major upstream regulator of PLA2G2A expression. The invasive ability of PLA2G2A-expressing AGS cells was enhanced by PLA2G2A silencing, whereas cellular migration in non–PLA2G2A-expressing N87 cells was inhibited by enforced PLA2G2A expression, indicating that PLA2G2A is both necessary and sufficient to function as an inhibitor of GC invasion in vitro. We provide evidence that antiinvasive effect of PLA2G2A occurs, at least in part, through its ability to inhibit the S100A4 metastasis mediator gene. Consistent with its invasion inhibitor role, PLA2G2A expression was elevated in primary gastric, colon, and prostrate early-stage tumors, but was decreased in metastatic and late-stage tumors. There was a strong association between PLA2G2A promoter methylation status and PLA2G2A expression, suggesting that the loss of PLA2G2A expression in late-stage cancers may be due to epigenetic silencing. Supporting this, among the non–PLA2G2A-expressing lines, pharmacologic inhibition of epigenetic silencing reactivated PLA2G2A in Wnt-active lines, but in non–Wnt-active lines, a combination of Wnt hyperactivation and inhibition of epigenetic silencing were both required for PLA2G2A reactivation. Our results highlight the complexity of PLA2G2A regulation and provide functional evidence for PLA2G2A as an important regulator of invasion and metastasis in GC. [Cancer Res 2008;68(11):4277–86]

Objective Type 2 diabetes mellitus and nonalcoholic fatty liver disease (NAFLD) are closely related, and antidiabetic medications have been shown to be potential therapeutics in NAFLD. Using a network meta-analysis, we sought to examine the effectiveness of antidiabetic agents for the treatment of NAFLD in patients with type 2 diabetes mellitus. Methods Medline and Embase were searched for randomized controlled trials relating to the use of antidiabetic agents, including sodium-glucose transport protein 2 (SGLT2) inhibitors, glucagon-like peptide-1 receptor agonists, and peroxisome proliferator-activated receptor gamma (PPARγ) agonists, biguanides, sulfonylureas and insulin, on NAFLD in patients with diabetes. The p-score was used as a surrogate marker of effectiveness. Results A total of 14 articles were included in the analysis. PPARγ agonists were ranked as the best treatment in steatosis reduction, resulting in the greatest reduction of steatosis. There was statistical significance between PPARγ agonists [mean difference (MD): −6.02%, confidence interval (CI): −10.37% to −1.67%] and SGLT2 inhibitors (MD: −2.60%, CI: −4.87% to −0.33%) compared with standard of care for steatosis reduction. Compared with PPARγ agonists, SGLT2 inhibitors resulted in a statistical significant reduction in fibrosis (MD: −0.06, CI: −0.10 to −0.02). Body mass index reduction was highest in SGLT2 inhibitors and glucagon-like peptide-1 receptor agonists. Additionally, SGLT2 inhibitors were ranked as the best treatment for increasing high-density lipoprotein and reducing low-density lipoprotein. Conclusion Glucagon-like peptide-1 receptor agonists and SGLT2 inhibitors were suitable alternatives for the treatment of NAFLD in those with type 2 diabetes mellitus with a reduction in body mass index, fibrosis, and steatosis. SGLT2 inhibitors also have the added benefit of lipid modulation.

