Nucleic Acid Level Research Articles

Tumor suppressor let-7a inhibits breast cancer cell proliferation, migration and invasion by targeting MAGE-A1.

Let-7 was one of the earliest discovered miRNAs and while it reportedly acts as a tumor suppressor in various solid tumors, its function in breast cancer has not been fully studied. Therefore, we examined let-7a and MAGE-A1 expression in breast tissues by qRT-PCR and found that let-7a expression significantly correlates with larger tumor size, higher histological grade (p<0.05) and is significantly lower in patients with Her-2-positive cancers and Ki-67 >14% (p=0.028 and p=0.023). MAGE-A1 expression incidence is 50.8% (33/65) and it inversely correlates with let-7a expression (p=0.008). let-7a inhibition of breast cancer cell proliferation, migration and invasion was also observed in in vitro cell culture experiments, and dual-luciferase reporter assays showed that melanoma-associated antigen A1 (MAGE-A1) was its target gene; the target comprised bases 451-457 of the 3'UTR region of the MAGE-A1 mRNA. RT-qPCR and Western blot analyses showed that let-7a inhibited MAGE-A1 expression at both the nucleic acid and protein levels. In our final co-transfection experiment, we targeted MAGE-A1 in a breast cancer cell line and observed that let-7a inhibited cell proliferation, migration and invasion. These combined results confirm that let-7a functions as a tumor suppressor by targeting MAGE-A1 in breast cancer and it therefore provides a novel target in breast cancer clinical treatment.

Coincidence cloning recovery of Brucella melitensis RNA from goat tissues: advancing the in vivo analysis of pathogen gene expression in brucellosis

BackgroundBrucella melitensis bacteria cause persistent, intracellular infections in small ruminants as well as in humans, leading to significant morbidity and economic loss worldwide. The majority of experiments on the transcriptional responses of Brucella to conditions inside the host have been performed following invasion of cultured mammalian cells, and do not address gene expression patterns during long-term infection.ResultsHere, we examine the application of the previously developed coincidence cloning methodology to recover and characterize B. melitensis RNA from the supramammary lymph node of experimentally-infected goats. Using coincidence cloning, we successfully recovered Brucella RNA from supramammary lymph nodes of B. melitensis-infected goats at both short-term (4 weeks) and long-term (38 weeks) infection time points. Amplified nucleic acid levels were sufficient for analysis of Brucella gene expression patterns by RNA-sequencing, providing evidence of metabolic activity in both the short-term and the long-term samples. We developed a workflow for the use of sequence polymorphism analysis to confirm recovery of the inoculated strain in the recovered reads, and utilized clustering analysis to demonstrate a distinct transcriptional profile present in samples recovered in long-term infection. In this first look at B. melitensis gene expression patterns in vivo, the subset of Brucella genes that was highly upregulated in long-term as compared to short-term infection included genes linked to roles in murine infection, such as genes involved in proline utilization and signal transduction. Finally, we demonstrated the challenges of qPCR validation of samples with very low ratios of pathogen:host RNA, as is the case during in vivo brucellosis, and alternatively characterized intermediate products of the coincidence cloning reaction.ConclusionsOverall, this study provides the first example of recovery plus characterization of B. melitensis RNA from in vivo lymph node infection, and demonstrates that the coincidence cloning technique is a useful tool for characterizing in vivo transcriptional changes in Brucella species. Genes upregulated in long-term infection in this data set, including many genes not previously demonstrated to be virulence factors in mice or macrophage experiments, are candidates of future interest for potential roles in Brucella persistence in natural host systems.

Abstract 2012: The generation of a NSCLC EMT reporter cell line for metastatic lung cancer drug discovery and development

