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Abstract The genetic contribution of protein-coding variants to immune-mediated diseases (IMDs) remains underexplored. Through whole exome sequencing of 40 IMDs in 350,770 UK Biobank participants, we identified 162 unique genes in 35 IMDs, among which 124 were novel genes. Several genes, including FLG which is associated with atopic dermatitis and asthma, showed converging evidence from both rare and common variants. 91 genes exerted significant effects on longitudinal outcomes (interquartile range of Hazard Ratio: 1.12-5.89). Mendelian randomization identified five causal genes, of which four were approved drug targets (CDSN, DDR1, LTA, and IL18BP). Proteomic analysis indicated that mutations associated with specific IMDs might also affect protein expression in other IMDs. For example, DXO (celiac disease-related gene) and PSMB9 (alopecia areata-related gene) could modulate CDSN (autoimmune hypothyroidism-, psoriasis-, asthma-, and Graves’ disease-related gene) expression. Identified genes predominantly impact immune and biochemical processes, and can be clustered into pathways of immune-related, urate metabolism, and antigen processing. Our findings identified protein-coding variants which are the key to IMDs pathogenesis and provided new insights into tailored innovative therapies.

Abstract The development of broad-spectrum coronavirus vaccines is essential to prepare for future respiratory virus pandemics. We demonstrated broad neutralization by a trivalent subunit vaccine, formulating the receptor-binding domains of SARS-CoV, MERS-CoV, and SARS-CoV-2 XBB.1.5 with Alum and CpG55.2. Vaccinated mice produced cross-neutralizing antibodies against all three human Betacoronaviruses and others currently exclusive to bats, indicating the epitope preservation of the individual antigens during co-formulation and the potential for epitope broadening.

Hepatocellular carcinoma (HCC) is one of the deadliest cancers. For patients with advanced HCC, liver function decompensation often occurs, which leads to poor tolerance to chemotherapies and other aggressive treatments. Therefore, it remains critical to develop effective therapeutic strategies for HCC. Etiological factors for HCC are complex and multifaceted, including hepatitis virus infection, alcohol, drug abuse, chronic metabolic abnormalities, and others. Thus, HCC has been categorized as a “genomically unstable” cancer due to the typical manifestation of chromosome breakage and aneuploidy, and oxidative DNA damage. In recent years, immunotherapy has provided a new option for cancer treatments, and the degree of genomic instability positively correlates with immunotherapy efficacies. This article reviews the endogenous and exogenous causes that affect the genomic stability of liver cells; it also updates the current biomarkers and their detection methods for genomic instabilities and relevant applications in cancer immunotherapies. Including genomic instability biomarkers in consideration of cancer treatment options shall increase the patients’ well-being.

Introduction The development of a yeast-expressed recombinant protein-based vaccine technology co-developed with LMIC vaccine producers and suitable as a COVID-19 vaccine for global access is described. The proof-of-concept for developing a SARS-CoV-2 spike protein receptor-binding domain (RBD) antigen as a yeast-derived recombinant protein vaccine technology is described. Areas Covered Genetic Engineering: The strategy is presented for the design and genetic modification used during cloning and expression in the yeast system. Process and Assay Development: A summary is presented of how a scalable, reproducible, and robust production process for the recombinant protein COVID-19 vaccine antigen was developed. Formulation and Pre-clinical Strategy: We report on the pre-clinical and formulation strategy used for the proof-of-concept evaluation of the SARS-CoV-2 RBD vaccine antigen. Technology Transfer and Partnerships: The process used for the technology transfer and co-development with LMIC vaccine producers is described. Clinical Development and Delivery: The approach used by LMIC developers to establish the industrial process, clinical development, and deployment is described. Expert Opinion Highlighted is an alternative model for developing new vaccines for emerging infectious diseases of pandemic importance starting with an academic institution directly transferring their technology to LMIC vaccine producers without the involvement of multinational pharma companies.

In response to emphasis for internationalization in higher education and cultivating workplace competencies, English has become an essential skill for college students nowadays. Although the university library provides abundant digital collection for English learning, not many college students use these resources frequently. Many students graduate with a lower level of English than what expected. This study takes students at Fu Jen Catholic University to explore their learning motivation for English, and the use of digital learning resources in library. The study adopted a questionnaire survey method to assess the responses of college students through 5-point Likert Scale. The items cover aspects of English learning experience, English learning motivation, English learning obstacles, the use of English digital learning resources in Fu Jen Catholic University Library, and expectations for the promotion of English digital learning resources. A total of 296 valid questionnaires were collected in the study. Based on the study results, suggestions were proposed for English teaching in higher education, students, libraries, and future research respectively.

IntroductionThe MET exon 14 skipping (METex14) mutation is an important oncogenic driver in lung cancer. We performed a retrospective analysis of clinical data from lung cancer patients with the METex14 mutation to analyze their survival outcomes and associated prognostic factors.MethodsA one-step reverse transcription-polymerase chain reaction to examine the presence of the METex14 mutation was performed using RNA samples from 1374 lung cancer patients with no detected EGFR and ALK mutations. Pathological features and immunohistochemistry (IHC) results for c-MET were analyzed in patients with METex14-positive tumors.ResultsMETex14 was identified in 69 patients with lung cancer, including 53 adenocarcinoma (ADC) and 16 non-ADC patients. In comparison with patients without the METex14 mutation, lung cancer patients harboring the METex14 mutation were generally elderly individuals, never-smokers, and had poor performance scores. A higher frequency of METex14 mutations was detected in pulmonary sarcomatoid carcinoma (PSC) patients (24.3%, n = 9/37). However, stage IV PSC patients with or without the METex14 mutations showed similarly poor overall survival (OS) (p = 0.429). For all 36 METex14-positive lung ADCs, multivariate analysis showed several poor prognostic factors, including strong c-MET IHC staining (p = 0.006), initial brain metastasis (p = 0.005), and administration of only supportive care (p < 0.001). After excluding seven patients who received only supportive care, we further analyzed 29 stage IV lung ADC patients with METex14 mutations who received anti-cancer treatment. Multivariate analysis showed that pemetrexed treatment (p = 0.003), lung radiotherapy (p = 0.020), initial brain metastasis (p = 0.005), and strong c-MET IHC staining (p = 0.012) were independent prognostic factors for OS in these patients.ConclusionsA higher frequency of METex14 mutations was detected in PSC patients. Stage IV PSC patients with or without the METex14 mutations had similarly poor overall survival. Pemetrexed-based chemotherapy, strong c-MET ICH staining, initial brain metastasis, and lung radiotherapy, may help predict survival outcomes in patients with advanced lung ADCs harboring the METex14 mutation.

