Tumor biomechanical stiffness by magnetic resonance elastography predicts surgical outcomes and identifies biomarkers in vestibular schwannoma and meningioma.

Variations in the biomechanical stiffness of brain tumors can not only influence the difficulty of surgical resection but also impact postoperative outcomes. In a prospective, single-blinded study, we utilize pre-operative magnetic resonance elastography (MRE) to predict the stiffness of intracranial tumors intraoperatively and assess the impact of increased tumor stiffness on clinical outcomes following microsurgical resection of vestibular schwannomas (VS) and meningiomas. MRE measurements significantly correlated with intraoperative tumor stiffness and baseline hearing status of VS patients. Additionally, MRE stiffness was elevated in patients that underwent sub-total tumor resection compared to gross total resection and those with worse postoperative facial nerve function. Furthermore, we identify tumor microenvironment biomarkers of increased stiffness, including αSMA + myogenic fibroblasts, CD163 + macrophages, and HABP (hyaluronic acid binding protein). In a human VS cell line, a dose-dependent upregulation of HAS1-3, enzymes responsible for hyaluronan synthesis, was observed following stimulation with TNFα, a proinflammatory cytokine present in VS. Taken together, MRE is an accurate, non-invasive predictor of tumor stiffness in VS and meningiomas. VS with increased stiffness portends worse preoperative hearing and poorer postoperative outcomes. Moreover, inflammation-mediated hyaluronan deposition may lead to increased stiffness.

Nursing Orientation During the COVID-19 Pandemic.

Traditional in-person delivery of nursing orientation programs at a large academic hospital could not occur because of the COVID-19 pandemic, with the need to limit group sizes and adhere to physical distancing guidelines. A nurse educator team pivoted the orientation program to a virtual model combined with the review of select clinical skills and buddy shifts. This model effectively met the nurses' needs required to practice safely on an inpatient environment.

Two-hybrid screening of FAM13A protein partners in lung epithelial cells.

OBJECTIVES: Family with sequence similarity 13 member A (FAM13A) genetic variants have been associated with several chronic respiratory diseases including chronic obstructive pulmonary disease (COPD), cystic fibrosis (CF), idiopathic pulmonary fibrosis (IPF) and lung cancer. The FAM13A protein includes a RhoGTPase activating protein (RhoGAP) domain known to participate in various cellular mechanisms including cell proliferation. While intensive genomic studies have been performed to reveal its involvement in lung diseases, the biological role of FAM13A protein is still not completely elucidated. RESULTS: We therefore performed a two-hybrid screening to identify protein partners of FAM13A using a human lung cancer cDNA library. We identified several protein partners with a high confidence score. Researchers in the field of chronic lung diseases may benefit from this two-hybrid screening data which may reveal new research pathways to decipher.

IL-13 Impairs Tight Junctions in Airway Epithelia.

Interleukin-13 (IL-13) drives symptoms in asthma with high levels of T-helper type 2 cells (Th2-cells). Since tight junctions (TJ) constitute the epithelial diffusion barrier, we investigated the effect of IL-13 on TJ in human tracheal epithelial cells. We observed that IL-13 increases paracellular permeability, changes claudin expression pattern and induces intracellular aggregation of the TJ proteins zonlua occludens protein 1, as well as claudins. Furthermore, IL-13 treatment increases expression of ubiquitin conjugating E2 enzyme UBE2Z. Co-localization and proximity ligation assays further showed that ubiquitin and the proteasomal marker PSMA5 co-localize with TJ proteins in IL-13 treated cells, showing that TJ proteins are ubiquitinated following IL-13 exposure. UBE2Z upregulation occurs within the first day after IL-13 exposure. Proteasomal aggregation of ubiquitinated TJ proteins starts three days after IL-13 exposure and transepithelial electrical resistance (TEER) decrease follows the time course of TJ-protein aggregation. Inhibition of JAK/STAT signaling abolishes IL-13 induced effects. Our data suggest that that IL-13 induces ubiquitination and proteasomal aggregation of TJ proteins via JAK/STAT dependent expression of UBE2Z, resulting in opening of TJs. This may contribute to barrier disturbances in pulmonary epithelia and lung damage of patients with inflammatory lung diseases.

Inflammation-induced upregulation of P2X4 expression augments mucin secretion in airway epithelia.

