Physiological ER stress caused by amylase production induces regulated Ire1-dependent mRNA decay in Aspergillus oryzae.

Regulated Ire1-dependent decay (RIDD) is a feedback mechanism in which the endoribonuclease Ire1 cleaves endoplasmic reticulum (ER)-localized mRNAs encoding secretory and membrane proteins in eukaryotic cells under ER stress. RIDD is artificially induced by chemicals that generate ER stress; however, its importance under physiological conditions remains unclear. Here, we demonstrate the occurrence of RIDD in filamentous fungus using Aspergillus oryzae as a model, which secretes copious amounts of amylases. α-Amylase mRNA was rapidly degraded by IreA, an Ire1 ortholog, depending on its ER-associated translation when mycelia were treated with dithiothreitol, an ER-stress inducer. The mRNA encoding maltose permease MalP, a prerequisite for the induction of amylolytic genes, was also identified as an RIDD target. Importantly, RIDD of malP mRNA is triggered by inducing amylase production without any artificial ER stress inducer. Our data provide the evidence that RIDD occurs in eukaryotic microorganisms under physiological ER stress.

ISOTOPE: ISOform-guided prediction of epiTOPEs in cancer.

Immunotherapies provide effective treatments for previously untreatable tumors and identifying tumor-specific epitopes can help elucidate the molecular determinants of therapy response. Here, we describe a pipeline, ISOTOPE (ISOform-guided prediction of epiTOPEs In Cancer), for the comprehensive identification of tumor-specific splicing-derived epitopes. Using RNA sequencing and mass spectrometry for MHC-I associated proteins, ISOTOPE identified neoepitopes from tumor-specific splicing events that are potentially presented by MHC-I complexes. Analysis of multiple samples indicates that splicing alterations may affect the production of self-epitopes and generate more candidate neoepitopes than somatic mutations. Although there was no difference in the number of splicing-derived neoepitopes between responders and non-responders to immune therapy, higher MHC-I binding affinity was associated with a positive response. Our analyses highlight the diversity of the immunogenic impacts of tumor-specific splicing alterations and the importance of studying splicing alterations to fully characterize tumors in the context of immunotherapies. ISOTOPE is available at https://github.com/comprna/ISOTOPE.

Pseudo-elevation of conduction system pacing threshold through parallel connection of an intracardiac electrogram recording system.

Parallel connection of an electrophysiology recording system (EP system) to equipment for conduction system pacing (CSP) has been widely used for fine monitoring of intracardiac electrograms and pacing evaluation. We experienced a case showing unexpected pacing threshold exacerbation under specific conditions when the EP system was connected in parallel. We evaluated the underlying mechanism using an ex vivo model. An ex vivo pacing and intracardiac electrogram monitoring model was generated using an oscilloscope, pacing system analyzer (PSA), EP system, and simulated heart. The discrepancy between expected output at the PSA and the actual measured output value at the simulated heart was measured under various conditions and using various combinations of pacing equipment. Parallel connection of the EP system was associated with reduced electrical output from the PSA as recorded at the simulated heart. The unexpected adverse effects were particularly noticeable when using an RMC-5000 EP system with the pacing function on. The trouble is completely resolved by simply turning off the pacing function of the system. There is a possibility that the EP system might increase the pacing threshold in CSP when the PSA and EP system is are deployed in parallel. The issue may provoke pseudo failure of CSP due to the high pacing threshold. When the RMC-5000 is used for conduction system pacing in parallel with a PSA for the pacing test, the pacing function of RMC-5000 should be turned off.

Applications of three-dimensional printers in prosthetic dentistry.

This narrative review aims to provide an overview of recent studies and case reports on three-dimensional (3D) printing, and to verify the applicability of 3D printers in the field of dental prostheses. This review was performed by conducting a search of PubMed. The clinical application of fabricating a prosthesis made with cobalt-chromium is considered possible depending on the material and hardware of the 3D printer. However, it is currently difficult to assess the clinical use of 3D-printed zirconia crowns. Further research is required, such as verification of materials used, margin morphology, and hardware. Clinically acceptable results have been reported for patterns using 3D printers. Interim restorations made using a 3D printer have been reported with good results that are considered clinically usable. Dentures made with 3D printers need further verification in terms of strength and deformation. Custom trays made with 3D printers are clinically useful, however, issues remain with design time and effort. Although several studies have reported the usefulness of 3D printers, further verification is required since 3D printers are still considered new technology.