Abstract Neoadjuvant immunotherapy and chemotherapy have improved the major pathological response (MPR) in patients with early-stage operable non-small cell lung cancer (NSCLC). This study aimed to assess whether the presence of targetable driver mutations affects the efficacy of the combination of immunotherapy and chemotherapy. We enrolled patients with early-stage operable NSCLC who received preoperative neoadjuvant therapy between January 1, 2017, and December 30, 2020. Neoadjuvant therapy was delivered with platinum-doublet chemotherapy; moreover, pembrolizumab was added at the attending physician’s discretion based on patient’s request. Pathological responses were assessed; moreover, disease-free survival was estimated. Next-generation sequencing was performed in case sufficient preoperative biopsy specimens were obtained. We included 23 patients; among them, 11 received a combination of neoadjuvant immunotherapy and chemotherapy while 12 received neoadjuvant chemotherapy alone. The MPR and pathological complete response rates were 54.5% and 27.3%, respectively, in patients who received a combination of neoadjuvant immunotherapy and chemotherapy. These rates were significantly higher than those in patients who only received neoadjuvant chemotherapy. Three patients in the combination group experienced disease recurrence during the follow-up period even though two of them showed an MPR. These three patients had targetable driver mutations, including an EGFR exon 20 insertion, EGFR exon 21 L858R substitution, and MET exon 14 skipping. Only one patient who remained disease-free had a targetable driver mutation. Among patients with early-stage operable NSCLC requiring neoadjuvant therapy, comprehensive genomic profiling is crucial before the administration of the combination of neoadjuvant immunotherapy and chemotherapy.

Microsatellite instability (MSI) is the primary predictive biomarker for therapeutic efficacies of cancer immunotherapies. Establishment of the MSI detection methods with high sensitivity and accessibility is important. Because MSI is mainly caused by defects in DNA mismatch repair (MMR), immunohistochemical (IHC) staining for the MMR proteins has been widely employed to predict the responses to immunotherapies. Thus, due to the high sensitivity of PCR, the MSI-PCR analysis has also been recommended as the primary approach as MMR IHC. This study aimed to develop a sensitive and convenient platform for daily MSI-PCR services. The routine workflow used a non-labeling QIAxcel capillary electrophoresis system which did not need the fluorescence labeling of the DNA products or usage of a multi-color fluorescence reader. Furthermore, the 15 and 1000 bp size alignment markers were used to precisely detect the size of the DNA product. A cohort of 336 CRC cases was examined by MSI-PCR on the five mononucleotide MSI markers recommended by ESMO. The PCR products were analyzed in the screening gels, followed by high-resolution gel electrophoresis for confirmation if needed. In the MSI-PCR tests, 90.1% (303/336) cases showed clear major shift patterns in the screening gels, and only 33 cases had to be re-examined using the high-resolution gels. The cohort was also analyzed by MMR IHC is, which revealed 98.5% (331/336) concordance with MSI-PCR. In the five discordant cases, 4 (3 MSI-L and 1 MSS) showed MSH6 loss. Besides, one case exhibited MSI-H but no loss in the MMR IHC. Further NGS analysis, in this case, found that missense and frameshift mutations in the PMS2 and MSH6 genes occurred, respectively. In conclusion, the non-labeling MSI-PCR capillary electrophoresis revealed high concordance with the MMR IHC analysis and is cost- and time-effective. Therefore, it shall be highly applicable in clinical laboratories.

Tc24 is a Trypanosoma cruzi-derived flagellar protein that, when formulated with a TLR-4 agonist adjuvant, induces a balanced immune response in mice, elevating IgG2a antibody titers and IFN-γ levels. Furthermore, vaccination with the recombinant Tc24 protein can reduce parasite levels and improve survival during acute infection. Although some mRNA vaccines have been proven to elicit a stronger immune response than some protein vaccines, they have not been used against T. cruzi. This work evaluates the immunogenicity of a heterologous prime/boost vaccination regimen using protein and mRNA-based Tc24 vaccines. Mice (C57BL/6) were vaccinated twice subcutaneously, three weeks apart, with either the Tc24-C4 protein + glucopyranosyl A (GLA)-squalene emulsion, Tc24 mRNA Lipid Nanoparticles, or with heterologous protein/mRNA or mRNA/protein combinations, respectively. Two weeks after the last vaccination, mice were euthanized, spleens were collected to measure antigen-specific T-cell responses, and sera were collected to evaluate IgG titers and isotypes. Heterologous presentation of the Tc24 antigen generated antigen-specific polyfunctional CD8+ T cells, a balanced Th1/Th2/Th17 cytokine profile, and a balanced humoral response with increased serum IgG, IgG1 and IgG2c antibody responses. We conclude that heterologous vaccination using Tc24 mRNA to prime and Tc24-C4 protein to boost induces a broad and robust antigen-specific immune response that was equivalent or superior to two doses of a homologous protein vaccine, the homologous mRNA vaccine and the heterologous Tc24-C4 Protein/mRNA vaccine.