Abstract Cancer metastasis continues to be the leading cause of mortality in cancer patients around the world. One of the prominent biological processes implicated in cancer metastasis is epithelial-to-mesenchymal transition (EMT). EMT has also been shown to have roles in many aspects of cancer initiation and progression, including tumorigenesis and drug resistance. Despite the accumulation of a large body of data on the association of EMT with cancer, to date, EMT has not been an active target for therapeutic development partly due to the lack of appropriate in vitro models. Utilizing some of the basic biology of EMT, we have created a novel advanced in vitro model for use in both basic research and discovery of new anti-EMT drugs. In lung cancer, vimentin (VIM) intermediate filament (IF) proteins are associated with EMT, and the metastatic spread of cancer. Vimentin expression is generally upregulated when epithelial cells transition to the mesenchymal phenotype. We capitalized on this biological phenomenon, using CRISPR/Cas9 gene editing to generate a VIM RFP (red fluorescent protein) reporter cell line in the A549 non-small cell lung cancer (NSCLC) cell line; one of the most widely used and recognized lung cancer cell lines. The A549 VIM RFP cell line harbors a C-terminal red fluorescent protein (RFP) tag on the vimentin gene which enables end-point or real-time tracking of the EMT status as cells transition from epithelial to mesenchymal phenotype under defined conditions. The EMT reporter cell line was verified at the nucleic acid (genomic and mRNA) and protein levels as well as in cell-based assays. Bio-functional evaluation of the A549 VIM RFP cell line shows sensitivity to metastatic NSCLC drugs PP1 (SRC inhibitor) and A83-01 (ALK5 inhibitor). These results provide the foundation for the use of this cell line in high throughput screening (HTS) applications including the identification of new anti-EMT drugs for metastatic NSCLC. Furthermore, the A549 Vim RFP reporter cell line is also a convenient and sensitive model for basic science research on the mechanisms of metastasis. Citation Format: Metewo S. Enuameh, Sangeeta Kumari, Chauzhong Zou, John Foulke, Elizabeth Turner, Weiguo Shu, Robert Newman. The generation of a NSCLC EMT reporter cell line for metastatic lung cancer drug discovery and development [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2012.

Comparing Photon and Charged Particle Therapy Using DNA Damage Biomarkers.

Treatment modalities for cancer radiation therapy have become increasingly diversified given the growing number of facilities providing proton and carbon-ion therapy in addition to the more historically accepted photon therapy. An understanding of high-LET radiobiology is critical for optimization of charged particle radiation therapy and potential DNA damage response. In this review, we present a comprehensive summary and comparison of these types of therapy monitored primarily by using DNA damage biomarkers. We focus on their relative profiles of dose distribution and mechanisms of action from the level of nucleic acid to tumor cell death.

Effectiveness of a flow-based device using riboflavin photochemistry in damaging blood-borne viral nucleic acids

BackgroundEffectiveness of a flow-based treatment device using riboflavin photochemistry was demonstrated by cytopathic effect method using indicator viruses. However, inactivation efficacy against real blood-borne viruses needs to be evaluated, especially at nucleic acid level. Material and MethodsSpecial plasma samples with varying concentrations of blood-borne virus were selected using a strict blood selection procedure and were treated with device treatment (DT). Nucleic acid test (NAT) using polymerase chain reaction fluorescence method was used to detect virus copies. ResultsThe NAT value of 4325 in plasma with high Hepatitis B Virus (HBV) concentrations decreased to 1330 with DT. After 100-fold dilution, the NAT value was below the NAT detection limits with DT compared with 23.0 that without DT. The NAT value of 61.9 in plasma with medium HBV concentrations decreased to 37.8 with DT, and after 10-fold dilution, the NAT value was below the NAT detection limits with DT compared with below 20 that without DT. The Ct values of plasma with low concentrations of blood-borne viruses were below the NAT detection limits with DT. ConclusionThere was a dose effect with DT which was effective in blood-borne viruses damaging nucleic acids to a level below the NAT detection limits.

Formation of high-order oligomers is required for functional bioactivity of an African bat henipavirus surface glycoprotein

