Establishment and application of Agrobacterium-delivered CRISPR/Cas9 system for wild tobacco (Nicotiana alata) genome editing.

Clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (CRISPR-Cas9) system has been widely applied in cultivated crops, but limited in their wild relatives. Nicotiana alata is a typical wild species of genus Nicotiana that is globally distributed as a horticultural plant and well-studied as a self-incompatibility model. It also has valuable genes for disease resistance and ornamental traits. However, it lacks an efficient genetic transformation and genome editing system, which hampers its gene function and breeding research. In this study, we developed an optimized hypocotyl-mediated transformation method for CRISPR-Cas9 delivery. The genetic transformation efficiency was significantly improved from approximately 1% to over 80%. We also applied the CRISPR-Cas9 system to target the phytoene desaturase (NalaPDS) gene in N. alata and obtained edited plants with PDS mutations with over 50% editing efficiency. To generate self-compatible N. alata lines, a polycistronic tRNA-gRNA (PTG) strategy was used to target exonic regions of allelic S-RNase genes and generate targeted knockouts simultaneously. We demonstrated that our system is feasible, stable, and high-efficiency for N. alata genome editing. Our study provides a powerful tool for basic research and genetic improvement of N. alata and an example for other wild tobacco species.

Characterization of the CMS genetic regulation through comparative complete mitochondrial genome sequencing in Nicotiana tabacum.

Mitochondrial genomes (mitogenomes) of flowering plants vary greatly in structure and size, which can lead to frequent gene mutation, rearrangement, or recombination, then result in the cytoplasmic male sterile (CMS) mutants. In tobacco (Nicotiana tabacum), suaCMS lines are widely used in heterosis breeding; however, the related genetic regulations are not very clear. In this study, the cytological observation indicated that the pollen abortion of tobacco suaCMS(HD) occurred at the very early stage of the stamen primordia differentiation. In this study, the complete mitochondrial genomes of suaCMS(HD) and its maintainer HD were sequenced using the PacBio and Illumina Hiseq technology. The total length of the assembled mitogenomes of suaCMS(HD) and HD was 494,317 bp and 430,694 bp, respectively. Comparative analysis indicated that the expanded 64 K bases in suaCMS(HD) were mainly located in noncoding regions, and 23 and 21 big syntenic blocks (>5000 bp) were found in suaCMS(HD) and HD with a series of repeats. Electron transport chain-related genes were highly conserved in two mitogenomes, except five genes (ATP4, ATP6, COX2, CcmFC, and SDH3) with substantial substitutions. Three suaCMS(HD)-specific genes, orf261, orf291, and orf433, were screened. Sequence analysis and RT-PCR verification showed that they were unique to suaCMS(HD). Further gene location analysis and protein property prediction indicated that all the three genes were likely candidates for suaCMS(HD). This study provides new insight into understanding the suaCMS mechanism and is useful for improving tobacco breeding.

NtERF4 promotes the biosynthesis of chlorogenic acid and flavonoids by targeting PAL genes in Nicotiana tabacum.

Chlorogenic acid (CGA) and flavonoids are important secondary metabolites, which modulate plant growth and development, and contribute to plant resistance to various environmental stresses. ERF4 has been shown to be a repressor of anthocyanin accumulation in grape, but its full roles in regulating the biosynthesis of other phenylpropanoid compounds still needs to be further studied. In the present study, two NtERF4 genes were identified from N. tabacum genome. The expression level of NtERF4a was higher than that of NtERF4b in all the tobacco tissues examined. Over-expression of NtERF4a significantly promoted the accumulation of CGA and flavonoids in tobacco leaves, while silencing of NtERF4a significantly repressed the biosynthesis of CGA and flavonoids. RNA-seq analysis of NtERF4a-OE and WT plants revealed 8 phenylpropanoids-related differentially expressed genes (DEGs), including 4 NtPAL genes that encode key enzymes in the phenylpropanoid pathway. Activation of NtERF4a-GR fusion protein in tobacco significantly induced the transcription of NtPAL1 and NtPAL2 in the presence of protein synthesis inhibitor. Chromatin immunoprecipitation and Dual-Luc assays further indicated that NtERF4a could bind to the GCC box presented in the promoters of NtPAL1 and NtPAL2, thereby activating their transcription. Moreover, ectopic expression of NtERF4a induced the transcription of NtGSK1, NtMYC2, and NtJAZ3 genes, and enhanced the resistance of tobacco seedlings to salt and drought stresses, indicating multiple roles of NtERF4a in plants. Our findings revealed new roles of NtERF4a in modulating the accumulation of phenylpropanoid compounds in tobacco, and provided a putative target for improving phenylpropanoids synthesis and stress resistance in plants.