Clonality assessment, which can detect neoplastic T cells by identifying the uniquely recombined T-cell receptor (TCR) genes, provides important support in the diagnosis of T-cell lymphoma (TCL). BIOMED-2 is the gold standard clonality assay and has proven to be effective in European TCL patients. However, we failed to prove its sensitivity in Taiwanese TCL patients, especially based on the TCRβ gene. To explore potential impact of genetic background in the BIOMED-2 test, we analyzed TCRβ sequences of 21 healthy individuals and two TCL patients. This analysis suggests that genetic variations in the BIOMED-2 primer sites could not explain the difference in sensitivity. The BIOMED-2 test results of the two TCL patients were positive and negative, respectively. Interestingly, a higher percentage (>81%) of non-recombined TCRβ sequences was observed in the test-negative patient than those of the test-positive patient and all healthy individuals (13~66%). The result suggests a new TCR target for enhancing TCL diagnosis. To further explore the hypothesis, we proposed a cost-effective digital PCR assay that quantifies the relative abundance of non-recombined TCRβ sequences containing a J2-2P~J2-3 segment. With the digital PCR assay, bone marrow specimens from TCL patients (n=9) showed a positive outcome (i.e., the relative abundance of the J2-2P~J2-3 sequences ≧5%), whereas non-TCL patients (n=6) gave a negative result. As five of nine TCL patients had a negative BIOMED-2 test result, the J2-2P~J2-3 sequences may improve TCL detection. This is the first report showing the capability of characterizing non-recombined TCR sequences as a supplementary strategy for the BIOMED-2 clonality test.

(1) Background: We previously reported the development of a recombinant protein SARS-CoV-2 vaccine, consisting of the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein, adjuvanted with aluminum hydroxide (alum) and CpG oligonucleotides. In mice and non-human primates, our wild-type (WT) RBD vaccine induced high neutralizing antibody titers against the WT isolate of the virus, and, with partners in India and Indonesia, it was later developed into two closely resembling human vaccines, Corbevax and Indovac. Here, we describe the development and characterization of a next-generation vaccine adapted to the recently emerging XBB variants of SARS-CoV-2. (2) Methods: We conducted preclinical studies in mice using a novel yeast-produced SARS-CoV-2 XBB.1.5 RBD subunit vaccine candidate formulated with alum and CpG. We examined the neutralization profile of sera obtained from mice vaccinated twice intramuscularly at a 21-day interval with the XBB.1.5-based RBD vaccine, against WT, Beta, Delta, BA.4, BQ.1.1, BA.2.75.2, XBB.1.16, XBB.1.5, and EG.5.1 SARS-CoV-2 pseudoviruses. (3) Results: The XBB.1.5 RBD/CpG/alum vaccine elicited a robust antibody response in mice. Furthermore, the serum from vaccinated mice demonstrated potent neutralization against the XBB.1.5 pseudovirus as well as several other Omicron pseudoviruses. However, regardless of the high antibody cross-reactivity with ELISA, the anti-XBB.1.5 RBD antigen serum showed low neutralizing titers against the WT and Delta virus variants. (4) Conclusions: Whereas we observed modest cross-neutralization against Omicron subvariants with the sera from mice vaccinated with the WT RBD/CpG/Alum vaccine or with the BA.4/5-based vaccine, the sera raised against the XBB.1.5 RBD showed robust cross-neutralization. These findings underscore the imminent opportunity for an updated vaccine formulation utilizing the XBB.1.5 RBD antigen.

BackgroundObesity has become a global health and socioeconomic problem because of an inadequate balance between energy intake and energy expenditure. Roux-en-Y gastric bypass (RYGB) and sleeve gastrectomy (SG) are the two most commonly used strategies for weight loss, which have been proven to benefit from gut microbiota restoration.MethodsRats received SG, RYGB, and sham operations for 10 weeks. At the end of the experiment, the fecal microbiota was analyzed using 16s rRNA gene sequencing. In addition, the shift in the plasma metabolism of rats that underwent RYGB surgery was analyzed using untargeted metabolomics. The crosstalk between microbiome and metabolites was revealed using metabolic pathway enrichment and integrated analysis.ResultThe SG surgery induced a modest shift in the gut microbiota relative to the RYGB. RYGB significantly decreased the alpha diversity and Firmicutes/Bacteroides (F/B) ratio and increased the proportion of Escherichia, Bacteroides, and Akkermansia genera compared to sham and SG operations. The predicted function of gut microbiota revealed that the RYGB surgery uniquely enhanced the capability of linoleic acid and sphingolipid metabolism. Furthermore, the circulating serine, phosphatidylcholine (PC) 20:5/22:5, riboflavin, L–carnitine, and linoleic acid were evaluated after RYGB surgery. In addition, the metabolic pathway enrichment and integrated analysis suggest that the RYGB induced Escherichia, Bacteroides, and Akkermansia might inhibit the sphingonine and phytosphingosine metabolisms from serine and promote the PC (20:5/22:5) metabolism to produce linoleic acid.ConclusionThis comprehensive analysis not only revealed the difference in the gut microbiota shifts after SG and RYGB but also discovered the perturbative changes in microbial communities and metabolic pathways after RYGB surgery, which provided clues for improving the beneficial effect of RYGB in metabolic disease intervention via regulating bacterial-metabolite crosstalk.

Telisotuzumab vedotin is a MET-targeting antibody–drug conjugate that has demonstrated a good treatment response in patients with EGFR wild-type MET-overexpressing non-squamous non-small cell lung cancer. However, patients have been reported to acquire resistance to this drug, and the subsequent therapy has not been standardized. Here, we present a case of a 56-year-old woman diagnosed with KIF5B-MET fusion-positive non-small cell lung cancer who had a durable response to capmatinib after acquired resistance to telisotuzumab vedotin.