Mucus clearance provides an essential innate defense mechanism to keep the airways and lungs free of particles and pathogens. Baseline and stimulated mucin secretion from secretory airway epithelial cells need to be tightly regulated to prevent mucus hypersecretion and mucus plugging of the airways. It is well established that extracellular ATP is a potent stimulus for regulated mucus secretion. Previous studies revealed that ATP acts via metabotropic P2Y2 purinoreceptors on goblet cells. Extracellular ATP, however, is also a potent agonist for ionotropic P2X purinoreceptors. Expression of several P2X isoforms has been reported in airways, but cell type-specific expression and the function thereof remained elusive. With this study, we now provide evidence that P2X4 is the predominant P2X isoform expressed in secretory airway epithelial cells. After IL-13 treatment of either human primary tracheal epithelial cells or mice, P2X4 expression is upregulated in vitro and in vivo under conditions of chronic inflammation, mucous metaplasia, and hyperplasia. Upregulation of P2X4 is strongest in MUC5AC-positive goblet cells. Moreover, activation of P2X4 by extracellular ATP augments intracellular Ca2+ signals and mucin secretion, whereas Ca2+ signals and mucin secretion are dampened by inhibition of P2X4 receptors. These data provide new insights into the purinergic regulation of mucin secretion and add to the emerging picture that P2X receptors modulate exocytosis of large secretory organelles and secretion of macromolecular vesicle cargo.

FAM13A is a modifier gene of cystic fibrosis lung phenotype regulating rhoa activity, actin cytoskeleton dynamics and epithelial-mesenchymal transition.

BACKGROUND: Cystic fibrosis (CF) lung disease severity is highly variable and dependent on several factors including genetic modifiers. Family with sequence similarity 13 member A (FAM13A) has been previously associated with lung function in the general population as well as in several chronic lung diseases, such as chronic obstructive pulmonary disease (COPD), we examined whether FAM13A is a modifier gene of CF lung phenotype. We also studied how FAM13A may contribute to the physiopathological mechanisms associated with CF. METHODS: We investigated the association of FAM13A with lung function in CF French patients (n=1222) by SNP-wise analysis and Versatile Gene Based Association Study. We also analyzed the consequences of FAM13A knockdown in A549 cells and primary bronchial epithelial cells from CF patients. RESULTS: We found that FAM13A is associated with lung function in CF patients. Utilizing lung epithelial A549 cells and primary human bronchial epithelial cells from CF patients we observed that IL-1ß and TGFß reduced FAM13A expression. Knockdown of FAM13A was associated with increased RhoA activity, induction of F-actin stress fibers and regulation of epithelial-mesenchymal transition markers such as E-cadherin, α-smooth muscle actin and vimentin. CONCLUSION: Our data show that FAM13A is a modifier gene of CF lung phenotype regulating RhoA activity, actin cytoskeleton dynamics and epithelial-mesenchymal transition.

Water Permeability Adjusts Resorption in Lung Epithelia to Increased Apical Surface Liquid Volumes.

The apical surface liquid (ASL) layer covers the airways and forms a first line of defense against pathogens. Maintenance of ASL volume by airway epithelia is essential for maintaining lung function. The proteolytic activation of epithelial Na+ channels is believed to be the dominating mechanism to cope with increases in ASL volumes. Alternative mechanisms, in particular increases in epithelial osmotic water permeability (Posm), have so far been regarded as rather less important. However, most studies mainly addressed immediate effects upon apical volume expansion (AVE) and increases in ASL. This study addresses the response of lung epithelia to long-term AVE. NCI-H441 cells and primary human tracheal epithelial cells, both cultivated in air-liquid interface conditions, were used as models for the lung epithelium. AVE was established by adding isotonic solution to the apical surface of differentiated lung epithelia, and time course of ASL volume restoration was assessed by the deuterium oxide dilution method. Concomitant ion transport was investigated in Ussing chambers. We identified a low resorptive state immediately after AVE, which coincided with proteolytic ion transport activation within 10-15 minutes after AVE. The main clearance of excess ASL occurred during a delayed (hours after AVE) high resorptive state, which did not correlate with ion transport activation. Instead, high resorptive state onset coincided with an increase in Posm, which depended on aquaporin up-regulation. In summary, our data demonstrate that, aside from ion transport activation, modulation of Posm is a major mechanism to compensate for long-term AVE in lung epithelia.