Gene Amplification-Associated Overexpression of the Selenoprotein tRNA Enzyme TRIT1 Confers Sensitivity to Arsenic Trioxide in Small-Cell Lung Cancer.

The alteration of RNA modification patterns is emerging as a common feature of human malignancies. If these changes affect key RNA molecules for mRNA translation, such as transfer RNA, they can have important consequences for cell transformation. TRIT1 is the enzyme responsible for the hypermodification of adenosine 37 in the anticodon region of human tRNAs containing serine and selenocysteine. Herein, we show that TRIT1 undergoes gene amplification-associated overexpression in cancer cell lines and primary samples of small-cell lung cancer. From growth and functional standpoints, the induced depletion of TRIT1 expression in amplified cells reduces their tumorigenic potential and downregulates the selenoprotein transcripts. We observed that TRIT1-amplified cells are sensitive to arsenic trioxide, a compound that regulates selenoproteins, whereas reduction of TRIT1 levels confers loss of sensitivity to the drug. Overall, our results indicate a role for TRIT1 as a small-cell lung cancer-relevant gene that, when undergoing gene amplification-associated activation, can be targeted with the differentiation agent arsenic trioxide.

BET-Inhibitor I-BET762 and PARP-Inhibitor Talazoparib Synergy in Small Cell Lung Cancer Cells.

Small cell lung cancer (SCLC) is an aggressive type of lung cancer with high mortality that is caused by frequent relapses and acquired resistance. Despite that several target-based approaches with potential therapeutic impact on SCLC have been identified, numerous targeted drugs have not been successful in providing improvements in cancer patients when used as single agents. A combination of targeted therapies could be a strategy to induce maximum lethal effects on cancer cells. As a starting point in the development of new drug combination strategies for the treatment of SCLC, we performed a mid-throughput screening assay by treating a panel of SCLC cell lines with BETi or AKi in combination with PARPi or EZH2i. We observed drug synergy between I-BET762 and Talazoparib, BETi and PARPi, respectively, in SCLC cells. Combinatorial efficacy was observed in MYCs-amplified and MYCs-wt SCLC cells over SCLC cells with impaired MYC signaling pathway or non-tumor cells. We indicate that drug synergy between I-BET762 and Talazoparib is associated with the attenuation HR-DSBR process and the downregulation of various players of DNA damage response by BET inhibition, such as CHEK2, PTEN, NBN, and FANCC. Our results provide a rationale for the development of new combinatorial strategies for the treatment of SCLC.

Accuracy and practicality of intraoral scanner in dentistry: A literature review.

PURPOSE: The digitization of the dental field has been vigorously promoted in recent years. An impression using an intraoral scanner is considered to significantly change future dental treatment. The purpose of this review is to evaluate accuracy and practicality of various intraoral scanners and verification method of intraoral scanners. STUDY SELECTION: This review was based on articles searched through the MEDLINE and PubMed databases. The main keywords that were employed during the search were "Oral Scanner, Intraoral Scanners, Desktop Scanner, and Digital Impression". RESULT: It was reported that illuminance and color temperature affected trueness and precision of intraoral scanners. The repeatability of intraoral scanners indicated the possibility of producing fixed prostheses within the range of being partially edentulous. It is considered difficult to use intraoral scanners in fabricating cross-arch fixed prostheses. However, with intraoral scanners, it may be considered possible to fabricate mouth guards and dentures equivalent to those of desktop scanners. Current intraoral scanner scans are considered more comfortable than traditional impressions that use irreversible hydrocolloid and elastomeric impression materials. CONCLUSION: Since the intraoral scanner is an evolving device, further improvement in accuracy is expected in the future. In addition, verification of the accuracy of intraoral scanners must be conducted accordingly.

Pipeline design to identify key features and classify the chemotherapy response on lung cancer patients using large-scale genetic data.