Introduction: Tocolytic treatment is beneficial to pregnant women with a risk of premature labor or miscarriage. However, previous reports have shown that progestogen might not be effective and ritodrine may increase the risk of maternal vascular-related diseases. Chinese herbal products (CHP) are used as alternative therapies for pregnant women. The goal was to evaluate the efficacy of combined tocolytic therapy and CHP therapy in pregnancy outcomes for pregnant women in Taiwan.Materials and Methods: We conducted a retrospective cohort study based on the National Health Insurance Research Database. A total of 47,153 pregnant women treated with tocolytics aged 18–50 years from 2001 to 2015 were selected from two million random samples. According to the medical use of tocolytics and CHP, we divided the users into two groups: western medicine (WM) only (n = 40,961) and WM/CHP (n = 6,192) groups. A propensity score (PS)-matched cohort (6,192 pairs) was established based on baseline confounders. All participants were followed up to perinatal outcomes. Conditional logistic regression analysis was used to examine the effects of CHP use on the odds of miscarriage and preterm birth.Results: The adjusted odds ratio (OR) for premature birth in the WM/CHP group (n = 411, 6.64%) was significantly lower than in the WM group (n = 471, 7,61%) (0,86, 95% confidence interval [CI], 0.74–0.99). Further subgroup analysis based on the usage of formulae that activate blood and remove stasis or purgative formulae, the adjusted OR of preterm birth of those using these formulae was significantly lower in the WM/CHP group (n = 215, 6.32%) than that in the WM group (n = 265, 7.77%) (OR: 0.79, 95% CI: 0.65–0.96).Conclusion: We found that the combination of CHP and tocolytics can be beneficial to pregnant women in the prevention of premature birth. Further research is required to investigate causal relationships.

Abstract Background Illumina sequencing of a marker gene is popular in metagenomic studies. However, Illumina paired-end (PE) reads sometimes cannot be merged into single reads for subsequent analysis. When mergeable PE reads are limited, one can simply use only first reads for taxonomy annotation, but that wastes information in the second reads. Presumably, including second reads should improve taxonomy annotation. However, a rigorous investigation of how best to do this and how much can be gained has not been reported. Results We evaluated two methods of joining as opposed to merging PE reads into single reads for taxonomy annotation using simulated data with sequencing errors. Our rigorous evaluation involved several top classifiers (RDP classifier, SINTAX, and two alignment-based methods) and realistic benchmark datasets. For most classifiers, read joining ameliorated the impact of sequencing errors and improved the accuracy of taxonomy predictions. For alignment-based top-hit classifiers, rearranging the reference sequences is recommended to avoid improper alignments of joined reads. For word-counting classifiers, joined reads could be compared to the original reference for classification. We also applied read joining to our own real MiSeq PE data of nasal microbiota of asthmatic children. Before joining, trimming low quality bases was necessary for optimizing taxonomy annotation and sequence clustering. We then showed that read joining increased the amount of effective data for taxonomy annotation. Using these joined trimmed reads, we were able to identify two promising bacterial genera that might be associated with asthma exacerbation. Conclusions When mergeable PE reads are limited, joining them into single reads for taxonomy annotation is always recommended. Reference sequences may need to be rearranged accordingly depending on the classifier. Read joining also relaxes the constraint on primer selection, and thus may unleash the full capacity of Illumina PE data for taxonomy annotation. Our work provides guidance for fully utilizing PE data of a marker gene when mergeable reads are limited.

BackgroundTo compare the testing costs and testing turnaround times of tissue-first, plasma-first, and complementary next-generation sequencing (NGS) approaches in patients with treatment-naïve metastatic lung adenocarcinoma.Materials and MethodsWe developed a decision tree model to compare three different approaches. Patients were entered into the model upon cancer diagnosis and those with both insufficient tissue specimens and negative liquid-based NGS were subjected to tissue re-biopsy. Actionable gene alterations with the U.S. Food and Drug Administration (FDA)-approved therapies included epidermal growth factor receptor (EGFR) mutation, anaplastic lymphoma kinase (ALK) gene rearrangement, ROS proto-oncogene 1 (ROS1) rearrangement, B-Raf proto-oncogene (BRAF) V600E mutation, rearranged during transfection (RET) gene rearrangement, mesenchymal-epithelial transition factor (MET) mutation, neurotrophic tyrosine receptor kinase (NTRK) gene rearrangement, K-Ras proto-oncogene (KRAS) G12C mutation, and human epidermal growth factor receptor 2 (HER2) mutation. Model outcomes were testing costs, testing turnaround times, and monetary losses taking both cost and time into consideration. We presented base-case results using probabilistic analysis. Stacked one-way and three-way sensitivity analyses were also performed.ResultsIn terms of testing costs, tissue-first approach incurred US$2,354($1,963–$2,779) and was the most cost-efficient strategy. Complementary approach testing turnaround time (days) of 12.7 (10.8 to 14.9) was found as the least time-consuming strategy. Tissue-first, complementary, and plasma-first approaches resulted in monetary losses in USD of $4,745 ($4,010–$5,480), $6,778 ($5,923–$7,600), and $7,006 ($6,047–$7,964) respectively, and identified the same percentage of patients with appropriate FDA-approved therapies. Costs for liquid-based NGS, EGFR mutation rates, and quantity of tissue specimens were the major determinants in minimizing monetary loss. Plasma-first approach would be the preferable strategy if its testing price was reduced in USD to $818, $1,343, and $1,869 for populations with EGFR mutation rates of 30%, 45%, and 60% respectively.ConclusionThe tissue-first approach is currently the best strategy in minimizing monetary loss. The complementary approach is an alternative for populations with a low EGFR mutation rate. The plasma-first approach becomes increasingly preferable as EGFR mutation rates gradually increase.

Airway and gut microbiota are important in asthma pathogenesis. Although several studies have revealed distinct microbiota in asthmatic airways at baseline compared to healthy controls, limited studies compared microbiota during acute exacerbation (AE) and in the recovery phase (RP) in the same asthmatic children. We aim to investigate association between microbiota and asthma status in children and explore their relationship with clinical features of asthma. We recruited 56 asthmatic children and investigated their nasal, throat, and stool microbiota during AE and in the RP. Totally, 320 samples were subjected to 16S rRNA sequencing. Although the microbial communities were clearly separated by body site, within each site the overall communities during AE and in the RP could not be distinguished. Most nasal microbiota were dominated by only one or two of six bacterial genera. The domination was associated with mite allergy and patient age only during AE but not in the RP. When moving into RP, the relative abundance of Staphylococcus increased while that of Moraxella decreased. Throat and stool microbiota were not associated with most of the clinical features. Interestingly, stool microbiota during AE was associated with ABO blood type and stool microbiota in the RP was associated with frequency of the subsequent exacerbations. In summary, the association between nasal microbiota and mite allergy only during AE suggests an altered local immunity and its interplay with nasal microbes. Our work provides a basis for studying microbes, and prevention or therapeutic strategy in childhood asthma, especially during AE.