Small bowel adenocarcinoma (SBA) is a rare neoplasm that is associated with Crohn’s disease (CD). This study aims to quantify the prevalence of CD-SBA, review the current evidence of histopathology and molecular analysis findings, and identify the clinical presentation and outcomes of CD-SBA. Electronic databases Medline and Embase were searched for articles describing SBA in inflammatory bowel disease patients. The histopathology, molecular analysis findings, clinical presentation, prevalence, and outcomes of CD-SBA were extracted, and results were pooled with random effects. In total, 33 articles were included in the analysis. Prevalence of SBA was 1.15 (CI: 0.31–2.33) per 1000 CD patients. Only 11% (CI: 0.04–0.21) of CD-SBA patients had observable radiological features. CD-SBA was most commonly found in the ileum (84%), diagnosed at stage 2 (36%), with main presenting complaints including obstruction, weight loss, and abdominal pain. Significant histopathological findings included adjacent epithelial dysplasia, and an equal distribution of well-differentiated (49%) and poorly differentiated subtypes (46%). Most prevalent genetic mutation was KRAS mutation (18%), followed by mismatch repair deficiency (9.7%). The 5-year overall survival for CD-SBA patients was 29% (CI: 0.18–0.41), and 33% (CI: 0.26–0.41) for de novo SBA. No statistically significant increase in risk for CD-SBA was noted for treatment with thiopurines, steroids, and 5-ASA. Our meta-analysis found the prevalence of CD-SBA to be 1.15 per 1000 CD patients. The 5-year overall survival for CD-SBA was poor. The presenting symptoms were non-specific, and therefore the diagnosis requires a high index of suspicion.

Gastric cancer cases are often diagnosed at an advanced stage with poor prognosis. Platinum-based chemotherapy has been internationally accepted as first-line therapy for inoperable or metastatic gastric cancer. To achieve greater benefits, selection of patients eligible for this treatment is critical. Although gene expression profiling has been widely used as a genomic classifier to identify molecular subtypes of gastric cancer and to stratify patients for different chemotherapy regimens, its prediction accuracy can be improved. Adenosine-to-inosine (A-to-I) RNA editing has emerged as a new player contributing to gastric cancer development and progression, offering potential clinical utility for diagnosis and treatment. Using a systematic computational approach followed by both in vitro validations and in silico validations in The Cancer Genome Atlas (TCGA), we conducted a transcriptome-wide RNA editing analysis of a cohort of 104 patients with advanced gastric cancer and identified an RNA editing (GCRE) signature to guide gastric cancer chemotherapy. RNA editing events stood as a prognostic and predictive biomarker in advanced gastric cancer. A GCRE score based on the GCRE signature consisted of 50 editing sites associated with 29 genes, predicting response to chemotherapy with a high accuracy (84%). Of note, patients demonstrating higher editing levels of this panel of sites presented a better overall response. Consistently, gastric cancer cell lines with higher editing levels showed higher chemosensitivity. Applying the GCRE score on TCGA dataset confirmed that responders had significantly higher levels of editing in advanced gastric cancer. Overall, this newly defined GCRE signature reliably stratifies patients with advanced gastric cancer and predicts response from chemotherapy. Significance: This study describes a novel A-to-I RNA editing signature as a prognostic and predictive biomarker in advanced gastric cancer, providing a new tool to improve patient stratification and response to therapy.

ObjectiveGastric cancer (GC) is a leading cause of cancer mortality, withARID1Abeing the second most frequently mutated driver gene in GC. We sought to decipherARID1A-specific GC regulatory networks and examine therapeutic vulnerabilities arising fromARID1Aloss.DesignGenomic profiling of GC patients including a Singapore cohort (>200 patients) was performed to derive mutational signatures ofARID1Ainactivation across molecular subtypes. Single-cell transcriptomic profiles ofARID1A-mutated GCs were analysed to examine tumour microenvironmental changes arising fromARID1Aloss. Genome-wide ARID1A binding and chromatin profiles (H3K27ac, H3K4me3, H3K4me1, ATAC-seq) were generated to identify gastric-specific epigenetic landscapes regulated by ARID1A. Distinct cancer hallmarks ofARID1A-mutated GCs were converged at the genomic, single-cell and epigenomic level, and targeted by pharmacological inhibition.ResultsWe observed prevalentARID1Ainactivation across GC molecular subtypes, with distinct mutational signatures and linked to a NFKB-driven proinflammatory tumour microenvironment.ARID1A-depletion caused loss of H3K27ac activation signals atARID1A-occupied distal enhancers, but unexpectedly gain of H3K27ac at ARID1A-occupied promoters in genes such asNFKB1andNFKB2. Promoter activation inARID1A-mutated GCs was associated with enhanced gene expression, increased BRD4 binding, and reduced HDAC1 and CTCF occupancy. Combined targeting of promoter activation and tumour inflammation via bromodomain and NFKB inhibitors confirmed therapeutic synergy specific toARID1A-genomic status.ConclusionOur results suggest a therapeutic strategy forARID1A-mutated GCs targeting both tumour-intrinsic (BRD4-assocatiated promoter activation) and extrinsic (NFKB immunomodulation) cancer phenotypes.