Hendra virus (HeV) and Nipah virus (NiV) are highly pathogenic henipaviruses originating from fruit bats in Australia and Asia that can cause severe infections in livestock and humans. In recent years, also African bat henipaviruses were identified at the nucleic acid level. To assess their potential to replicate in non-bat species, several studies were performed to characterize the two surface glycoproteins required for virus entry and spread by cell-cell fusion. It has been shown that surface expression and fusion-helper function of the receptor-binding G protein of Kumasi virus (KV), the prototypic Ghanaian bat henipavirus, is reduced compared to other non-African henipavirus G proteins. Immunostainings and pulse-chase analysis revealed a delayed export of KV G from the ER. As defects in oligomerization of viral glycoproteins can be responsible for limited surface transport thereby restricting the bioactivity, we analyzed the oligomerization pattern of KV G. In contrast to HeV and NiV whose G proteins are known to be expressed at a dimer-tetramer ratio of 1:1, KV G almost exclusively formed stable tetramers or higher oligomers. KV G also showed less stringent requirements for defined stalk cysteines to form dimers and tetramers. Interestingly, any changes in the oligomeric forms negatively affected the fusion-helper activity although surface expression and receptor binding was unchanged. This clearly indicates that the formation of mostly higher oligomeric KV G forms is not a deficiency responsible for ER retention, but is rather a basic structural feature essential for the bioactivity of this African bat henipavirus glycoprotein.

Functional Somatic Noncoding ncRNA of Extracelular Vesicles [EV] for Vascular Epigenetics: A Genomic Crossword Puzzle in Adaptation of Cells under Stress

OBJECTIVEFormation, remodeling and adaptation of different blood and lymphatic vessel phenotypes result from vasculo‐, angio‐, arterio‐ and lymph‐angiogenesis by complex interplay of various cells and factors [proteins, RNA, metal ions, metabolites, hormones, environment, stress]. Beyond genetics, by vascular epigenetics, diversity, complexity and tolerance of vascular phenotypes are vice versa requisites for function and survival of networked cells with interfaces upon intrinsic and extrinsic [environmental] needs and changes. It may be manifested beneficial or maladaptive entangled with, e.g. development, healing, implants, cardiovascular, immune or cancer‐angiogenesis‐tolerance reactions. This investigation aimed at whether formation and structure of some angio‐morphogen RNA sequences are from inherited germline [Mendelian] origin or somatic non‐Mendelian derivatives. The latter are functional RNA which retranslation do not anymore fit to inherited genes.METHODSFor search, angiotropin‐related RNA [ARNA] and MIR126 genes of microRNA‐126 [miR‐126] structures were used.RESULTSmiR‐126 are small Mendelian split products of MIR126 genes. Angiotropin is one of several exosomal RNP angiomorphogens from stressed mononuclear cells [shear, exercise stress, hypoxia, etc.]. It was sequenced after isolation from EV fluids where it is active in vitro and in tissue. By metal ions [Cu, Ca, Na, K], a Mendelian‐coded angiotropin‐related protein [S100‐A12] folds to a stable complex with somatic non‐Mendelian functional 5′end‐phosphorylated, edited, modified, redox‐ and metallo‐regulated non‐coding ARNA [75n] with 5′CUG3′‐hairpin loop. Its modified bases isoG/adenosine‐N1‐oxide are from Fenton‐type redox‐OH*/NO*‐radical RNA modification of adenosine in mesomeric/tautomeric complex with copper ions. It is shown that ARNA are formed by stressed mononuclear cells by rearrangement, recombination, mutation, editing, modification, redox‐ and metalloregulation of inherited MIR‐126 gene and its complementary derivative chain segments.CONCLUSIONSThe results suggest novel targets for vascular therapy. Some aspects of non‐Mendelian functional ncRNA resemble the process of somatic generation of immune diversity, as shown by Tonegawa. In this special case, all is achieved on the DNA and mRNA level, namely for coding new [immune] proteins by somatic cells under antigen attack stress. Here, a general principle is disclosed operated by somatic cells under stress on all nucleic acid, protein and carbohydrate levels to manage any diversity and complexity problems with their limited set of the inherited DNA in response to environmental chance reactions. New non‐Mendelian functional somatic noncoding RNA sequences for vascular epigenetics result from a genomic crossword puzzle by combined mechanisms of rearrangement, recombination, mutation, editing, modification, redox‐ and metalloregulation of inherited ncRNA of genes and their complementary derivative chain segments, i.e. by shuffling together segments of gene ncRNA and reuse of its non‐coding complementary sequence for tissue survival and adaptation to extraordinary environmental stress situations.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

DNA silencing by prokaryotic Argonaute proteins adds a new layer of defense against invading nucleic acids.