Glucocorticoids Regulate Tight Junction Permeability of Lung Epithelia by Modulating Claudin 8.

The lung epithelium constitutes a selective barrier that separates the airways from the aqueous interstitial compartment. Regulated barrier function controls water and ion transport across the epithelium and is essential for maintaining lung function. Tight junctions (TJs) seal the epithelial barrier and determine the paracellular transport. The properties of TJs depend especially on their claudin composition. Steroids are potent drugs used to treat a variety of airway diseases. Therefore, we addressed whether steroid hormones directly act on TJ properties in lung epithelia. Primary human tracheal epithelial cells and NCI-H441 cells, both cultivated under air-liquid interface conditions, were used as epithelial cell models. Our results demonstrate that glucocorticoids, but not mineralocorticoids, decreased paracellular permeability and shifted the ion permselectivity of TJs toward Cl(-). Glucocorticoids up-regulated claudin 8 (cldn8) expression via glucocorticoid receptors. Silencing experiments revealed that cldn8 is necessary to recruit occludin at the TJs. Immunohistochemistry on human lung tissue showed that cldn8 is specifically expressed in resorptive epithelia of the conducting and respiratory airways but not in the alveolar epithelium. We conclude that glucocorticoids enhance lung epithelia barrier function and increase paracellular Cl(-) selectivity via modulation of cldn8-dependent recruitment of occludin at the TJs. This mode of glucocorticoid action on lung epithelia might be beneficial to patients who suffer from impaired lung barrier function in various diseased conditions.

Translating the genetics of cystic fibrosis to personalized medicine.

Cystic fibrosis (CF) is the most common life-threatening recessive genetic disease in the Caucasian population. This multiorgan disease is caused by mutations in the gene encoding the CF transmembrane conductance regulator (CFTR) protein, a chloride channel recognized as regulating several apical ion channels. The gene mutations result either in the lack of the protein at the apical surface or in an improperly functioning protein. Morbidity and mortality because of the mutation of CFTR are mainly attributable to lung disease resulting from chronic infection and inflammation. Since its discovery as the causative gene in 1989, much progress has been achieved not only in clinical genetics but also in basic science studies. Recently, combinations of these efforts have been successfully translated into development and availability for patients of new therapies targeting specific CFTR mutations to correct the CFTR at the protein level. Current technologies such as next gene sequencing and novel genomic editing tools may offer new strategies to identify new CFTR variants and modifier genes, and to correct CFTR to pursue personalized medicine, which is already developed in some patient subsets. Personalized medicine or P4 medicine ("personalized," "predictive," "preventive," and "participatory") is currently booming for CF. The various current and future challenges of personalized medicine as they apply to the issues faced in CF are discussed in this review.

Amiloride-sensitive fluid resorption in NCI-H441 lung epithelia depends on an apical Cl(-) conductance.

Proper apical airway surface hydration is essential to maintain lung function. This hydration depends on well-balanced water resorption and secretion. The mechanisms involved in resorption are still a matter of debate, especially as the measurement of transepithelial water transport remains challenging. In this study, we combined classical short circuit current (I SC) measurements with a novel D2O dilution method to correlate ion and water transport in order to reveal basic transport mechanisms in lung epithelia. D2O dilution method enabled precise analysis of water resorption with an unprecedented resolution. NCI-H441 cells cultured at an air-liquid interface resorbed water at a rate of 1.5 ± 0.4 µL/(h cm(2)). Water resorption and I SC were reduced by almost 80% in the presence of the bulk Cl(-) channel inhibitor 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) or amiloride, a specific inhibitor of epithelial sodium channel (ENaC). However, water resorption and I SC were only moderately affected by forskolin or cystic fibrosis transmembrane regulator (CFTR) channel inhibitors (CFTRinh-172 and glybenclamide). In line with previous studies, we demonstrate that water resorption depends on ENaC, and CFTR channels have only a minor but probably modulating effect on water resorption. However, the major ENaC-mediated water resorption depends on an apical non-CFTR Cl(-) conductance.

Surfactant secretion in LRRK2 knock-out rats: changes in lamellar body morphology and rate of exocytosis.