The extension of lifespan by means of calorie restriction (CR) is one of the most replicable mechanisms across a range of different taxa. However, the effects of this on the next generation are less studied due to the complexities associated with such an experiment. In this study, the effects of CR and ad libitum (AL) feeding on lifespan, duration of ovipositional stages, and fecundity of a predatory mite, Neoseiulus cucumeris were investigated. The egg volume, sex ratio, and survival rate of the offspring were then compared across the different treatments. The three treatments were: (1) 40 prey Tyrophagus putrescentiae eggs per day representing the AL treatment; (2) 10 eggs per day representing medium CR; and (3) 5 eggs per day being high CR. To assess for transgenerational effects, the eggs produced under the three different treatments were fed AL and reared until adulthood or death. Mothers under the medium CR treatment had a lifespan that almost doubled the lifespan of mothers fed AL. Contrastingly, a higher reproductive rate was seen in mothers under AL, which also had a significantly higher total fecundity when compared with both CR treatments. The AL mothers had a shorter gestational period and produced larger eggs. There was no difference between the sex ratio of offspring for the medium CR and AL treatments. These results indicate the different effects of the two levels of CR and highlight the trade-offs that are accompanied with a longer lifespan due to caloric restriction.

Background: DNA methylation signatures describing distinct histological subtypes of oesophageal cancer have been reported. We studied DNA methylation in samples from the MRC OE02 phase III trial, which randomised patients with resectable oesophageal cancer to surgery alone (S) or neoadjuvant chemotherapy followed by surgery (CS).Aim: The aim of the study was to identify epigenetic signatures predictive of chemotherapy benefit in patients with oesophageal adenocarcinoma (OAC) from the OE02 trial and validate the findings in an independent cohort.Methods: DNA methylation was analysed using the Illumina GoldenGate platform on surgically resected OAC specimens from patients in the OE02 trial. Cox proportional hazard analysis was performed to select probes predictive of survival in the CS arm. Non-negative matrix factorisation was used to perform clustering and delineate DNA methylation signatures. The findings were validated in an independent cohort of patients with gastroesophageal adenocarcinoma treated with neoadjuvant chemotherapy.Results: A total of 229 patients with OAC were analysed from the OE02 trial (118 in the CS arm and 111 in the S arm). There was no difference in DNA methylation status between the CS and S arms. A metagene signature was created by dichotomising samples into two clusters. In cluster 1, patients in the CS arm had significant overall survival (OS) benefit (median OS CS: 931 days vs. S: 536 days [HR: 1.54, P = 0.031]). In cluster 2, patients in the CS arm had similar (or worse) OS compared with patients in the S arm (CS: 348 days vs. S: 472 days [HR: 0.70, P = 0.1], and test of interaction was significant (p = 0.005). In the validation cohort (n = 13), there was no difference in DNA methylation status in paired pre- and post-treatment samples. When the epigenetic signature was applied, cluster 1 samples had better OS (median OS, cluster 1: 1174 days vs. cluster 2: 392 days, HR: 3.47, p = 0.059)Conclusions: This is the first and largest study of DNA methylation in patients with OAC uniformly treated in a randomised phase III trial. We identified an epigenetic signature that may serve as a predictive biomarker for chemotherapy benefit in OAC. (C) 2019 Elsevier Ltd. All rights reserved.