BACKGROUND: During the last decade, the interest to apply machine learning algorithms to genomic data has increased in many bioinformatics applications. Analyzing this type of data entails difficulties for managing high-dimensional data, class imbalance for knowledge extraction, identifying important features and classifying individuals. In this study, we propose a general framework to tackle these challenges with different machine learning algorithms and techniques. We apply the configuration of this framework on lung cancer patients, identifying genetic signatures for classifying response to drug treatment response. We intersect these relevant SNPs with the GWAS Catalog of the National Human Genome Research Institute and explore the Regulomedb, GTEx databases for functional analysis purposes. RESULTS: The machine learning based solution proposed in this study is a scalable and flexible alternative to the classical uni-variate regression approach to analyze large-scale data. From 36 experiments executed using the machine learning framework design, we obtain good classification performance from the top 5 models with the highest cross-validation score and the smallest standard deviation. One thousand two hundred twenty four SNPs corresponding to the key features from the top 20 models (cross validation F1 mean >= 0.65) were compared with the GWAS Catalog finding no intersection with genome-wide significant reported hits. From these, new genetic signatures in MAE, CEP104, PRKCZ and ADRB2 show relevant biological regulatory functionality related to lung physiology. CONCLUSIONS: We have defined a machine learning framework using data with an unbalanced large data-set of SNP-arrays and imputed genotyping data from a pharmacogenomics study in lung cancer patients subjected to first-line platinum-based treatment. This approach found genome signals with no genome-wide significance in the uni-variate regression approach (GWAS Catalog) that are valuable for classifying patients, only few of them with related biological function. The effect results of these variants can be explained by the recently proposed omnigenic model hypothesis, which states that complex traits can be influenced mostly by genes outside not only by the "core genes", mainly found by the genome-wide significant SNPs, but also by the rest of genes outside of the "core pathways" with apparent unrelated biological functionality.

Genomic profiling in advanced stage non-small-cell lung cancer patients with platinum-based chemotherapy identifies germline variants with prognostic value in SMYD2.

OBJECTIVE: The aim of the study was to investigate the relationship between germline variations as a prognosis biomarker in patients with advanced Non-Small-Cell-Lung-Cancer (NSCLC) subjected to first-line platinum-based treatment. MATERIALS AND METHODS: We carried out a two-stage genome-wide-association study in non-small-cell lung cancer patients with platinum-based chemotherapy in an exploratory sample of 181 NSCLC patients from Caucasian origin, followed by a validation on 356 NSCLC patients from the same ancestry (Valencia, Spain). RESULTS: We identified germline variants in SMYD2 as a prognostic factor for survival in patients with advanced NSCLC receiving chemotherapy. SMYD2 alleles are associated to a decreased overall survival and with a reduced Time to Progression. In addition, enrichment pathway analysis identified 361 variants in 40 genes to be involved in poorer outcome in advanced-stage NSCLC patients. CONCLUSION: Germline SMYD2 alleles are associated with bad clinical outcome of first-line platinum-based treatment in advanced NSCLC patients. This result supports the role of SMYD2 in the carcinogenic process, and might be used as prognostic signature directing patient stratification and the choice of therapy. MICROABSTRACT: A two-Stage Genome wide association study in Caucasian population reveals germline genetic variation in SMYD2 associated to progression disease in first-line platinum-based treatment in advanced NSCLC patients. SMYD2 profiling might have prognostic / predictive value directing choice of therapy and enlighten current knowledge on pathways involved in human carcinogenesis as well in resistance to chemotherapy.

A Nucleolar Stress-Specific p53-miR-101 Molecular Circuit Functions as an Intrinsic Tumor-Suppressor Network.