An ultrasmall organic synapse for neuromorphic computing.

High-performance organic neuromorphic devices with miniaturized device size and computing capability are essential elements for developing brain-inspired humanoid intelligence technique. However, due to the structural inhomogeneity of most organic materials, downscaling of such devices to nanoscale and their high-density integration into compact matrices with reliable device performance remain challenging at the moment. Herein, based on the design of a semicrystalline polymer PBFCL10 with ordered structure to regulate dense and uniform formation of conductive nanofilaments, we realize an organic synapse with the smallest device dimension of 50 nm and highest integration size of 1 Kb reported thus far. The as-fabricated PBFCL10 synapses can switch between 32 conductance states linearly with a high cycle-to-cycle uniformity of 98.89% and device-to-device uniformity of 99.71%, which are the best results of organic devices. A mixed-signal neuromorphic hardware system based on the organic neuromatrix and FPGA controller is implemented to execute spiking-plasticity-related algorithm for decision-making tasks.

CRISPR/Cas9-mediated seamless gene replacement in protoplasts expands the resistance spectrum to TMV-U1 strain in regenerated Nicotiana tabacum.

CRISPR/Cas-based genome editing is now extensively used in plant breeding and continues to evolve. Most CRISPR/Cas current applications in plants focus on gene knock-outs; however, there is a pressing need for new methods to achieve more efficient delivery of CRISPR components and gene knock-ins to improve agronomic traits of crop cultivars. We report here a genome editing system that combines the advantages of protoplast technologies with recent CRISPR/Cas advances to achieve seamless large fragment insertions in the model Solanaceae plant Nicotiana tabacum. With this system, two resistance-related regions of the N' gene were replaced with homologous fragments from the N'alata gene to confer TMV-U1 resistance in the T0 generation of GMO-free plants. Our study establishes a reliable genome-editing tool for efficient gene modifications and provides a detailed description of the optimization process to assist other researchers adapt this system for their needs.

CCT3 as a Diagnostic and Prognostic Biomarker in Cervical Cancer.

The chaperonin-containing TCP1 complex subunit 3 (CCT3) has been reported to be involved in the development and prognosis of many tumors, including cervical cancer (CC). This study aimed to analyze the expression and prognostic value of CCT3 in CC by bioinformatics and retrospective study. CCT3 gene expression profiles and clinical information in CC were downloaded from the cancer genome atlas (TCGA) and gene expression omnibus (GEO) databases. CCT3 expression was verified by quantitative real-time polymerase chain reaction (RT-qPCR), Western blot, and immunohistochemistry (IHC). Logistic regression and chi-square testing were used to analyze the relationship between CCT3 expression and the clinical characteristics of CC. Kaplan-Meier and Cox analyses were used to evaluate whether CCT3 affects the prognosis of CC. Nomogram and calibration curves were used to test the predictive value of CCT3. The expression of CCT3 in CC tissues was significantly upregulated compared with that in adjacent benign tissues, and was related to HPV16/18 infection, grade, and positive lymph nodes. High expression of CCT3 is associated with poor prognosis of CC and can be used as an independent risk factor for CC. The prognostic model based on CCT3 and CC clinical features has good predictive ability. CCT3 is overexpressed in CC, which is related to poor prognosis and expected to become a biomarker for CC.

The Mechanism of Dendrite Formation in a Solid-State Transformation of High Aluminum Fe-Al Alloys.