UV/cold shock-mediated frostbite involves non-apoptotic nuclear bubbling cell death (BCD) and participation of functional WWOX in cells (WWOXf). In contrast, cells with WWOX deficiency or dysfunction (WWOXd) undergo pop-out explosion death (POD). Here, by time-lapse microscopy, when WWOXf cells were exposed to UV or UV/cold shock and then incubated at room temperature, these cells rapidly and sequentially underwent: 1) loss of mitochondrial membrane potential, 2) formation of a nitric oxide (NO)-containing nuclear bubble per cell, 3) WWOX-dependent increase in calcium (Ca2+) influx, 4) shutdown of mRNA and protein synthesis machinery, as determined by RT/PCR and gene chip analysis, and 5) eventual cell death without caspase activation, stress fiber formation and chromosomal DNA fragmentation. In contrast, WWOXd cells exhibited a faster kinetics of stress fiber formation, explosion and death without NO production. Ectopic WWOX restored calcium influx and nuclear bubbling in WWOXd cells. In hairless mice, UV/cold shock rapidly downregulated protein expression in the skin and then liver, which may lead to organ damages. UV/cold shock induced complex formation of antiapoptotic TRAF2 and proapoptotic WWOX and their co-translocation to the nucleus, where the complex dissociation occurred. The observations suggest that WWOX and TRAF2 dissociation is needed for nuclear bubbling and death.

Small bowel adenocarcinoma (SBA) is a rare neoplasm that is associated with Crohn's disease (CD). This study aims to quantify the prevalence of CD-SBA, review the current evidence of histopathology and molecular analysis findings, and identify the clinical presentation and outcomes of CD-SBA.Electronic databases Medline and Embase were searched for articles describing SBA in inflammatory bowel disease patients. The histopathology, molecular analysis findings, clinical presentation, prevalence, and outcomes of CD-SBA were extracted, and results were pooled with random effects.In total, 33 articles were included in the analysis. Prevalence of SBA was 1.15 (CI: 0.31-2.33) per 1000 CD patients. Only 11% (CI: 0.04-0.21) of CD-SBA patients had observable radiological features. CD-SBA was most commonly found in the ileum (84%), diagnosed at stage 2 (36%), with main presenting complaints including obstruction, weight loss, and abdominal pain. Significant histopathological findings included adjacent epithelial dysplasia, and an equal distribution of well-differentiated (49%) and poorly differentiated subtypes (46%). Most prevalent genetic mutation was KRAS mutation (18%), followed by mismatch repair deficiency (9.7%). The 5-year overall survival for CD-SBA patients was 29% (CI: 0.18-0.41), and 33% (CI: 0.26-0.41) for de novo SBA. No statistically significant increase in risk for CD-SBA was noted for treatment with thiopurines, steroids, and 5-ASA.Our meta-analysis found the prevalence of CD-SBA to be 1.15 per 1000 CD patients. The 5-year overall survival for CD-SBA was poor. The presenting symptoms were non-specific, and therefore the diagnosis requires a high index of suspicion.

ObjectiveGastric cancer (GC) is a leading cause of cancer mortality. Previous studies have shown that hepatocyte nuclear factor-4α (HNF4α) is specifically overexpressed in GC and functionally required for GC development. In this study, we investigated, on a genome-wide scale, target genes of HNF4α and oncogenic pathways driven by HNF4α and HNF4α target genes.DesignWe performed HNF4α chromatin immunoprecipitation followed by sequencing across multiple GC cell lines, integrating HNF4α occupancy data with (epi)genomic and transcriptome data of primary GCs to define HNF4α target genes of in vitro and in vivo relevance. To investigate mechanistic roles of HNF4α and HNF4α targets, we performed cancer metabolic measurements, drug treatments and functional assays including murine xenograft experiments.ResultsGene expression analysis across 19 tumour types revealed HNF4α to be specifically upregulated in GCs. Unbiased pathway analysis revealed organic acid metabolism as the top HNF4α-regulated pathway, orthogonally supported by metabolomic analysis. Isocitrate dehydrogenase 1 (IDH1) emerged as a convergent HNF4α direct target gene regulating GC metabolism. We show that wild-type IDH1 is essential for GC cell survival, and that certain GC cells can be targeted by IDH1 inhibitors.ConclusionsOur results highlight a role for HNF4α in sustaining GC oncogenic metabolism, through the regulation of IDH1. Drugs targeting wild-type IDH1 may thus have clinical utility in GCs exhibiting HNF4α overexpression, expanding the role of IDH1 in cancer beyond IDH1/2 mutated malignancies.