BACKGROUND: Activation of intrinsic p53 tumor-suppressor (TS) pathways is an important principle underlying cancer chemotherapy. It is necessary to elucidate the precise regulatory mechanisms of these networks to create new treatment strategies. METHODS: Comprehensive analyses were carried out by microarray. Expression of miR-101 was analyzed by clinical samples of lung adenocarcinomas. FINDINGS: We discovered a functional link between p53 and miR-101, which form a molecular circuit in response to nucleolar stress. Inhibition of RNA polymerase I (Pol I) transcription resulted in the post-transcriptional activation of miR-101 in a p53-dependent manner. miR-101 induced G2 phase-specific feedback regulation of p53 through direct repression of its target, EG5, resulting in elevated phosphorylation of ATM. In lung cancer patients, low expression of miR-101 was associated with significantly poorer prognosis exclusively in p53 WT cases. miR-101 sensitized cancer cells to Pol I transcription inhibitors and strongly repressed xenograft growth in mice. Interestingly, the most downstream targets of this circuit included the inhibitor of apoptosis proteins (IAPs). Repression of cIAP1 by a selective inhibitor, birinapant, promoted activation of the apoptosis induced by Pol I transcription inhibitor in p53 WT cancer cells. INTERPRETATION: Our findings indicate that the p53-miR-101 circuit is a component of an intrinsic TS network formed by nucleolar stress, and that mimicking activation of this circuit represents a promising strategy for cancer therapy. FUND: National Institute of Biomedical Innovation, Ministry of Education, Culture, Sports & Technology of Japan, Japan Agency for Medical Research and Development.

BET inhibition is an effective approach against KRAS-driven PDAC and NSCLC.

Effectively treating KRAS-driven tumors remains an unsolved challenge. The inhibition of downstream signaling effectors is a way of overcoming the issue of direct targeting of mutant KRAS, which has shown limited efficacy so far. Bromodomain and Extra-Terminal (BET) protein inhibition has displayed anti-tumor activity in a wide range of cancers, including KRAS-driven malignancies. Here, we preclinically evaluate the effect of BET inhibition making use of a new BET inhibitor, BAY 1238097, against Pancreatic Ductal Adenocarcinoma (PDAC) and Non-Small Cell Lung Cancer (NSCLC) models harboring RAS mutations both in vivo and in vitro. Our results demonstrate that BET inhibition displays significant therapeutic impact in genetic mouse models of KRAS-driven PDAC and NSCLC, reducing both tumor area and tumor grade. The same approach also causes a significant reduction in cell number of a panel of RAS-mutated human cancer cell lines (8 PDAC and 6 NSCLC). In this context, we demonstrate that while BET inhibition by BAY 1238097 decreases MYC expression in some cell lines, at least in PDAC cells its anti-tumorigenic effect is independent of MYC regulation. Together, these studies reinforce the use of BET inhibition and prompt the optimization of more efficient and less toxic BET inhibitors for the treatment of KRAS-driven malignancies, which are in urgent therapeutic need.

A Case of a Cardiac Resynchronization Therapy-Defibrillator Exhibiting a Lower and Alternately Variable Basic Rate.

A cardiac resynchronization therapy defibrillator (CRT-D) (Medtronic Inc. Protecta XT) was implanted in a 67-year-old man who had cardiac sarcoidosis with extremely low cardiac function. He had ventricular tachycardia which was controlled by catheter ablation, medication and pacing. The programmed mode was DDI, lower rate was 90 beats/minute, paced AV delay was 150 ms, and the noncompetitive atrial pacing (NCAP) function was programmed as 300 ms.After his admission for pneumonia and heart failure, we changed his DDI mode to a DDD mode because he had atrial tachycardia, which led to inadequate bi-ventricular pacing. After a while, there were cycle lengths which were longer than his device setting and alternately varied. We were able to avoid this phenomenon with AV delay of 120 ms and NCAP of 200 ms.NCAP is an algorithm which creates a gap above a certain period after the detection of an atrial signal during the postventricular atrial refractory period of the pacemaker. This is to prevent atrial tachycardia and repetitive non-reentrant ventriculoatrial (VA) synchrony in the presence of retrograde VA conduction. But in this case, NCAP algorithm induced much lower rate than the programmed basic lower rate. This situation produced some arrhythmias and exacerbated symptoms of heart failure. This had to be paid attention to, especially when the device was programmed at high basic heart rate.

Significantly mutated genes and regulatory pathways in SCLC-a meta-analysis.