The mechanism of solid-state dendrite formation in high-aluminum Fe-Al alloys is not clear. Applying an in-situ observation technique, the real-time formation and growth of FeAl solid-state dendrites during the eutectoid decomposition of the high-temperature phase Fe5Al8 is visualized. In-situ experiments by HT-CSLM reveal that proeutectoid FeAl usually does not preferentially nucleate at grain boundaries regardless of rapid or slow cooling conditions. The critical radii for generating morphological instability are 1.2 µm and 0.9 µm for slow and rapid cooling, respectively. The morphology after both slow and rapid cooling exhibits dendrites, while there are differences in the size and critical instability radius Rc, which are attributed to the different supersaturation S and the number of protrusions l. The combination of crystallographic and thermodynamic analysis indicates that solid-state dendrites only exist on the hypoeutectoid side in high-aluminum Fe-Al alloys. A large number of lattice defects in the parent phase provides an additional driving force for nucleation, leading to coherent nucleation from the interior of the parent phase grains based on the orientation relationship {3¯30}Fe5Al8//{1¯10}FeAl, <111¯>Fe5Al8//<111¯>FeAl. The maximum release of misfit strain energy leads to the preferential growth of the primary arm of the nucleus along <111¯> {1¯10}. During the rapid cooling process, a large supersaturation is induced in the matrix, driving the Al atoms to undergo unstable uphill diffusion and causing variations in the concentration gradient as well as generating constitutional undercooling, ultimately leading to morphological instability and the growth of secondary arms.

A New Strategy on Designing Fluxes for Aluminum Alloy Melt Refinement.

With the aim of obtaining a refining flux that is stable and provides effective refining of aluminum melt, a new strategy of designing the flux composition has been proposed. Ten fluxes were designed, by selecting ten molten salt compounds according to their thermophysical parameters, physical properties, and thermodynamic analysis. The melting points of the ten fluxes, and the phases transformation of the fluxes after melting, were studied by DSC and XRD, respectively. The contact angles between four groups of fluxes and alumina at refinement temperatures were studied, and the effect of refinement was characterized by a metallographic microscope. The process of the fluxes removing inclusions and degassing was analyzed thermodynamically. The research findings indicate that flux #10 (11.0 wt.%NaF, 29.5 wt.%NaCl, 46.5 wt.%Na2CO3, 3.0 wt.%CaF2, 10.0 wt.%Na3AlF6) has a melting point (562.2 °C) below the refining temperature. At the refining temperature (760 °C), flux #10 has the lowest contact angle, of 12.78°. In addition, compared to that of flux STJ-A3, currently used in practice, flux #10 has a better refining effectiveness, with the pores and inclusions content of the sample being reduced to 1.11% from 2.96%.

A Novel Glycolysis-Related Long Noncoding RNA Signature for Predicting Overall Survival in Gastric Cancer.

Background: The aim of this study was to construct a glycolysis-related long noncoding RNA (lncRNA) signature to predict the prognosis of patients with gastric cancer (GC). Methods: Glycolysis-related genes were obtained from the Molecular Signatures Database (MSigDB), lncRNA expression profiles and clinical data of GC patients were obtained from The Cancer Genome Atlas database (TCGA). Furthermore, univariate Cox regression analysis, Least Absolute Shrinkage and Selection Operator (LASSO) and multivariate Cox regression analysis were used to construct prognostic glycolysis-related lncRNA signature. The specificity and sensitivity of the signature was verified by receiver operating characteristic (ROC) curves. We constructed a nomogram to predict the 1-year, 3-year, and 5-year survival rates of GC patients. Besides, the relationship between immune infiltration and the risk score was analyzed in the high and low risk groups. Multi Experiment Matrix (MEM) was used to analyze glycolysis-related lncRNA target genes. R "limma" package was used to analyze the mRNA expression levels of the glycolysis-related lncRNA target genes in TCGA. Gene set enrichment analysis (GSEA) was employed to further explore the biological pathways in the high-risk group and the glycolysis-related lncRNA target gene. Results: A prognostic signature was conducted based on nine glycolysis-related lncRNAs, which are AL391152.1, AL590705.3, RHOXF1-AS1, CFAP61-AS1, LINC00412, AC005165.1, AC110995.1, AL355574.1 and SCAT1. The area under the ROC curve (AUC) values at 1-year, 3-year, and 5-year were 0.765, 0.828 and 0.707 in the training set, and 0.669, 740 and 0.807 in the testing set, respectively. In addition, the nomogram could efficaciously predict the 1-year, 3-year, and 5-year survival rates of the GC patients. Then, we discovered that GC patients with high-risk scores were more likely to respond to immunotherapy. GSEA revealed that the signature was mainly associated with the calcium signaling pathway, extracellular matrix (ECM) receptor interaction, and focal adhesion in high-risk group, also indicated that SBSPON is related to aminoacyl-tRNA biosynthesis, citrate cycle, fructose and mannose metabolism, pentose phosphate pathway and pyrimidine metabolism. Conclusion: Our study shows that the signature can predict the prognosis of GC and may provide new insights into immunotherapeutic strategies.