Argonaute (Ago) proteins are encoded in all three domains of life and are responsible for the regulation of intracellular nucleic acid levels. Whereas some Ago variants are able to cleave target nucleic acids by their endonucleolytic activity, others only bind to their target nucleic acids while target cleavage is mediated by other effector proteins. Although all Ago proteins show a high degree of overall structural homology, the nature of the nucleic acid binding partners differs significantly. Recent structural and functional data have provided intriguing new insights into the mechanisms of archaeal and bacterial Ago variants demonstrating the mechanistic diversity within the prokaryotic Ago family with astonishing differences in nucleic acid selection and nuclease specificity. In this review, we provide an overview of the structural organisation of archaeal Ago variants and discuss the current understanding of their biological functions that differ significantly from their eukaryotic counterparts.

Pathogens and Disease Play Havoc on the Host Epiproteome-The "First Line of Response" Role for Proteomic Changes Influenced by Disorder.

Organisms face stress from multiple sources simultaneously and require mechanisms to respond to these scenarios if they are to survive in the long term. This overview focuses on a series of key points that illustrate how disorder and post-translational changes can combine to play a critical role in orchestrating the response of organisms to the stress of a changing environment. Increasingly, protein complexes are thought of as dynamic multi-component molecular machines able to adapt through compositional, conformational and/or post-translational modifications to control their largely metabolic outputs. These metabolites then feed into cellular physiological homeostasis or the production of secondary metabolites with novel anti-microbial properties. The control of adaptations to stress operates at multiple levels including the proteome and the dynamic nature of proteomic changes suggests a parallel with the equally dynamic epigenetic changes at the level of nucleic acids. Given their properties, I propose that some disordered protein platforms specifically enable organisms to sense and react rapidly as the first line of response to change. Using examples from the highly dynamic host-pathogen and host-stress response, I illustrate by example how disordered proteins are key to fulfilling the need for multiple levels of integration of response at different time scales to create robust control points.

Phylogeny of dermatophytes with genomic character evaluation of clinically distinct Trichophyton rubrum and T.violaceum.

Trichophyton rubrum and T. violaceum are prevalent agents of human dermatophyte infections, the former being found on glabrous skin and nail, while the latter is confined to the scalp. The two species are phenotypically different but are highly similar phylogenetically. The taxonomy of dermatophytes is currently being reconsidered on the basis of molecular phylogeny. Molecular species definitions do not always coincide with existing concepts which are guided by ecological and clinical principles. In this article, we aim to bring phylogenetic and ecological data together in an attempt to develop new species concepts for anthropophilic dermatophytes. Focus is on the T. rubrum complex with analysis of rDNA ITS supplemented with LSU, TUB2, TEF3 and ribosomal protein L10 gene sequences. In order to explore genomic differences between T. rubrum and T. violaceum, one representative for both species was whole genome sequenced. Draft sequences were compared with currently available dermatophyte genomes. Potential virulence factors of adhesins and secreted proteases were predicted and compared phylogenetically. General phylogeny showed clear gaps between geophilic species of Arthroderma, but multilocus distances between species were often very small in the derived anthropophilic and zoophilic genus Trichophyton. Significant genome conservation between T. rubrum and T. violaceum was observed, with a high similarity at the nucleic acid level of 99.38 % identity. Trichophyton violaceum contains more paralogs than T. rubrum. About 30 adhesion genes were predicted among dermatophytes. Seventeen adhesins were common between T. rubrum and T. violaceum, while four were specific for the former and eight for the latter. Phylogenetic analysis of secreted proteases reveals considerable expansion and conservation among the analyzed species. Multilocus phylogeny and genome comparison of T. rubrum and T. violaceum underlined their close affinity. The possibility that they represent a single species exhibiting different phenotypes due to different localizations on the human body is discussed.