BackgroundCurrent guideline recommends both surgery and drug treatment for primary aldosteronism. Treatment effects on the cardiac structure and function remain under investigation.ObjectiveWe performed a prospective study in patients with primary aldosteronism to compare effects of surgery and drug treatment on the cardiac structure and function as assessed by the left ventricular (LV) pressure-strain loop, a novel echocardiographic technique that incorporates myocardial deformation and LV pressure.MethodsOur study included 39 and 28 patients treated with surgery and a mineralocorticoid antagonist, respectively. We performed conventional and speckle tracking echocardiography at baseline and 3 and 6 months of follow-up.ResultsDuring follow-up, both surgery and drug treatment normalized serum potassium concentration and significantly reduced blood pressure. Both treatments significantly and similarly decreased LV mass index and left atrial volume index. However, only in the surgery group did global wasted work significantly decrease (200.8 ± 86.7 at baseline vs. 142.1 ± 58.1 mmHg% at 6 months) and global work efficiency (91.5 ± 3.1 vs. 93.6 ± 2.3%) and global longitudinal strain (−18.3 ± 2.7 vs. −19.2 ± 1.9%) significantly (p < 0.01) increase at 6 months of follow-up. The corresponding differences from the changes in the drug treatment group were 39.5 mmHg% (95% CI, 17.1, 62.0 mmHg%), −1.64% (95% CI, −2.56, −0.71%), and −0.85% (95% CI, −1.51, −0.20%), respectively. In addition, the changes in global wasted work at 6 months of follow-up was significantly correlated with that in 24-h urinary aldosterone excretion in the drug treatment group (r = 0.54) and two groups combined (r = 0.55), but not the surgery group.ConclusionIn spite of similar serum potassium normalization and blood pressure control, surgical removal of an adrenal gland, but not mineralocorticoid receptor antagonism, showed early improvement in cardiac function.

Cancer stem cells (CSCs) are the main cause of tumor generation, recurrence, metastasis, and therapy failure in various malignancies including colorectal cancer (CRC). Accumulating evidence suggests that tumor cells can acquire CSC characteristics through the epithelial-mesenchymal transition (EMT) process. However, the molecular mechanism of CSCs remains unclear. OCT4B1 is a transcript of OCT4, which is initially expressed in embryonic stem and carcinoma cells, and is involved in the regulation and maintenance of an undifferentiated state of stem cells. In this study, three-dimensional (3D) microspheres were confirmed as CRC stem cells. Compared with that of parental cells, their self-renewal ability was significantly increased, and OCT4B1 expression was increased and promoted the EMT process. The knockdown of OCT4B1 decreased the self-renewal of CSCs and reversed EMT. Moreover, OCT4B1 induced the expression of Polo-like kinase 1 (PLK1), which is a key regulator of EMT in tumor cells. Further examination showed that OCT4B1 regulated the miR-8064/PLK1 balance to exert its function. Taken together, our data suggest that OCT4B1 may be involved in regulating the self-renewal of colorectal CSCs through EMT, which is at least partially due to the miR-8064/PLK1 balance. This study indicates that OCT4B1 is a potential therapeutic target for CRC by targeting CSCs. Keywords: OCT4B1, EMT, cancer stem cell, colorectal cancer, self-renewal, PLK1, miR-8064

Abstract Background Osimertinib yields significant tumor responses and durations of progression‐free survival (PFS) in patients with acquired T790M mutations. However, the evidence supporting liquid biopsy‐guided treatment is still limited. This study examined the real‐world benefits of osimertinib in patients with tissue or plasma T790M mutations. Methods From January 2016 to June 2018, a total of 183 non‐small‐cell lung cancer patients were enrolled. The presence of the T790M mutation was assessed by either tissue or plasma. The PFS, overall survival, and tumor response rates of the patients were calculated and compared with those of previous clinical trials. Results T790M mutations were detected in 51.5% of the patients, including 64 of 140 (45.7%) who underwent liquid biopsies and 23 of 29 (79.3%) who underwent tumor biopsies. After excluding those in clinical trials, 46 patients received osimertinib, including 33 with positive plasma and 13 with positive tissue results for T790M mutations. The median PFS was 11.3 months (interquartile range: 5.2‐NR) in all the T790M‐positive patients and 10.1 months (interquartile range: 5.9‐NR) in the plasma T790M‐positive patients. The overall survival, meanwhile, was not reached, whereas the one‐year survival rate was 66.1% in all the patients and 61.4% in those who were plasma T790M‐positive. The objective response rate and disease control rate were 37.8% and 91.9% in all the patients and 34.6% and 92.3% in the plasma T790M‐positive group, respectively. Using a Cox proportional hazards regression, we determined that male gender was a poor prognostic factor for PFS. Conclusions In this retrospective real‐world analysis, it was determined that both tissue and plasma T790M mutations can be used to guide treatment with osimertinib. Similar disease control rates and survival durations were observed in comparison to those of phase 3 clinical trials.