Purpose: The oxaliplatin plus S-1 and cisplatin plus S-1 regimens are interchangeably used in the management of advanced gastric cancer. The previously reported G-intestinal (G1) and G-diffuse (G2) intrinsic gene expression signatures showed promise for stratifying patients according to their tumor sensitivity to oxaliplatin or cisplatin. Experimental Design: The proof-of-concept, multicenter, open-label phase II “3G” trial was done to prospectively evaluate the feasibility and efficacy of using genomic classifiers to tailor treatment in gastric cancer. Patients’ tumors were classified as “G1” or “G2” using a nearest-prediction template method, or “G3” (unclear assignment) when FDR ≥ 0.05. The first 30 patients in the “G1” cohort were assigned oxaliplatin plus S-1 (SOX) chemotherapy; thereafter, subsequently recruited “G1” patients were treated with cisplatin plus S-1 (SP) chemotherapy. “G2” patients and “G3” patients were treated with SP and SOX chemotherapy, respectively. Results: A total of 48, 21, and 12 patients, respectively, were given “G1,” “G2,” and “G3” genomic assignments. Median turnaround time was 7 days (IQR, 5–9). Response rates were 44.8%, 8.3%, 26.7%, and 55.6% for the “G1-SOX,” “G1-SP,” “G2,” “G3” cohorts, respectively; and was higher in G1 patients treated with SOX compared with SP (P = 0.033). Exploratory analyses using the genomic classifier of Lei and colleagues validated the utility of the metabolic signature as a biomarker for predicting benefit from chemotherapy (log-rank P = 0.004 for PFS), whereas the Asian Cancer Research Group classifier did not demonstrate any predictive value. Conclusions: This bench-to-bedside effort establishes a reasonable turnaround time for gene expression profiling and possible utility of genomic classifiers in gastric cancer treatment stratification. Clin Cancer Res; 24(21); 5272–81. ©2018 AACR.

ObjectiveNon-alcoholic fatty liver disease is highly prevalent in patients with type 2 diabetes mellitus. Studies on glucagon-like peptide-1 receptor agonists for the treatment of non-alcoholic fatty liver disease have reported promising results. Despite this, there has been limited evidence of its efficacy in non-alcoholic fatty liver disease patients with type 2 diabetes mellitus. This meta-analysis examined existing evidence on the efficacy of glucagon-like peptide-1 receptor agonists on the management of non-alcoholic fatty liver disease in patients with type 2 diabetes mellitus.MethodsMedline, Embase and Cochrane Central Register of Controlled Trials (CENTRAL) were searched for articles discussing the efficacy of glucagon-like peptide-1 receptor agonists on non-alcoholic fatty liver disease in patients with type 2 diabetes mellitus. Values of standardized mean differences (SMD) and risk ratio (RR) were determined for continuous outcomes and dichotomous outcomes respectively.Results8 studies involving 1,454 patients from 5 randomized controlled trials and 3 cohort studies were included in the analysis. Our analysis found significant improvements in hepatic fat content, liver biochemistry, body composition, glucose parameters, lipid parameters, insulin sensitivity and inflammatory markers following glucagon-like peptide-1 receptor agonist treatment. Glucagon-like peptide-1 receptor agonists significantly decreased hepatic fat content compared to metformin and insulin-based therapies. Glucagon-like peptide-1 receptor agonists also improved fibrosis markers, but this did not reach statistical significance.ConclusionWith a high prevalence of obesity and non-alcoholic fatty liver disease among patients with type 2 diabetes mellitus, glucagon-like peptide-1 receptor agonist treatment shows promise in improving both diabetes and non-alcoholic fatty liver disease phenotype.