Investigating the effect of biosynthesized silver nanoparticles as antibiofilm on bacterial clinical isolates

Silver nanoparticles showed enhanced biofilm inhibitory activity of clinical pathogens. Eleven isolates (45.8%) of E. coli bacteria were obtained from 24 wound specimens. Silver nanoparticles biosynthesized by E. coli culture supernatant with exhibition dark brown color after 24 hr of incubation. Scanning electron microscopy showed that Ag-NPs spherical particles and its size were (14.2–67.8) nm and its average was 33.6 nm. X-ray diffraction shows one high peak at 2ø (32.5°) compared with standard data. Fourier transform infrared spectroscopy analysis of Ag-NPs exposed the strong band at 1367.53 corresponds to OH-bend which influences the synthesis and stability of Ag-NPs, whereas the stretch for Ag-NPs found at 518.58 cm−1. The antibacterial effect of Ag-Nps against E. coli, P. aeruginosa, K. pneumoniae and S. aureus showed the inhibition zone of 10, 11, 13, and 10 mm, respectively. Strong biofilm formed by isolates of E. coli exhibited as black colonies on Congo red agar, while pink colonies on it with Ag-Nps, indicating a loss of biofilm formation ability in all tested bacterial isolates. Antibiofilm of 10 mM Ag-Nps by Microtiter plate exhibited lower biofilm inhibition against S. aureus reached to 22.2%, while 36.2% against E. coli, 30.4% for K. pneumonae, and P. aeruginosa 94.7%. Analysis of biofilm components after exposure to Ag-NPs by FT-IR reveals a low level of proteins, polysaccharides, lipids and nucleic acids compared with controls, then glucose formation measured and exhibited reduction of absorbance (0.146 nm) after treatment with Ag-Nps compared with control (0.347 nm), while the percentage of protein decreased completely (0%) compared with control (0.25%). The combined effect of Ag-NPs with antibiotics enhanced the antibiofilm activity which tested under inverted microscope (40X).

Effects of dielectric barrier discharge plasma on the inactivation of Zygosaccharomyces rouxii and quality of apple juice

This work aimed to evaluate the effects of dielectric barrier discharge (DBD) plasma on inactivation of spoilage yeast Zygosaccharomyces rouxii (Z. rouxii), in apple juice. Results showed that DBD plasma treatment at 90 W for 140 s resulted in about 5-log reduction of Z. rouxii in apple juice. The levels of extracellular nucleic acids and proteins as well as contents of H2O2 and NO2− in yeast extract-peptone-dextrose (YPD) medium increased significantly after DBD plasma treatment at 90 W for 40–200 s. The increases in membrane permeability and generation of reactive species would likely contribute to DBD plasma-mediated inactivation of Z. rouxii. DBD plasma caused significant changes in pH, titratable acidity, and certain color parameters of apple juice, but had no effect on the contents of total soluble solids, reducing sugar, and total phenolics. This study provides key implications for the application of DBD plasma in fruit juice processing.

Urinary cell-free nucleic acid IQGAP3: a new non-invasive diagnostic marker for bladder cancer.

BackgroundThere is growing interest in developing new non-invasive diagnostic tools for bladder cancer (BC) that have better sensitivity and specificity than cystoscopy and cytology. This study examined the value of urinary cell-free nucleic acid (NA) as a diagnostic marker for BC.Material and methodsA total of 81 patients (74 BC and 7 normal controls) were used for a tissue set, and 212 patients (92 BC and 120 normal controls) were used as a urine set. Expression of tissue mRNA and urinary cell-free NAs was then examined.ResultsFour candidate genes were top-ranked in the tissue microarray. Expression levels of two of these (IQGAP3 and TOP2A) in BC tissue and urine samples from BC patients were significantly higher than those in samples from the control groups. Binary logistic regression analysis of cell-free NA levels in urine samples revealed that IQGAP3 was significantly associated with BC: PicoGreen-adjusted odds ratio (OR), 3.434; confidence interval (CI), 2.999–4.180; P<0.001; RiboGreen-adjusted OR, 2.242; CI, 1.793–2.840; P<0.001. Further analysis of IQGAP3 urinary cell-free NAs with respect to tumor invasiveness and grade also yielded a high AUC, suggesting that IQGAP3 can discriminate between BC patients and non-cancer patients with hematuria.ConclusionsLevels of IQGAP3 urinary cell-free NA in BC patients were significantly higher than those in normal controls or patients with hematuria. High levels of IQGAP3 urinary cell-free NA also reflected high expression in BC tissues. Therefore, IQGAP3 urinary cell-free NA may be a complementary diagnostic biomarker for BC.