Upregulation of FGD6 Predicts Poor Prognosis in Gastric Cancer.

Background: The aim of this study was to investigate the prognostic significance of faciogenital dysplasia 6 (FGD6) in gastric cancer (GC). Methods: The data of GC patients from The Cancer Genome Atlas (TCGA) database were used for the primary study. Then, our data were validated by the GEO database and RuiJin cohort. The relationship between the FGD6 level and various clinicopathological features was analyzed by logistic regression and univariate Cox regression. Multivariate Cox regression analysis was used to evaluate whether FGD6 was an independent prognostic factor for survival of patients with GC. The relationship between FGD6 and overall survival time was explored by the Kaplan-Meier method. In addition, gene set enrichment analysis (GSEA) was performed to investigate the possible biological processes of FGD6. Results: The FGD6 level was significantly overexpressed in GC tissues, compared with adjacent normal tissues. The high expression of FGD6 was related to a high histological grade, stage, and T classification and poor prognosis of GC. Multivariate Cox regression analysis showed that FGD6 was an independent prognostic factor for survival of patients with GC. GSEA identified that the high expression of FGD6 was mainly enriched in regulation of actin cytoskeleton. Conclusion: FGD6 may be a prognostic biomarker for predicting the outcome of patients with GC.

The complete chloroplast genome sequence of Nicotiana debneyi (Solanaceae).

In present study, we report the complete chloroplast genome of Nicotiana debneyi, a species endemic to eastern coast of Australia. The total genome size of N.debneyi is 156,073 bp in length, containing a large single-copy region of 86,672 bp, a small single-copy region of 18,581 bp, and two inverted repeat regions of 25,410 bp. The all GC content of N.debneyi chloroplast genome is 38.4%. It encodes a total of 129 unique genes, including 84 protein-coding genes, 37 tRNA genes, and eight rRNA genes, of which seven tRNA, four rRNA and seven protein-coding genes are duplicated in the IR. Sixteen genes contain a single intron, and only two genes have two introns. Phylogenetic analysis results strongly supported that N.debneyi was closely related to Nicotiana sylvestris and Nicotiana tabacum.

Identification of two additional plasmodesmata localization domains in the tobacco mosaic virus cell-to-cell-movement protein.

Despite decades of intensive studies, the failure to identify plasmodesmata (PD) localization sequences has constrained our understanding of Tobacco mosaic virus (TMV) movement. Recently, we identified the first PD localization signal (major PLS) in the TMV movement protein (MP), which encompasses the first 50 amino acid residues of the MP. Although the major PLS is sufficient for PD targeting, the efficiency is lower than the full-length TMV MP. To address this efficiency gap, we identified two additional PLS domains encompassing amino acid residues 61 to 80, and 147 to 170 of the MP and showed that these two domains target to PD, but do not transit to adjacent cells. We also demonstrated that the MP61-80 fragment interacts with Arabidopsis synaptotagmin A, which was also shown to interact with the major TMV MP PLS. Therefore, our findings have provided new insights to more fully understand the mechanism underlying plasmodesmal targeting of TMV MP.

Construction of a high-density genetic map with whole genome sequencing in Nicotiana tabacum L.

Tobacco (Nicotiana tabacum L.) is an essential commercial crop and an ideal model plant for biological mechanism studies. As an allopolyploid species, tobacco harbors a massive and complex genome, which makes the application of molecular markers complicated and challenging. In our study, we performed whole-genome sequencing of an intraspecific recombinant inbred line (RIL) population, a F1 generation and their parents. With the Nicotiana tabacum (K326 cultivar) genome as reference, a total of 45,081 markers were characterized to construct the genetic map, which spanned a genetic distance of 3486.78 cM. Evaluation of a two-dimensional heat map proved the high quality of the genetic map. We utilized these markers to anchor scaffolds and analyzed the ancestral genome origin of linkage groups (LGs). Furthermore, such a high-density genetic map will be applied for quantitative trait locus (QTL) detection, gene localization, genome-wide association studies (GWAS), and marker-assisted breeding in tobacco.

Non-Structural Protein NSm of Tomato Spotted Wilt Virus Is an Avirulence Factor Recognized by Resistance Genes of Tobacco and Tomato via Different Elicitor Active Sites.