Flower-like phosphorus-doped nickel oxide (P-NiO) is proposed as a counter electrode (CE) for dye-sensitized solar cells (DSSCs). The flower-like nickel oxide essentially serves as the matrix for the CE, which is expected to promote a two-dimensional electron transport pathway. The phosphorus is intended to improve the catalytic ability by creating more active sites in the NiO for the catalysis of triiodide ions (I3−) to iodide ions (I−) on the surface of the CE. The P-NiO is controlled by a sequencing of precursor concentration, which allows the P-NiO to possess different features. The debris aggregation occurs in the P-NiO-1, while the P-NiO-0.75 leads to the incomplete flower-like nanosheets. The complete flower-like morphology can be observed in the P-NiO-0.5, P-NiO-0.25 and P-NiO-0.1 catalytic electrodes. The DSSC with the P-NiO-0.5 CE achieves a power conversion efficiency (η) of 9.05%, which is better than that of the DSSC using a Pt CE (η = 8.51%); it also performs better than that with the Pt CE, even under rear illumination and dim light conditions. The results indicate the promising potential of the P-NiO CE to replace the expensive Pt CE.

Targeted therapy with combined dabrafenib and trametinib has been proven to provide clinical benefits in patients with NSCLC with a BRAF V600E mutation. Nevertheless, the treatment strategy for patients with NSCLC with BRAF non-V600E mutations remains limited. Here, we present a patient with NSCLC with a BRAF K601E mutation, a class II BRAF mutation, who had a durable response to targeted therapy with combined dabrafenib and trametinib.

Summary: Background: Screening for atrial fibrillation before onset of symptoms and the subsequent initiation of oral anticoagulants could prevent stroke and death. The most cost-effective strategy to screen for atrial fibrillation in a population at high risk aged 65 years and older is unknown. Therefore, we aimed to investigate whether more frequent electrocardiography (ECG) recordings would significantly improve the detection of atrial fibrillation compared with annual ECG screenings. Methods: We did a randomised controlled trial that compared different screening frequencies of 30 s single-lead ECG (AliveCor Heart Monitor) in the detection of atrial fibrillation in Chinese residents (≥65 years) in five community health centres in Shanghai, China. Only participants without history of atrial fibrillation and without atrial fibrillation rhythm at baseline were eligible for inclusion in the trial. Random assignment was done with the use of a random number table and stratified for study site. Participants were randomly assigned in a 1:1 ratio to annual or quarterly screening groups. The quarterly screening group was further randomly assigned in a 3:1 ratio to subgroups of quarterly screening and quarterly screening plus (which involved ECG screening once per week for the first month of follow-up, then quarterly for the remainder of follow-up). The primary outcome was the detection rate of atrial fibrillation. The intention-to-treat analysis was done for all randomly assigned patients who had at least one ECG recording during follow-up. This trial was registered at ClinicalTrials.gov, NCT02990741, and terminated on Oct 31, 2020. Findings: Between April 17, 2017, and June 26, 2018, 8240 participants were randomly assigned to annual screening (n=4120), quarterly screening (n=3090), and quarterly screening plus (n=1030), with a mean number of ECG recordings of 1·6 (SD 0·5) for annual screening, 3·5 (1·5) for quarterly screening, and 5·2 (2·9) for quarterly screening plus during a median of 2·1 years follow-up (13 284 person-years). 73 incident cases of atrial fibrillation occurred: 26 in the annual screening group (4·1 per 1000 person-years) and 47 in the quarterly screening group (6·7 per 1000 person-years. Quarterly screening was associated with a significant increase in the detection rate of atrial fibrillation, compared with annual screening (hazard ratio [HR] 1·71; 95% CI 1·06–2·76; p=0·029). 40 incident cases were detected in quarterly screening (7·2 per 1000 person-years; HR compared to annual screening, 1·83; 95% CI 1·12–3·00; p=0·017) and seven in the quarterly screening plus group (4·8 per 1000 person-years; HR compared with annual screening, 1·24; 0·54–2·86; p=0·61). No significant difference was noted between quarterly screening and the quarterly screening plus group (HR of quarterly screening plus compared with quarterly screening, 0·68; 0·30–1·52; p=0·35). Interpretation: Quarterly 30 s single-lead ECG screening was associated with a significantly higher detection rate of incident atrial fibrillation compared with annual screening, but additional once per week screenings in the first month did not yield an added predictive value. Quarterly screening might be considered in a general population at a high risk of atrial fibrillation, such as those aged 65 years and older. Funding: Bayer Healthcare Company.

Approximately 5-7% of non-small cell lung cancer (NSCLC) cases harbor an anaplastic lymphoma kinase (ALK) fusion gene and may benefit from ALK inhibitor therapy. To detect ALK fusion genes, we developed a novel test using reverse transcription polymerase chain reaction (RT-PCR) for the ALK kinase domain (KD). Since ALK expression is mostly silenced in the adult with the exception of neuronal tissue, the normal lung tissue, mesothelial lining, and inflammatory cells are devoid of ALK transcript, making ALK KD RT-PCR an ideal surrogate test for ALK fusion transcripts in lung or pleural effusion. The test was designed with a short PCR product (197 bp) to work for both malignant pleural effusion (MPE) and formalin-fixed, paraffin-embedded (FFPE) NSCLC samples. Using ALK IHC as a reference, the sensitivity of the test was 100% for both MPE and FFPE. The specificity was 97.6% for MPE and 97.4% for FFPE. Two false positive cases were found. One was a metastatic brain lesion which should be avoided in the future due to intrinsic ALK expression in the neuronal tissue. The other one resulted from ALK gene amplification. Due to potential false positivity, subsequent confirmation tests such as fluorescence in situ hybridization or multiplex PCR would be preferable. Nevertheless, the test is simple and inexpensive with no false negativity, making it a desirable screening test. It also offers an advantage over multiplex RT-PCR with the capability to detect novel ALK fusions. Indeed through the screening test, we found a novel ALK fusion partner (sperm antigen with calponin homology and coiled-coil domains 1 like gene, SPECC1L) with increased sensitivity to crizotinib in vitro. In summary, a novel RNA-based ALK KD analysis was developed for ALK rearrangement screening in MPE and FFPE specimens of NSCLC. This simple inexpensive test can be implemented as routine diagnostics.