Background CIMP (CpG island methylator phenotype) is an epigenetic molecular subtype, observed in multiple malignancies and associated with the epigenetic silencing of tumor suppressors. Currently, for most cancers including gastric cancer (GC), mechanisms underlying CIMP remain poorly understood. We sought to discover molecular contributors to CIMP in GC, by performing global DNA methylation, gene expression, and proteomics profiling across 14 gastric cell lines, followed by similar integrative analysis in 50 GC cell lines and 467 primary GCs. Results We identify the cystathionine beta-synthase enzyme (CBS) as a highly recurrent target of epigenetic silencing in CIMP GC. Likewise, we show that CBS epimutations are significantly associated with CIMP in various other cancers, occurring even in premalignant gastroesophageal conditions and longitudinally linked to clinical persistence. Of note, CRISPR deletion of CBS in normal gastric epithelial cells induces widespread DNA methylation changes that overlap with primary GC CIMP patterns. Reflecting its metabolic role as a gatekeeper interlinking the methionine and homocysteine cycles, CBS loss in vitro also causes reductions in the anti-inflammatory gasotransmitter hydrogen sulfide (H2S), with concomitant increase in NF-κB activity. In a murine genetic model of CBS deficiency, preliminary data indicate upregulated immune-mediated transcriptional signatures in the stomach. Conclusions Our results implicate CBS as a bi-faceted modifier of aberrant DNA methylation and inflammation in GC and highlights H2S donors as a potential new therapy for CBS-silenced lesions.

Peptide vaccines are safe, and aim to elicit and expand tumor-specific immunity so as to eradicate tumors. However, achieving strong and long-lasting anti-tumor immunity with peptide vaccines for the antigen-specific treatment of cancer is challenging, in part because their efficacy depends on strong adjuvants or immunomodulators. We approached this problem by conjugating an epitope-based cancer vaccine with a lipidated sequence (an immunomodulator) to elicit a strong immune response. Lipidated and non-lipidated polyepitope proteins were generated that contained the universal T helper cell epitope (pan-DR), B cell epitopes, and the extended loop sequence of extracellular domain 2 of tumor-associated antigen L6 (TAL6). We show that the lipidated polyepitope cancer vaccine can activate bone marrow-derived dendritic cells, and trigger effective antigen-specific antibody and T helper cell responses, more effectively than the non-lipidated vaccine. Moreover, potent T cell immune responses were elicited in mice inoculated with the lipidated polyepitope cancer vaccine, providing protective antitumor immunity in mice bearing TAL6 tumors. Our study demonstrates that a lipidated polyepitope cancer vaccine could be employed to generate potent anti-tumor immune responses, including humoral and cellular immunity, which could be beneficial in the treatment of TAL6+ cancer.