Tomato spotted wilt virus (TSWV) is one of the most destructive viral pathogens of plants. Recently, a single dominant gene conferring complete resistance to TSWV (RTSW) was identified in Nicotina alata and introgressed into cultivated tobacco (N. tabacum). However, whether the TSWV carries an avirulence (Avr) factor directed against RTSW remains obscure. In the present study, we identified the non-structural protein (NSm), the movement protein of TSWV, which is an RTSW-specific Avr factor, by using two different transient expression systems. Using amino acid (aa) substitution mutants, we demonstrated the ability to induce RTSW-mediated hypersensitive response (HR) of NSm is independent of its movement function. Moreover, key substitutions (C118Y and T120N), a 21-aa viral effector epitope, and different truncated versions of NSm, which are responsible for the recognition of the Sw-5b resistance gene of tomato, were tested for their ability to trigger HR to TSWV in tobacco. Together, our results demonstrated that RTSW-mediated resistance is triggered by NSm in the same way as by Sw-5b, however, via different elicitor active sites. Finally, an Avr gene-based diagnostic approach was established and used to determine the presence and effectiveness of resistance genes in tobacco.

Large-scale development of SSR markers in tobacco and construction of a linkage map in flue-cured tobacco.

Tobacco (Nicotiana tabacum L.), particularly flue-cured tobacco, is one of the most economically important nonfood crops and is also an important model system in plant biotechnology. Despite its importance, only limited molecular marker resources are available for genome analysis, genetic mapping, and breeding. Simple sequence repeats (SSR) are one of the most widely-used molecular markers, having significant advantages including that they are generally co-dominant, easy to use, abundant in eukaryotic organisms, and produce highly reproducible results. In this study, based on the genome sequence data of flue-cured tobacco (K326), we developed a total of 13,645 mostly novel SSR markers, which were working in a set of eighteen tobacco varieties of four different types. A mapping population of 213 backcross (BC1) individuals, which were derived from an intra-type cross between two flue-cured tobacco varieties, Y3 and K326, was selected for mapping. Based on the newly developed SSR markers as well as published SSR markers, we constructed a genetic map consisting of 626 SSR loci distributed across 24 linkage groups and covering a total length of 1120.45 cM with an average distance of 1.79 cM between adjacent markers, which is the highest density map of flue-cured tobacco till date.

Nanostructure of aluminum oxide inclusion and formation of hydrogen bubbles in molten aluminum.

Hydrogen in molten aluminum is one of the major factors that lead to pore formation in the solidification process of aluminum castings. Previous research showed that aluminum oxide inclusion had a close correlation with the hydrogen content in molten aluminum. Though some researchers thought there must have been a relationship between surface morphology of the inclusion and hydrogen concentration in molten aluminum, very few documents have reported on the surface property of aluminum oxide inclusion. In the present work, AFM (Atomic Force Microscope) was first used to investigate surface morphology of aluminum oxide inclusion in molten aluminum. It was found that there were a large number of nanoscale micropores on the surface of aluminum oxide inclusion. The interior of the micropores was approximated as a tapered shape. These micropores were thought to be helpful to heterogeneous nucleation for hydrogen atoms aggregation because they provided necessary concentration fluctuation and energy undulation for the growth of hydrogen bubbles. Based on the nanostructure observed on the surface of aluminum oxide inclusion, a theoretical model was developed to describe the hydrogen pore formation in aluminum casting under the condition of heterogeneous nucleation.

Activation of Stat1, IRF-1, and NF-kappaB is required for the induction of uridine phosphorylase by tumor necrosis factor-alpha and interferon-gamma.