Three-dimensional Culture of Human Airway Epithelium in Matrigel for Evaluation of Human Rhinovirus C and Bocavirus Infections

ObjectiveNewly identified human rhinovirus C (HRV-C) and human bocavirus (HBoV) cannot propagate in vitro in traditional cell culture models; thus obtaining knowledge about these viruses and developing related vaccines are difficult. Therefore, it is necessary to develop a novel platform for the propagation of these types of viruses. MethodsA platform for culturing human airway epithelia in a three-dimensional (3D) pattern using Matrigel as scaffold was developed. The features of 3D culture were identified by immunochemical staining and transmission electron microscopy. Nucleic acid levels of HRV-C and HBoV in 3D cells at designated time points were quantitated by real-time polymerase chain reaction (PCR). Levels of cytokines, whose secretion was induced by the viruses, were measured by ELISA. ResultsProperties of bronchial-like tissues, such as the expression of biomarkers CK5, ZO-1, and PCK, and the development of cilium-like protuberances indicative of the human respiration tract, were observed in 3D-cultured human airway epithelial (HAE) cultures, but not in monolayer-cultured cells. Nucleic acid levels of HRV-C and HBoV and levels of virus-induced cytokines were also measured using the 3D culture system. ConclusionOur data provide a preliminary indication that the 3D culture model of primary epithelia using a Matrigel scaffold in vitro can be used to propagate HRV-C and HBoV.

Tissue-Based Universal Virus Detection (TUViD-VM) Protocol for Viral Metagenomics.

The protocol for tissue-based universal virus detection (TUViD-VM) was developed to allow for the detection of yet unknown viruses from infected tissues. Tissues are a challenging source when it comes to virus detection. The very rapid degradation rate of tissue and the high level of host nucleic acids are the biggest challenges when examining tissues. However, to overcome these challenges, we had to decrease the amount of host nucleic acids rigorously in order to increase the amount of viral nucleic acids. We compared modern and common virological approaches to find the optimal conditions. The final TUViD-VM protocol was extensively validated by using real-time PCR and next-generation sequencing. We could increase the amount of detectable virus nucleic acids and improved the detection of viruses <75,000-fold compared with other approaches tested. The TUViD-VM protocol can be used in metagenomic and virome studies to increase the likelihood of detecting viruses from any biological source. The protocol has been widely used to detect viruses in different tissues with great success. Here we provide the bench-top protocol version of the TUViD-VM protocol.

Effect of Helium-Neon Laser and Sodium Hypochlorite on Calf Thymus Double-Stranded Deoxyribonucleic Acid Molecule: An in Vitro Experimental Study

Introduction: Low-energy helium-neon (He-Ne) laser beam lightis used in combination with sodium hypochlorite (Na2HOCl3) for clinical purposes. Regarding this, the present study aimed to investigate the effect of He-Ne laser (632.8 nm) and sodium hypochlorite on the calf thymus double-stranded deoxyribonucleic acid (ctdsDNA) molecule. Materials and Methods: For the purpose of the study, ctdsDNA solutions (30µg/ml) were exposed to He-Ne laser (632.8 nm) light in the absence and presence of different concentrations of sodium hypochlorite for up to 60 sec. The levels of nucleic acids released as uncontaminated and contaminated proteins were considered as the markers of DNA damage in terms of hypochromasia (i.e., DNA strand breakage) and hyperchromasia (i.e., of DNA strands separation). Results: The mean concentration of nucleic acids insignificantly (P > 0.05) decreased after exposure to laser light irradiation (hypochromic effect). Furthermore, laser irradiation insignificantly and inconsistency protected the ctdsDNA molecules from the effect of sodium hypochlorite. Sodium hypochlorite at concentrations of 1 and 3 mmol reduced the levels of the nucleic acids released from contaminated protein by 29.2% and 78.3% of the pre-incubated levels (hyporchromasia effect). The He-Ne laser (632.8 nm) irradiation induced hypochromic effect on the uncontaminated and contaminated proteins, while sodium hypochlorite induced a remarkable hyperchromic effect at higher concentrations. Conclusion: As the finding indicated, a short time He-Ne laser light (632.8 nm) irradiation exerted minor significant effect on the ctdsDNA molecule. This laser light did not interact with sodium hypochlorite as a synergistic combination against the ctdsDNA molecule.