Small cell lung cancer (SCLC) accounts for approximately 15% of all lung cancers and demands effective targeted therapeutic strategies. In this meta-analysis study, we aim to identify significantly mutated genes and regulatory pathways to help us better understand the progression of SCLC and to identify potential biomarkers. Besides ranking genes based on their mutation frequencies, we sought to identify statistically significant mutations in SCLC with the MutSigCV software. Our analysis identified several genes with relatively low mutation frequency, including PTEN, as highly significant (p < 0.001), suggesting these genes may play an important role in the progression of SCLC. Our results also indicated mutations in genes involved in the axon guidance pathways likely play an important role in SCLC progression. In addition, we observed that the mutation rate was significantly higher in samples with RB1 gene mutated when compared to samples with wild type RB1, suggesting that RB1 status has significant impact on the mutation profile and disease progression in SCLC.

Design of a Lentiviral Vector for the Inducible Expression of MYC: A New Strategy for Construction Approach.

Lentiviral vectors are powerful tools for gene expression studies. Here we report the construction of pTIJ, a vector for inducible gene expression. pTIJ was generated from pTRIPZ backbone, which is designed for the inducible expression of shRNA sequences, by the introducing of a multiple cloning site upstream of the Tet promoter and the removal of miR30 flanking sequences. To evaluate pTIJ as a tool for the inducible expression of genes of interest, we introduced MYC cDNA into pTIJ and infected two small cell lung cancer cell lines, H209 and H345. Induction of MYC expression by doxycycline was detectable in both cell lines by real-time PCR and western blot analysis. This study highlights the relevance of pTIJ vector to allow the inducible expression of any gene of interest. In our belief, pTIJ will be an extremely useful tool to simplify the generation of genetically engineered cell lines for the inducible expression of cDNA sequences in biological studies. Furthermore, we report the generation of a pTIJ-MYC vector for the inducible expression of the oncogene MYC.

Genomic Profiling of Patient-Derived Xenografts for Lung Cancer Identifies B2M Inactivation Impairing Immunorecognition.

Purpose: We aimed to maximize the performance of detecting genetic alterations in lung cancer using high-throughput sequencing for patient-derived xenografts (PDXs).Experimental Design: We undertook an integrated RNA and whole-exome sequencing of 14 PDXs. We focused on the genetic and functional analysis of ß2-microglobulin (B2M), a component of the HLA class-I complex.Results: We identified alterations in genes involved in various functions, such as B2M involved in immunosurveillance. We extended the mutational analysis of B2M to about 230 lung cancers. Five percent of the lung cancers carried somatic mutations, most of which impaired the correct formation of the HLA-I complex. We also report that genes such as CALR, PDIA3, and TAP1, which are involved in the maturation of the HLA-I complex, are altered in lung cancer. By gene expression microarrays, we observed that restitution of B2M in lung cancer cells upregulated targets of IFNα/IFNγ. Furthermore, one third of the lung cancers lacked the HLA-I complex, which was associated with lower cytotoxic CD8+ lymphocyte infiltration. The levels of B2M and HLA-I proteins correlated with those of PD-L1. Finally, a deficiency in HLA-I complex and CD8+ infiltration tended to correlate with reduced survival of patients with lung cancer treated with anti-PD-1/anti-PD-L1.Conclusions: Here, we report recurrent inactivation of B2M in lung cancer. These observations, coupled with the mutations found at CALR, PDIA3, and TAP1, and the downregulation of the HLA-I complex, indicate that an abnormal immunosurveillance axis contributes to lung cancer development. Finally, our observations suggest that an impaired HLA-I complex affects the response to anti-PD-1/anti-PD-L1 therapies. Clin Cancer Res; 23(12); 3203-13. ©2016 AACR.

Cellular responses to the expression of unstable secretory proteins in the filamentous fungus Aspergillus oryzae.