In recent years, bilingual education has become a critical index, and with its growth, internationalization has been significantly improved in the Republic of China (Taiwan). Therefore, countries worldwide are promoting all-English teaching, taking English as a medium of instruction and indicator of university education while moving toward an internationalized curriculum and teaching excellence. Finding the required number of bilingual teachers is also one of the keys to increasing the index. If information technology can be adopted to reduce the time and cost of bilingual teacher training, it will significantly improve the effectiveness of bilingual education promotion. However, the current traditional online training system only focuses on the delivery of one-way training content, and it cannot assist in judging the training effectiveness of the trainees. In this paper, we integrate the pose estimation technology into the online teacher training system to analyze the interactive content of the trainees during the training. We can assist in recording and interpreting the teaching demonstration process in classroom observation. We also verify the result by comparing our method’s efficiency and judgment accuracy with the traditional way. The results show that our approach is more convenient and cost-efficient.

Mature cystic teratomas (MCTs) are common benign tumors of the ovary. Coexistence of a carcinoid tumor inside an MCT is extremely rare. We present a 40-year-old woman who had intermittent abdominal pain and urinary frequency. An adnexal mass was detected during physical examination. Ultrasound indicated a 12.3 cm × 10.7 cm left ovarian mass. Surgical excision of the mass was performed, and histopathological examination revealed an MCT with three germ layers. The microcarcinoid tumor arising from an MCT showed a cord to trabecular pattern. The pathological diagnosis was a trabecular carcinoid tumor arising in an MCT. The patient did not present with carcinoid syndrome, and no recurrence was observed after 12 months of follow-up.

Deep sequencing of T-cell receptor (TCR) genes is powerful at profiling immune repertoire. To prepare a TCR sequencing library, multiplex polymerase chain reaction (mPCR) is widely applied and is highly efficient. That is, most mPCR products contain the region critical for antigen recognition, which also indicates regular V(D)J recombination. Multiplex PCR, however, may suffer from primer bias. A promising alternative is 5'-RACE, which avoids primer bias by applying only one primer pair. In 5'-RACE data, however, non-regular V(D)J recombination (e.g., TCR sequences without a V gene segment) has been observed and the frequency varies (30-80%) between studies. This suggests that the cause of or how to reduce non-regular TCR sequences is not yet well known by the science community. Although it is possible to speculate the cause by comparing the 5'-RACE protocols, careful experimental confirmation is needed and such a systematic study is still not available. Here, we examined the 5'-RACE protocol of a commercial kit and demonstrated how a modification increased the fraction of regular TCR-β sequences to >85%. We also found a strong linear correlation between the fraction of short DNA fragments and the percentage of non-regular TCR-β sequences, indicating that the presence of short DNA fragments in the library was the main cause of non-regular TCR-β sequences. Therefore, thorough removal of short DNA fragments from a 5'-RACE library is the key to high data efficiency. We highly recommend conducting a fragment length analysis before sequencing, and the fraction of short DNA fragments can be used to estimate the percentage of non-regular TCR sequences. As deep sequencing of TCR genes is still relatively expensive, good quality control should be valuable.

Third-generation tyrosine kinase inhibitors (TKIs) were developed to overcome T790M-mediated resistance to earlier generations of epidermal growth factor receptor (EGFR)-targeted TKIs. We compared four well-established and one in-house method for the analysis of the EGFR T790M mutation in plasma cell-free DNA (cfDNA), in hope to find a better way to select non-small cell lung cancer (NSCLC) patients appropriate for 3rd-generation TKI therapy. For sensitivity levels of each method, plasmid DNA with EGFR T790M mutations was serially diluted with cfDNA from healthy controls with wild type EGFR. The clinical performance was analyzed in a clinical cohort of EGFR mutation-positive NSCLC patients with acquired EGFR TKI resistance (n = 40). All methods except the therascreen kit (Qiagen) had a sensitivity level of 10 copies of T790M plasmid DNA in the spiked specimen. The detection rates of the EGFR T790M mutation in plasma cfDNA from the clinical cohort were 42.5, 35, 32.5, 22.5, and 17.5% for the in-house ARMS method, Bio-Rad droplet digital PCR, PANAMutyper, Therascreen EGFR Plasma RGQ PCR Kit and Cobas EGFR Mutation kit (with suboptimal template amounts), respectively. Osimertinib was given to 17 of 20 patients with EGFR T790M mutations. The best treatment responses, based on the RECIST criteria, included 6 partial responses (PR) and 7 stable diseases (SD). The PANAMutyper and the Bio-Rad droplet digital PCR were comparable, the Cobas EGFR Mutation kit required significantly more template for testing. The best combination would be the in-house ARMS method plus the PANAMutyper or Bio-Rad droplet digital PCR, which would have a detection rate of 50% (20/40) and a disease control rate of 76% (13/17).

Objective: Preimplantation genetic diagnosis (PGD) offers an alternative for women to carry an unaffected fetus risk of hereditary diseases. Trophectoderm biopsy may provide more cells for accurate diagnosis. However, the time allowed for transportation of the specimens to the laboratory and performance of molecular diagnosis is limited. We designed a PGD program of trophectoderm biopsy, vitrification of blastocysts, whole genome amplification (WGA), double confirmatory genotypings, and thawed embryo transfer. Case Report: We conducted this strategy for a woman of familial neurofibromatosis type I (NF-1). She had a genotype of heterozygous c.6709C>T mutation of NF1 gene. Trophectoderm biopsies were performed on 13 blastocysts. Then, individual blastocyst was vitrified. WGA was performed for the samples, followed by genotypings with both real-time polymerase chain reaction and sequencing. Eight embryos were diagnosed as unaffected, four were affected, and one was inconclusive because of allele drop-out. In the next cycle, two unaffected blastocysts were thawed and transferred, that resulted in a singleton pregnancy. The pregnancy was confirmed as unaffected by means of chorionic villi sampling. Conclusion: We first demonstrate successful application of blastocyst biopsy, vitrification, WGA, and thawed embryo transfer for PGD of a monogenic disease. Vitrification of blastocysts after biopsy permits sufficient time for shipment of samples and operation of molecular diagnosis.