Leucine-rich repeat kinase 2 (LRRK2) is known to play a role in the pathogenesis of various diseases including Parkinson disease, morbus Crohn, leprosy and cancer. LRRK2 is suggested to be involved in a number of cell biological processes such as vesicular trafficking, transcription, autophagy and lysosomal pathways. Recent histological studies of lungs of LRRK2 knock-out (LRRK2 -/-) mice revealed significantly enlarged lamellar bodies (LBs) in alveolar type II (ATII) epithelial cells. LBs are large, lysosome-related storage organelles for pulmonary surfactant, which is released into the alveolar lumen upon LB exocytosis. In this study we used high-resolution, subcellular live-cell imaging assays to investigate whether similar morphological changes can be observed in primary ATII cells from LRRK2 -/- rats and whether such changes result in altered LB exocytosis. Similarly to the report in mice, ATII cells from LRRK2 -/- rats contained significantly enlarged LBs resulting in a >50% increase in LB volume. Stimulation of ATII cells with ATP elicited LB exocytosis in a significantly increased proportion of cells from LRRK2 -/- animals. LRRK2 -/- cells also displayed increased intracellular Ca(2+) release upon ATP treatment and significant triggering of LB exocytosis. These findings are in line with the strong Ca(2+)-dependence of LB fusion activity and suggest that LRRK2 -/- affects exocytic response in ATII cells via modulating intracellular Ca(2+) signaling. Post-fusion regulation of surfactant secretion was unaltered. Actin coating of fused vesicles and subsequent vesicle compression to promote surfactant expulsion were comparable in cells from LRRK2 -/- and wt animals. Surprisingly, surfactant (phospholipid) release from LRRK2 -/- cells was reduced following stimulation of LB exocytosis possibly due to impaired LB maturation and surfactant loading of LBs. In summary our results suggest that LRRK2 -/- affects LB size, modulates intracellular Ca(2+) signaling and promotes LB exocytosis upon stimulation of ATII cells with ATP.

A new role for P2X4 receptors as modulators of lung surfactant secretion.

In recent years, P2X receptors have attracted increasing attention as regulators of exocytosis and cellular secretion. In various cell types, P2X receptors have been found to stimulate vesicle exocytosis directly via Ca(2+) influx and elevation of the intracellular Ca(2+) concentration. Recently, a new role for P2X4 receptors as regulators of secretion emerged. Exocytosis of lamellar bodies (LBs), large storage organelles for lung surfactant, results in a local, fusion-activated Ca(2+) entry (FACE) in alveolar type II epithelial cells. FACE is mediated via P2X4 receptors that are located on the limiting membrane of LBs and inserted into the plasma membrane upon exocytosis of LBs. The localized Ca(2+) influx at the site of vesicle fusion promotes fusion pore expansion and facilitates surfactant release. In addition, this inward-rectifying cation current across P2X4 receptors mediates fluid resorption from lung alveoli. It is hypothesized that the concomitant reduction in the alveolar lining fluid facilitates insertion of surfactant into the air-liquid interphase thereby "activating" it. These findings constitute a novel role for P2X4 receptors in regulating vesicle content secretion as modulators of the secretory output during the exocytic post-fusion phase.

Exome and whole-genome sequencing of esophageal adenocarcinoma identifies recurrent driver events and mutational complexity.

The incidence of esophageal adenocarcinoma (EAC) has risen 600% over the last 30 years. With a 5-year survival rate of ~15%, the identification of new therapeutic targets for EAC is greatly important. We analyze the mutation spectra from whole-exome sequencing of 149 EAC tumor-normal pairs, 15 of which have also been subjected to whole-genome sequencing. We identify a mutational signature defined by a high prevalence of A>C transversions at AA dinucleotides. Statistical analysis of exome data identified 26 significantly mutated genes. Of these genes, five (TP53, CDKN2A, SMAD4, ARID1A and PIK3CA) have previously been implicated in EAC. The new significantly mutated genes include chromatin-modifying factors and candidate contributors SPG20, TLR4, ELMO1 and DOCK2. Functional analyses of EAC-derived mutations in ELMO1 identifies increased cellular invasion. Therefore, we suggest the potential activation of the RAC1 pathway as a contributor to EAC tumorigenesis.

Fusion-activated cation entry (FACE) via P2X4 couples surfactant secretion and alveolar fluid transport.