Filamentous fungi are often used as cell factories for recombinant protein production because of their ability to secrete large quantities of hydrolytic enzymes. However, even using strong transcriptional promoters, yields of nonfungal proteins are generally much lower than those of fungal proteins. Recent analyses revealed that expression of certain nonfungal secretory proteins induced the unfolded protein response (UPR), suggesting that they are recognized as proteins with folding defects in filamentous fungi. More recently, however, even highly expressed endogenous secretory proteins were found to evoke the UPR. These findings raise the question of whether the unfolded or misfolded state of proteins is selectively recognized by quality control mechanisms in filamentous fungi. In this study, a fungal secretory protein (1,2-α-D-mannosidase; MsdS) with a mutation that decreases its thermostability was expressed at different levels in Aspergillus oryzae. We found that, at moderate expression levels, wild-type MsdS was secreted to the medium, while the mutant was not. In the strain with a deletion for the hrdA gene, which is involved in the endoplasmic reticulum-associated degradation pathway, mutant MsdS had specifically increased levels in the intracellular fraction but was not secreted. When overexpressed, the mutant protein was secreted to the medium to a similar extent as the wild-type protein; however, the mutant underwent hyperglycosylation and induced the UPR. Deletion of α-amylase (the most abundant secretory protein in A. oryzae) alleviated the UPR induction by mutant MsdS overexpression. These findings suggest that misfolded MsdS and unfolded species of α-amylase might act synergistically for UPR induction.

A Two-Gene Prognostic Classifier for Early-Stage Lung Squamous Cell Carcinoma in Multiple Large-Scale and Geographically Diverse Cohorts.

INTRODUCTION: There are no validated molecular methods that prospectively identify patients with surgically resected lung squamous cell carcinoma (SCC) at high risk for recurrence. By focusing on the expression of genes with known functions in development of lung SCC and prognosis, we sought to develop a robust prognostic classifier of early-stage lung SCC. METHODS: The expression of 253 genes selected by literature search was evaluated in microarrays from 107 stage I/II tumors. Associations with survival were evaluated by Cox regression and Kaplan-Meier survival analyses in two independent cohorts of 121 and 91 patients with SCC, respectively. A classifier score based on multivariable Cox regression was derived and examined in six additional publicly available data sets of stage I/II lung SCC expression profiles (n = 358). The prognostic value of this classifier was evaluated in meta-analysis of patients with stage I/II (n = 479) and stage I (n = 326) lung SCC. RESULTS: Dual specificity phosphatase 6 gene (DUSP6) and actinin alpha 4 gene (ACTN4) were associated with prognostic outcome in two independent patient cohorts. Their expression values were utilized to develop a classifier that identified patients with stage I/II lung SCC at high risk for recurrence (hazard ratio [HR] = 4.7, p = 0.018) or cancer-specific mortality (HR = 3.5, p = 0.016). This classifier also identified patients at high risk for recurrence (HR = 2.7, p = 0.008) or death (HR = 2.2, p = 0.001) in publicly available data sets of stage I/II and in meta-analysis of stage I patients. CONCLUSIONS: We have established and validated a prognostic classifier to inform clinical management of patients with lung SCC after surgical resection.

Genomic Amplification of CD274 (PD-L1) in Small-Cell Lung Cancer.

Purpose: Programmed death ligand-1 (PD-L1), encoded by the CD274 gene, is a target for immune checkpoint blockade; however, little is known about genomic CD274 alterations. A subset of small-cell lung cancer (SCLC) exhibits increased copy number of chromosome 9p24, on which CD274 resides; however, most SCLCs show low expression of PD-L1. We therefore examined whether CD274 is a target of recurrent genomic alterations.Experimental Design: We examined somatic copy number alterations in two patient cohorts by quantitative real-time PCR in 72 human SCLC cases (cohort 1) and SNP array analysis in 138 human SCLC cases (cohort 2). Whole-genome sequencing revealed the detailed genomic structure underlying focal amplification. PD-L1 expression in amplified cases from cohorts 1 and 2 was further examined by transcriptome sequencing and immunohistochemical (IHC) staining.Results: By examining somatic copy number alterations in two cohorts of primary human SCLC specimens, we observed 9p24 copy number gains (where CD274 resides) and focal, high-level amplification of CD274 We found evidence for genomic targeting of CD274, suggesting selection during oncogenic transformation. CD274 amplification was caused by genomic rearrangements not affecting the open reading frame, thus leading to massively increased CD274 transcripts and high level expression of PD-L1.Conclusions: A subset (4/210, 1.9%) of human SCLC patient cases exhibits massive expression of PD-L1 caused by focal amplification of CD274 Such tumors may be particularly susceptible to immune checkpoint blockade. Clin Cancer Res; 23(5); 1220-6. ©2016 AACR.