Analyzing EGFR mutations and detecting ALK gene fusion are indispensable when planning to treat pulmonary adenocarcinoma. Malignant pleural effusion (MPE) is a devastating complication of lung cancer and sometimes the only source for mutation analysis. The percentage of tumor cells in the pleural effusion may be low; therefore, mutant enrichment is required for a successful analysis. The EGFR mutation status in MPE was determined using three methods: (1) PCR sequencing of genomic DNA (direct sequencing), (2) mutant-enriched PCR sequencing of genomic DNA using peptide nucleic acid (PNA-sequencing), and (3) PCR sequencing of cDNA after reverse transcription for cellular RNA (RNA-sequencing). RT-PCR was also used to test cases for ALK gene fusion. PNA-sequencing and RNA-sequencing had similar analytical sensitivities (< 1%), which indicates similar enrichment capabilities. The clinical sensitivity in 133 cases when detecting the common EGFR exon 19 and exon 21 mutations was 56.4% (75/133) for direct sequencing, 63.2% (84/133) for PNA-sequencing, and 65.4% (87/133) for RNA-sequencing. RT-PCR and sequencing showed 5 cases (3.8%) with ALK gene fusion. All had wild-type EGFR. For EGFR analysis of MPE, RNA-sequencing is at least as sensitive as PNA-sequencing but not limited to specific mutations. Detecting ALK fusion can be incorporated in the same RNA workflow. Therefore, RNA is a better source for comprehensive molecular diagnoses in MPE.

Endometrial cancer (EC) is a complex disease involving multiple gene-gene and gene-environment interactions. TGF-β signaling plays pivotal roles in EC development. This study aimed to investigate whether the genetic polymorphisms of TGF-β signaling related genes TGFB1, TGFBR1, SNAI1 and TWIST1 contribute to EC susceptibility. Using the TaqMan Genotyping Assay, 19 tagging-SNPs of these four genes were genotyped in 516 EC cases and 707 controls among Chinese Han women. Logistic regression (LR) showed that the genetic variants of TGFB1 rs1800469, TGFBR1 rs6478974 and rs10733710, TWIST1 rs4721745 were associated with decreased EC risk, and these four loci showed a dose-dependent effect (Ptrend < 0.0001). Classification and regression tree (CART) demonstrated that women carrying both the genotypes of TGFBR1 rs6478974 TT and rs10512263 TC/CC had the highest risk of EC (aOR = 7.86, 95% CI = 3.42-18.07, P

We present a full-term female infant with a difficult delivery course complicated with Escherichia coli sepsis and bilateral adrenal abscesses. She developed renal failure and received peritoneal dialysis. Peritonitis of Candida parapsilosis developed later. The infant was successfully treated with hemofiltration and a combination of antifungal agents.

Tendon injury may occur suddenly or progressively, and can be divided into tendon rupture or tendinopathy based on the severity of injury. It is frequently found in professional or nonprofessional people who are making repetitive movements. In aged people, tendon degeneration becomes obvious; their tendon injuries are then frequently evident. No effective therapies for tendon injury are currently available. In this article, we review the tendon structure, mechanisms of tendon injury, and tendon healing process. More importantly, cell-based therapies for tendon injury are fully addressed, which will play an important role for tendon therapy in the near future.

Oral squamous cell carcinoma (OSCC) accounts for 5.8% of all malignancies in Taiwan and the incidence of OSCC is on the rise. OSCC is also a common malignancy worldwide and the five-year survival rate remains poor. Therefore, new and effective treatments are needed to control OSCC. In the present study we have investigated the efficacy and associated mechanisms of polyenylpyrroles and their analogs in both in vitro cell culture and in vivo nude mice xenografts. Auxarconjugatin B (compound 1a) resulted in cell cycle arrest in the G2/M phase and caspase-dependent apoptosis in OEC-M1 and HSC-3 cells by activating DNA damage and mitochondria dysfunction through the loss of mitochondrial membrane potential, release of cytochrome c, increase in B-cell lymphoma-2-associated X protein level, and decrease in B-cell lymphoma-2 level. Compound 1a-induced generation of intracellular reactive oxygen species through cytochrome P450 1A1 was identified as a major mechanism of its effect for DNA damage, mitochondria dysfunction and apoptosis, which was reversed by antioxidant N-acetylcysteine as well as cytochrome P450 1A1 inhibitor and specific siRNA. Furthermore, compound 1a-treated nude mice showed a reduction in the OEC-M1 xenograft tumor growth and an increase in the caspase-3 activation in xenograft tissue. These results provide promising insights as to how compound 1a mediates cytotoxicity and may prove to be a molecular rationale for its translation into a potential therapeutic against OSCC.

The CCNB1 and CDK1 genes encode the proteins of CyclinB1 and CDK1 respectively, which interact with each other and are involved in cell cycle regulation, centrosome duplication and chromosome segregation. This study aimed to investigate whether the genetic variants in these two genes may affect breast cancer (BC) susceptibility, progression, and survival in Chinese Han population using haplotype-based analysis. A total of ten tSNPs spanning from 2kb upstream to 2kb downstream of these genes were genotyped in 1204 cases and 1204 age-matched cancer-free controls. The haplotype blocks were determined according to our genotyping data and linkage disequilibrium (LD) status of these SNPs. For CCNB1, rs2069429 was significantly associated with increased BC susceptibility under recessive model (OR=2.352, 95%CI=1.480-3.737), so was the diplotype TAGT/TAGT (OR=1.947 95%CI=1.154-3.284, P=0.013). In addition, rs164390 was associated with Her2-negative BC. For CDK1, rs2448343 and rs1871446 were significantly associated with decreased BC risk under dominant models, so was the haplotype ATATT. These two SNPs also showed a dose-dependent effect on BC susceptibility. Using stratified association analysis, we found that women with the heterozygotes or minor allele homozygotes of rs2448343 had much less BC susceptibility among women with BMI

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