To examine risk factors that might be associated with thyroid eye disease (TED) in patients with Graves' disease (GD), which may guide physicians in the prevention and management of TED.Medline and Embase were searched for articles discussing risk factors of TED. Comparisons were made between GD patients with and without TED, and between active and inactive TED GD patients. Weighted mean differences (WMDs) and odds ratios (ORs) were determined for continuous and dichotomous outcomes, respectively. Results were pooled with random effects using the DerSimonian and Laird model.Fifty-six articles were included in the analysis. Smoking, inclusive of current and previous smoking status, was a significant risk factor for TED (OR: 2.401; CI: 1.958-2.945; P.001). Statistical significance was found upon meta-regression between male sex and the odds of smoking and TED (β = 1.195; SE = 0.436; P = .013). Other risk factors were also examined, and patients with TED were significantly older than those without TED (WMD: 1.350; CI: 0.328-2.372; P = .010). While both age (WMD: 5.546; CI: 3.075-8.017; P.001) and male sex (OR: 1.819; CI: 1.178-2.808; P = .007) were found to be significant risk factors for active TED patients compared to inactive TED patients, no statistical significance was found for family history, thyroid status, cholesterol levels, or body mass index.Factors such as smoking, sex, and age predispose GD patients to TED, and TED patients to active TED. A targeted approach in the management of GD and TED is required to reduce the modifiable risk factor of smoking.

Synthetic Zfra4-10 and WWOX7-21 peptides strongly suppress cancer growth in vivo. Hypothetically, Zfra4-10 binds to the membrane Hyal-2 of spleen Z cells and activates the Hyal-2/WWOX/SMAD4 signaling for cytotoxic Z cell activation to kill cancer cells. Stimulation of membrane WWOX in the signaling complex by a WWOX epitope peptide, WWOX7-21, is likely to activate the signaling. Here, mice receiving Zfra4-10 or WWOX7-21 peptide alone exhibited an increased binding of endogenous tumor suppressor WWOX with ERK, C1qBP, NF-&kappa, B, Iba1, p21, CD133, JNK1, COX2, Oct4, and GFAP in the spleen, brain, and/or lung which led to cancer suppression. However, when in combination, Zfra4-10 and WWOX7-21 reduced the binding of WWOX with target proteins and allowed tumor growth in vivo. In addition to Zfra4-10 and WWOX7-21 peptides, stimulating the membrane Hyal-2/WWOX complex with Hyal-2 antibody and sonicated hyaluronan (HAson) induced Z cell activation for killing cancer cells in vivo and in vitro. Mechanistically, Zfra4-10 binds to membrane Hyal-2, induces dephosphorylation of WWOX at pY33 and pY61, and drives Z cell activation for the anticancer response. Thus, Zfra4-10 and WWOX7-21 peptides, HAson, and the Hyal-2 antibody are of therapeutic potential for cancer suppression.

Objective Thyroid eye disease (TED) is a debilitating condition that frequently manifests in patients suffering from Graves' disease (GD). This study aims to analyse the prevalence of TED among GD patients, with a focus on geographical region-specific rates. Methods Medline and Embase were searched for articles examining TED prevalence on April 2020, and articles were retrieved and sieved. Statistical analysis was performed after Freeman-Tukey double arcsine transformation. Thereafter, results were pooled with random effects by DerSimonian and Laird model. Results Fifty-seven articles involving 26,804 patients were included in the review. The overall pooled prevalence of TED was 40% (CI: 0.32 to 0.48) and by continent was 38% (CI: 0.31 to 0.46) for Europe, 44% (CI: 0.32 to 0.56) for Asia, 27% (CI: 0.06 to 0.56) for North America and 58% (CI: 0.55 to 0.61) for Oceania. The prevalence of TED in Southeast Asia was 35% (CI: 0.24 to 0.47) and Middle East 48% (CI: 0.19 to 0.78). Subgroup analysis showed regions with predominantly Caucasians (37%; CI: 0.28 to 0.46) had a lower prevalence of TED compared to Asians (45%; CI: 0.33 to 0.58). The pooled prevalence of lid retraction was 57% (CI: 0.39 to 0.74), proptosis 57% (CI: 0.48 to 0.65), diplopia 36% (CI: 0.24 to 0.48) and ocular hypertension 13% (CI: 0.06 to 0.19). Conclusion A substantial proportion of patients with GD have TED and often manifest as lid retraction, proptosis and diplopia. Early detection through active screening might help to mitigate the progression of TED and its associated complications.