Uridine phosphorylase (UPase) has been shown to be induced in various human and murine tumors and could potentially serve as a specific target for the modulation of tumor-selectivity of fluoropyrimidines. However, the signaling mechanisms underlying the regulation of UPase gene expression have not been determined. In this study, we investigated the effects of IFN-gamma on the regulation of TNF-alpha-induced UPase activity and have uncovered the molecular mechanisms of this potentiation, utilizing murine EMT6 breast cancer cells. Our data has shown that IFN-gamma can significantly increase UPase mRNA expression and the enzymatic activity induced by TNF-alpha in a dose-dependent manner, resulting in an enhanced sensitivity to 5-fluorouracil (5-FU) and 5'-Deoxy-5-fluorouridine (5'DFUR). We have previously shown that TNF-alpha activates NF-kappaB through increased translocation of NF-kappaB p65 from the cytoplasm into the nuclei. Exposure to IFN-gamma mainly affects nuclear IRF-1 and STAT1 in EMT6, but inhibits NF-kappaB p65 activity, indicating that the cooperative stimulation was the result of the independent activation of NF-kappaB, STAT1 and IRF-1 transcriptional factors through binding to their unique sites in the UPase promoter. Notably, the activation of NF-kappaB and STAT1 in human breast tissues is consistent with UPase activity; signifying their role in the up-regulation of the UPase gene expression in human tumors.

Enhanced angiogenesis and reduced contraction in thrombospondin-2-null wounds is associated with increased levels of matrix metalloproteinases-2 and -9, and soluble VEGF.

Thrombospondin-2 (TSP2) is an inhibitor of angiogenesis with pro-apoptotic and anti-proliferative effects on endothelial cells. Mice deficient in this matricellular protein display improved recovery from ischemia and accelerated wound healing associated with alterations in angiogenesis and extracellular matrix remodeling. In this study, we probed the function of TSP2 by performing a detailed analysis of dermal wounds and wound-derived fibroblasts. Specifically, we analyzed incisional wounds by tensiometry and found no differences in strength recovery between wild-type and TSP2-null mice. In addition, analysis of full-thickness excisional wounds by terminal deoxynucleotidyl transferase-mediated 2'-deoxyuridine 5'-triphosphate nick-end labeling stain and MIB-5 immunohistochemistry revealed similar numbers of apoptotic and proliferating cells, respectively. In contrast, the levels of matrix metalloproteinase (MMP)-2, MMP-9, tissue inhibitors of metalloproteinase (TIMP)-1, TIMP-2, and soluble vascular endothelial growth factor were increased in wounds of TSP2-null mice. Evaluation of the ability of TSP2-null wound fibroblasts to contract collagen gels revealed that it was compromised, even though TSP2-null wounds displayed normal myofibroblast content. Therefore, we conclude that the lack of TSP2 leads to aberrant extracellular matrix remodeling, increased neovascularization, and reduced contraction due in part to elevated levels of MMP-2 and MMP-9. These observations provide in vivo supporting evidence for a newly proposed function of TSP2 as a modulator of extracellular matrix remodeling.

Modulation of uridine phosphorylase gene expression by tumor necrosis factor-alpha enhances the antiproliferative activity of the capecitabine intermediate 5'-deoxy-5-fluorouridine in breast cancer cells.

Uridine phosphorylase (UPase) has been shown to play an important role in the antineoplastic activity of 5-fluorouracil (5-FU) and in the anabolism of its oral prodrug, capecitabine, through the conversion of 5'-deoxy-5-fluorouridine (5'-DFUR) into 5-FU. In this study, we investigated the effect of tumor necrosis factor-alpha (TNF-alpha) on UPase gene expression and 5'-DFUR antiproliferative activity and elucidated the involved signal transduction pathway. Our data indicate that TNF-alpha significantly induced UPase mRNA expression and its enzymatic activity in EMT6 murine breast cancer cells, leading to an enhanced cytotoxicity of 5'-DFUR. This is further confirmed by an increased incorporation of 5'-DFUR-originated 5-FU nucleotides into nucleic acids. To clarify the mechanism of TNF-alpha-induced UPase expression, we first observed the effect of TNF-alpha on the UPase promoter activity with a series of 5'-deleted promoter-luciferase constructs. Transient transfection analysis showed that the TNF-alpha-inductive pattern in EMT6 cells was consistent with the presence of a nuclear factor-kappaB (NF-kappaB) binding element (-1332/-1312 bp) in the UPase promoter region. Furthermore, electrophoretic mobility shift assays, supershift, and cotransfection assays revealed that the activation of p65 was responsible for UPase induction by TNF-alpha. Finally, the induction of UPase by TNF-alpha could be suppressed by PS-341, a NF-kappaB inhibitor. In summary, TNF-alpha efficiently induces UPase gene expression through a NF-kappaB subunit p65-dependent pathway enhancing cell sensitivity to 5'-DFUR. The elucidation of this regulation mechanism may aid in the clinical use of 5-FU-based chemotherapy.