Cell death and immunity in cancer: From danger signals to mimicry of pathogen defense responses.

The immunogenicity of cancer cells is an emerging determinant of anti-cancer immunotherapy. Beyond developing immunostimulatory regimens like dendritic cell-based vaccines, immune-checkpoint blockers, and adoptive T-cell transfer, investigators are beginning to focus on the immunobiology of dying cancer cells and its relevance for the success of anticancer immunotherapies. It is currently accepted that cancer cells may die in response to anti-cancer therapies through regulated cell death programs, which may either repress or increase their immunogenic potential. In particular, the induction of immunogenic cancer cell death (ICD), which is hallmarked by the emission of damage-associated molecular patterns (DAMPs); molecules analogous to pathogen-associated molecular patterns (PAMPs) acting as danger signals/alarmins, is of great relevance in cancer therapy. These ICD-associated danger signals favor immunomodulatory responses that lead to tumor-associated antigens (TAAs)-directed T-cell immunity, which paves way for the removal of residual, treatment-resistant cancer cells. It is also emerging that cancer cells succumbing to ICD can orchestrate "altered-self mimicry" i.e. mimicry of pathogen defense responses, on the levels of nucleic acids and/or chemokines (resulting in type I interferon/IFN responses or pathogen response-like neutrophil activity). In this review, we exhaustively describe the main molecular, immunological, preclinical, and clinical aspects of immunosuppressive cell death or ICD (with respect to apoptosis, necrosis and necroptosis). We also provide an extensive historical background of these fields, with special attention to the self/non-self and danger models, which have shaped the field of cell death immunology.

Potential of Aqueous Humor as a Surrogate Tumor Biopsy for Retinoblastoma

Retinoblastoma (Rb) is one of the first tumors to have a known genetic etiology. However, because biopsy of this tumor is contraindicated, it has not been possible to define the effects of secondary genetic changes on the disease course. To investigate whether the aqueous humor (AH) of Rb eyes has sufficient tumor-derived DNA to perform genetic analysis of the tumor, including DNA copy number alterations. This investigation was a case series study at a tertiary care hospital (Children's Hospital Los Angeles) with a large Rb treatment center. Cell-free DNA (cfDNA) was isolated from 6 AH samples from 3 children with Rb, including 2 after primary enucleation and 1 undergoing multiple intravitreous injections of melphalan for vitreous seeding. Samples were taken between December 2014 and September 2015. Measurable levels of nucleic acids in the AH and identification of tumor-derived DNA copy number variation in the AH. The AH was analyzed for DNA, RNA, and micro-RNA using Qubit high-sensitivity kits. Cell-free DNA was isolated from the AH, and sequencing library protocols were optimized. Shallow whole-genome sequencing was performed on an Illumina platform, followed by genome-wide chromosomal copy number variation profiling to assess the presence of tumor DNA fractions in the AH cfDNA of the 3 patients. One child's cfDNA from the AH and tumor DNA were subjected to Sanger sequencing to isolate the RB1 mutation. Six AH samples were obtained from 3 Rb eyes in 3 children (2 male and 1 female; diagnosed at ages 7, 20, and 28 months). A corroborative pattern between the chromosomal copy number variation profiles of the AH cfDNA and tumor-derived DNA from the enucleated samples was identified. In addition, a nonsense RB1 mutation (Lys→STOP) from 1 child was also identified from the AH samples obtained during intravitreous injection of melphalan, which matched the tumor sample postsecondary enucleation. Sanger sequencing of the AH cfDNA and tumor DNA with polymerase chain reaction primers targeting RB1 gene c.1075A demonstrated this same RB1 mutation. In this study evaluating nucleic acids in the AH from Rb eyes undergoing salvage therapy with intravitreous injection of melphalan, the results suggest that the AH can serve as a surrogate tumor biopsy when Rb tumor tissue is not available. This novel method will allow for analyses of tumor-derived DNA in Rb eyes undergoing salvage therapy that have not been enucleated.

Extracellular vesicles: nature's nanoparticles for improving gene transfer with adeno-associated virus vectors.