Two fundamental mechanisms within alveoli are essential for lung function: regulated fluid transport and secretion of surfactant. Surfactant is secreted via exocytosis of lamellar bodies (LBs) in alveolar type II (ATII) cells. We recently reported that LB exocytosis results in fusion-activated cation entry (FACE) via P2X4 receptors on LBs. We propose that FACE, in addition to facilitating surfactant secretion, modulates alveolar fluid transport. Correlative fluorescence and atomic force microscopy revealed that FACE-dependent water influx correlated with individual fusion events in rat primary ATII cells. Moreover, ATII cell monolayers grown at air-liquid interface exhibited increases in short-circuit current (Isc) on stimulation with ATP or UTP. Both are potent agonists for LB exocytosis, but only ATP activates FACE. ATP, not UTP, elicited additional fusion-dependent increases in Isc. Overexpressing dominant-negative P2X4 abrogated this effect by ∼50%, whereas potentiating P2X4 lead to ∼80% increase in Isc. Finally, we monitored changes in alveolar surface liquid (ASL) on ATII monolayers by confocal microscopy. Only stimulation with ATP, not UTP, led to a significant, fusion-dependent, 20% decrease in ASL, indicating apical-to-basolateral fluid transport across ATII monolayers. Our data support the first direct link between LB exocytosis, regulation of surfactant secretion, and transalveolar fluid resorption via FACE.

Human airway ciliary dynamics.

Airway cilia depend on precise changes in shape to transport the mucus gel overlying mucosal surfaces. The ciliary motion can be recorded in several planes using video microscopy. However, cilia are densely packed, and automated computerized systems are not available to convert these ciliary shape changes into forms that are useful for testing theoretical models of ciliary function. We developed a system for converting planar ciliary motions recorded by video microscopy into an empirical quantitative model, which is easy to use in validating mathematical models, or in examining ciliary function, e.g., in primary ciliary dyskinesia (PCD). The system we developed allows the manipulation of a model cilium superimposed over a video of beating cilia. Data were analyzed to determine shear angles and velocity vectors of points along the cilium. Extracted waveforms were used to construct a composite waveform, which could be used as a standard. Variability was measured as the mean difference in position of points on individual waveforms and the standard. The shapes analyzed were the end-recovery, end-effective, and fastest moving effective and recovery with mean (± SE) differences of 0.31(0.04), 0.25(0.06), 0.50(0.12), 0.50(0.10), µm, respectively. In contrast, the same measures for three different PCD waveforms had values far outside this range.

Mapping the hallmarks of lung adenocarcinoma with massively parallel sequencing.

Lung adenocarcinoma, the most common subtype of non-small cell lung cancer, is responsible for more than 500,000 deaths per year worldwide. Here, we report exome and genome sequences of 183 lung adenocarcinoma tumor/normal DNA pairs. These analyses revealed a mean exonic somatic mutation rate of 12.0 events/megabase and identified the majority of genes previously reported as significantly mutated in lung adenocarcinoma. In addition, we identified statistically recurrent somatic mutations in the splicing factor gene U2AF1 and truncating mutations affecting RBM10 and ARID1A. Analysis of nucleotide context-specific mutation signatures grouped the sample set into distinct clusters that correlated with smoking history and alterations of reported lung adenocarcinoma genes. Whole-genome sequence analysis revealed frequent structural rearrangements, including in-frame exonic alterations within EGFR and SIK2 kinases. The candidate genes identified in this study are attractive targets for biological characterization and therapeutic targeting of lung adenocarcinoma.

Combined atomic force microscopy-fluorescence microscopy: analyzing exocytosis in alveolar type II cells.

Hybrid atomic force microscopy (AFM)-fluorescence microscopy (FM) investigation of exocytosis in lung epithelial cells (ATII cells) allows the detection of individual exocytic events by FM, which can be simultaneously correlated to structural changes in individual cells by AFM. Exocytosis of lamellar bodies (LBs) represents a slow form of exocytosis found in many non-neuronal cells. Exocytosis of LBs, following stimulation with adenosine-5'-triphosphate (ATP) and phorbol 12-myristate 13-acetate (PMA), results in a cation influx via P2X(4) receptors at the site of LB fusion with the plasma membrane (PM), which should induce a temporary increase in cell height/volume. AFM measurements were performed in single-line scans across the cell surface. Five minutes after stimulation, ATII cells revealed a cell height and volume increase of 13.7% ± 4.1% and 15.9 ± 4.8% (N = 9), respectively. These transient changes depend on exocytic LB-PM fusion. Nonstimulated cells and cells lacking LB fusions did not show a significant change in cell height/volume (N = 8). In addition, a cell height decrease was observed in ATII cells stimulated by uridine-5'-triphosphate (UTP) and PMA, agonists inducing LB fusion with the PM, but not activation of P2X(4) receptors. The cell height and volume decreased by -8.6 ± 3.6% and -11.2 ± 3.9% (N = 5), respectively. Additionally, low force contact and dynamic mode AFM imaging of cell areas around the nucleus after stimulation with ATP/PMA was performed. Fused LBs are more pronounced in AFM topography images compared to nonfused LBs, concluding that different "dynamic states" of LBs or locations from the PM are captured during imaging.