Due to issues with establishment and persistence of natural enemies in biological control, the provision of alternative food sources and oviposition sites are important factors to enhance pest control. In this study, three different supplementation treatments were examined for their ability to increase the populations of the predatory mite Amblydromalus limonicus, and its implications for greenhouse whitefly control on peppers and eggplants. These were: (1) pollen (Typha orientalis), (2) pollen and thread, (3) pollen, thread, and a substrate mixture of buckwheat, gorse, and rice husks, which were compared to a control treatment that had no supplementation. Significant treatment effects were found on pepper for A. limonicus (mite eggs p = 0.008, mobile mites p =

Membrane hyaluronidase Hyal-2 supports cancer cell growth. Inhibition of Hyal-2 by specific antibody against Hyal-2 or pY216-Hyal-2 leads to cancer growth suppression and prevention in vivo. By immunoelectron microscopy, tumor suppressor WWOX is shown to be anchored, in part, in the cell membrane by Hyal-2. Alternatively, WWOX undergoes self-polymerization and localizes in the cell membrane. Proapoptotic pY33-WWOX binds Hyal-2, and TGF-&beta, induces internalization of the pY33-WWOX/Hyal-2 complex to the nucleus for causing cell death. In contrast, when pY33 is downregulated and pS14 upregulated in WWOX, pS14-WWOX supports cancer growth in vivo. Here, we investigated whether membrane WWOX receives extracellular signals via surface-exposed epitopes, especially at the S14 area, that signals for cancer growth suppression and prevention. By using a simulated 3-dimentional structure and generated specific antibodies, WWOX epitopes were determined at amino acid #7 to 21 and #286 to 299. Synthetic WWOX7-21 peptide, or truncation to 5-amino acid WWOX7-11, significantly suppressed and prevented the growth and metastasis of melanoma and skin cancer cells in mice. Time-lapse microscopy revealed that WWOX7-21 peptide potently enhanced the explosion and death of 4T1 breast cancer stem cell spheres by ceritinib. This is due to rapid upregulation of proapoptotic pY33-WWOX, downregulation of prosurvival pERK, prompt increases in Ca2+ influx, and disruption of the IkB&alpha, /WWOX/ERK prosurvival signaling. In contrast, pS14-WWOX7-21 peptide dramatically increased cancer growth in vivo and protected cancer cells from ceritinib-mediated apoptosis in vitro, due to a prolonged ERK phosphorylation. Further, specific antibody against pS14-WWOX significantly enhanced the ceritinib-induced apoptosis. Together, the N-terminal epitopes WWOX7-21 and WWOX7-11 are potent in blocking cancer growth in vivo. WWOX7-21 and WWOX7-11 peptides and pS14-WWOX antibody are of therapeutic values in suppressing and preventing cancer growth in vivo.

Introduction Zinc finger-like protein that regulates apoptosis (Zfra) is a naturally occurring 31-amino-acid protein. Synthetic peptides Zfra1–31 and Zfra4–10 are known to effectively block the growth of many types of cancer cells. Methods Ten-month-old triple-transgenic (3×Tg) mice for Alzheimer's disease (AD) received synthetic Zfra peptides via tail vein injections, followed by examining restoration of memory deficits. Results Zfra significantly downregulated TRAPPC6AΔ, SH3GLB2, tau, and amyloid β (Αβ) aggregates in the brains of 3×Tg mice and effectively restored their memory capabilities. Zfra inhibited melanoma-induced neuronal death in the hippocampus and plaque formation in the cortex. Mechanistically, Zfra blocked the aggregation of amyloid β 42 and many serine-containing peptides in vitro, suppressed tumor necrosis factor–mediated NF-κB activation, and bound cytosolic proteins for accelerating their degradation in ubiquitin/proteasome-independent manner. Discussion Zfra peptides exhibit a strong efficacy in blocking tau aggregation and amyloid Αβ formation and restore memory deficits in 3×Tg mice, suggesting its potential for treatment of AD.