Gene therapy, the ability to treat a disease at the level of nucleic acid, has journeyed from science fiction, to hard lessons learned from early clinical trials, to improved technologies with efficacy in patients for several diseases. Adeno-associated virus (AAV) vectors are currently a leader for direct in vivo gene therapy. To date, AAV is safe in patients, with clinical benefit in trials to treat blindness, hemophilia, and a lipid disorder, with many more trials underway. Despite this remarkable progress, barriers exist for AAV vectors to be effective gene transfer vehicles in all organ/cell targets, as well as patient subpopulations. Extracellular vesicles (EVs, e.g., exosomes, microvesicles) are natural lipid particles released by many cell types. They have been reported to mediate cell to cell communication via transferred contents including proteins, nucleic acids, and metabolites. These properties of EV attracted our attention to help solve certain gene transfer issues encountered by AAV vectors. We made the initial discovery that a subpopulation of AAV vectors isolated from media directly interacted with EVs [referred to as exosome-associated AAV (exo-AAV)]. In following reports, we have demonstrated that exo-AAV has advantages over the conventional AAV vector in areas such as anti-AAV antibody evasion and transduction of cells of the eye and cochlea in preclinical models. The work of others using EVs as therapeutics as well as our continued development of the exo-AAV platform may advance the field towards useful clinical applications. This article is categorized under: Therapeutic Approaches and Drug Discovery > Emerging Technologies Biology-Inspired Nanomaterials > Lipid-Based Structures Therapeutic Approaches and Drug Discovery > Nanomedicine for Neurological Disease.

Abstract 1626: Technical variability in NGS immune gene expression and mutation profiling has a nominal effect on tumor classification

Abstract Background: A custom NGS cancer immune gene expression assay was developed which measures the transcript level of &amp;gt;350 genes involved in T-cell receptor signaling (TCRS), tumor infiltrating lymphocyte (TILs) complement as well as other key targets expected to predict the likelihood of patient response to checkpoint inhibitors (CPI). In parallel to the gene expression assay, mutational profiling was carried out using the 409 gene Comprehensive Cancer Panel (ThermoFisher). As variability between runs is common when performing NGS assays a detailed comparison of specific technical variations were assessed for their ability to effect gene expression and mutation profiles of clinical FFPE samples. Methods: Studies were designed to characterize the analytical performance of the immune response NGS assay using RNA and DNA from a subset of 300 FFPE tissues representing NSCLC, melanoma, renal cell carcinoma and bladder cancer. As part of the study, we tested the impact of variability in RNA and DNA input quantity at the library preparation step, sample batch size which affects mapped reads/sample and depth of coverage, and linearity of expression and sensitivity of mutation profiling through serial dilutions of pico-molar (pM) input of normalized library. PCA and unsupervised clustering was performed on samples with checkpoint inhibition, TCRS and TILs genes as well as mutational profiling to reveal sample groups with three distinct immune signatures (low, indeterminate and high). Further correlation and over-representation analysis was performed to determine impact of technical characteristics on these three immune signatures. Results: Immune signatures including mutation profiles and gene expression levels were maintained throughout variable RNA/DNA input amounts at the library generation level as well as with diminution of pM levels of library pooled at the sequencing step. Increase in the number of mapped reads and sequencing depth through decreasing the number of batched samples per sequencing run also did not affect the gene expression and mutation profile signatures of the FFPE derived samples. Conclusion: The gene expression and mutation profiles responsible for classifying FFPE samples using NGS are not affected by variation normally introduced in the technical workflow commonly associated with these platforms. The analytical assessment of input at the nucleic acid, library, and sample size level has shown the plasticity available when using amplicon based NGS technologies for classifying the immune gene expression signature as well as mutational profiles of FFPE derived clinical tumor samples. This flexibility increases the strength and utility of NGS-base gene expression profiling and mutational analysis of tumor samples for both basic research and clinical applications. Citation Format: Sean Glenn, Jeffrey Conroy, Blake Burgher, Sarabjot Pabla, Maochun Qin, Jon Andreas, Vincent Giamo, Marc Ernstoff, Mary Nesline, Ji He, Mark Gardner, Carl Morrison. Technical variability in NGS immune gene expression and mutation profiling has a nominal effect on tumor classification [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1626. doi:10.1158/1538-7445.AM2017-1626