Sequence analysis of mutations and translocations across breast cancer subtypes.

Breast carcinoma is the leading cause of cancer-related mortality in women worldwide, with an estimated 1.38 million new cases and 458,000 deaths in 2008 alone. This malignancy represents a heterogeneous group of tumours with characteristic molecular features, prognosis and responses to available therapy. Recurrent somatic alterations in breast cancer have been described, including mutations and copy number alterations, notably ERBB2 amplifications, the first successful therapy target defined by a genomic aberration. Previous DNA sequencing studies of breast cancer genomes have revealed additional candidate mutations and gene rearrangements. Here we report the whole-exome sequences of DNA from 103 human breast cancers of diverse subtypes from patients in Mexico and Vietnam compared to matched-normal DNA, together with whole-genome sequences of 22 breast cancer/normal pairs. Beyond confirming recurrent somatic mutations in PIK3CA, TP53, AKT1, GATA3 and MAP3K1, we discovered recurrent mutations in the CBFB transcription factor gene and deletions of its partner RUNX1. Furthermore, we have identified a recurrent MAGI3-AKT3 fusion enriched in triple-negative breast cancer lacking oestrogen and progesterone receptors and ERBB2 expression. The MAGI3-AKT3 fusion leads to constitutive activation of AKT kinase, which is abolished by treatment with an ATP-competitive AKT small-molecule inhibitor.

2'-Deoxy purine, 2'-O-methyl pyrimidine (dRmY) aptamers as candidate therapeutics.

Aptamers are short oligonucleotides that fold into well-defined three-dimensional architectures thereby enabling specific binding to molecular targets such as proteins. To be successful as a novel therapeutic modality, it is important for aptamers to not only bind their targets with high specificity and affinity, but also to exhibit favorable properties with respect to in vivo stability, cost-effective synthesis, and tolerability (i.e., safety). We describe methods for generating aptamers comprising 2 - deoxy purines and 2 -O-methyl pyrimidines (dRmY) that broadly satisfy many of these additional constraints. Conditions under which dRmY transcripts can be efficiently synthesized using mutant T7 RNA polymerases have been identified and used to generate large libraries from which dRmY aptamers to multiple target proteins, including interleukin (IL)-23 and thrombin, have been successfully discovered using the SELEX process. dRmY aptamers are shown to be highly nuclease-resistant, long-lived in vivo, efficiently synthesized, and capable of binding protein targets in a manner that inhibits their biologic activity with K(D) values in the low nM range. We believe that dRmY aptamers have considerable potential as a new class of therapeutic aptamers.

Co-expression of anti-NFkappaB RNA aptamers and siRNAs leads to maximal suppression of NFkappaB activity in mammalian cells.

The specific down-regulation of gene expression in cells is a powerful method for elucidating a gene's function. A common method for suppressing gene expression is the elimination of mRNA by RNAi or antisense. Alternatively, oligonucleotide-derived aptamers have been used as protein-directed agents for the specific knock-down of both intracellular and extracellular protein activity. Protein-directed methods offer the advantage of more closely mimicking small molecule therapeutics' mechanism of activity. Furthermore, protein-directed methods may synergize with RNA-directed methods since the two methods attack gene expression at different levels. Here we have knocked down a well-characterized intracellular protein's activity, NFkappaB, by expressing either aptamers or small interfering RNAs (siRNAs). Both methods can diminish NFkappaB's activity to similar levels (from 29 to 64%). Interestingly, expression of both aptamers and siRNAs simultaneously, suppressed NFkappaB activity better than either method alone (up to 90%). These results demonstrate that the expression of intracellular aptamers is a viable alternative to siRNA knock-down. Furthermore, for the first time, we show that the use of aptamers and siRNA together can be the most effective way to achieve maximal knock-down of protein activity.