Adherence and intracellular parasitism of Paracoccidioides brasiliensis in Vero cells

Paracoccidioides brasiliensis is a dimorphic fungus known to produce invasive systemic disease in humans. The 43-kDa glycoprotein of P, brasiliensis is the major diagnostic antigen of paracoccidioidomycosis and may act as a virulence factor, since it is a receptor for laminin. Very little is known about early interact-ions between this fungus and the host cells, so we developed in vitro a model system employing cultured mammalian cells (Vero cells), in order to investigate the factors and virulence mechanisms of P. brasiliensis related to the adhesion and invasion process. We found that there is a permanent interaction after 30 min of contact between the fungus and the cells. The yeasts multiply in the cells for between 5 and 24 h. Different strains of P, brasiliensis were compared, and strain 18 thigh virulence) was the most strongly adherent, followed by strain 113 (virulent), 265 (considered of low virulence) and 113M(mutant obtained by ultraviolet radiation, deficient in gp43). P. brasiliensis adhered to the epithelial cells by a narrow tube, while depressions were noticed in the cell surface, suggesting an active cavitation process. An inhibition assay was performed and it was verified that anti-gp43 serum and a pool of sera from individuals with paracoccidioidomycosis were able to inhibit the adhesion of P. brasiliensis to the Vero cells. Glycoprotein 43 (gp43) antiserum abolished 85 % of the binding activity of P. brasiliensis. This fungus can also invade the Vero cells, and intraepithelial parasitism could be an escape mechanism in paracoccidioidomycosis. (C) 2000 Editions scientifiques et medicales Elsevier SAS.

Biocompatibility of gutta-percha solvents using in vitro mammalian test-system

Objectives. Taking into consideration that DNA damage and cellular death play important roles during carcinogenesis, the purpose of the present study was to evaluate in vitro genotoxic or cytotoxic effects of chloroform and eucalyptol by single cell gel (comet) assay and trypan blue exclusion test, respectively.Study design. Chloroform and eucalyptol were exposed to Chinese hamster ovary cells in culture directly for 3 hours at 37 degrees C at final concentrations ranging from 1.25 to 10 mu L/mL. The negative control group was treated with vehicle control (phosphate-buffered solution), and the positive control group was treated with methyl metasulfonate (MMS, at 1 mu g/mL concentration). All data were analyzed by the Kruskal-Wallis nonparametric test followed by the Dunn test.Results. The results showed that both gutta-percha solvents were cytotoxic at concentrations of 2.5, 5, and 10 mu L/mL (P < .05). on the other hand, both solvents did not induce DNA breakage at 1.25 mu L/mL concentration.Conclusions. These results suggest that both chloroform or eucalyptol are strong cytotoxicants, but they may not be a factor that increases the level of DNA lesions in mammalian cells.

Infection and apparent invasion of Vero cells by Paracoccidioides brasiliensis

Paracoccidioides brasiliensis probably uses many different mechanisms to establish itself in the host and cause disease. In this work, we assess an in vitro model system which uses cultured mammalian cells to investigate the virulence factors of P. brasiliensis. We were able to demonstrate an invasion process of the yeast form of this fungus in Vero cell cultures. We deduced that the overall invasive process involved three steps: adhesion, followed by invasion of individual epithelial cells and spread to adjacent cells.

In vitro cytotoxic and non-genotoxic effects of gutta-percha solvents on mouse lymphoma cells by single cell gel (comet) assay

Chloroform and eucalyptol are widely used in clinical dentistry as gutta-percha solvents. However, these compounds may represent a hazard to human health, especially by causing injury to genetic apparatus and/or inducing cellular death. In this study, the genotoxic and cytotoxic potentials associated with exposure to chloroform and eucalyptol were assessed on mouse lymphoma cells in vitro by the single cell gel (comet) assay and trypan blue exclusion test, respectively. Both gutta-percha solvents proved to be cytotoxic at the same levels in concentrations of 2.5, 5 and 10 μL/mL (p<0.05). On the other hand, neither of the solvents induced DNA breakage. Taken together, these results suggest that although both tested compounds (chloroform and eucalyptol) are strong cytotoxicants, it seems that they are not likely to increase the level of DNA damage on mammalian cells.

Transient expression of rabies virus G-glycoprotein using BHK-21 cells cultured in suspension

Objective To assess the expression of rabies virus G-glycoprotein (RVGP) expression using Semliki Forest virus as a vector in combination with BHK-21 cells cultured in suspension. Results A multilevel factorial design was used to quantify effects of temperature (33–37 C), fresh medium addition after the viral adsorption step (100–200 % with respect to the initial cell suspension volume before infection) and harvest time (8–40 h) on RVGP production. Experimental runs were performed in 24-well cell culture plates at a multiplicity of infection (MOI) of 16. An additional experiment in spinner-flask was performed at MOI of 9, using the optimal conditions determined in cell culture plates. Values for temperature, fresh medium addition and harvest time of 33 C, 100 % and 16 h, respectively, ensured the optimal RVGP production in culture plates. The volumetric yield (239 ng ml-1 ) in these conditions was higher than that reported previously for adherent cell culture. In spinner-flasks, the volumetric yield was improved (559 ng ml-1 ). Conclusion These results establish the basis for designing bioprocess to produce RVGP.

Evaluation of the genotoxicity of waters impacted by domestic and industrial effluents of a highly industrialized region of So Paulo State, Brazil, by the comet assay in HTC cells

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Efficient RNAi-induced Protein Knockdown in Somatic Cells Using Diced or Chemically Produced Small Interfering RNAs (siRNA)

Background: RNA interference (RNAi) is a post-transcriptional gene silencing process in which double-stranded RNA (dsRNA) directs the degradation of a specific corresponding target mRNA. The mediators of this process are small dsRNAs of approximately 21 to 23 bp in length, called small interfering RNAs (siRNAs), which can be prepared in vitro and used to direct the degradation of specific mRNAs inside cells. Hence, siRNAs represent a powerful tool to study and control gene and cell function. Rapid progress has been made in the use of siRNA as a means to attenuate the expression of any protein for which the cDNA sequence is known. Individual siRNAs can be chemically synthesized, in vitro-transcribed, or expressed in cells from siRNA expression vectors. However, screening for the most efficient siRNAs for post-transcriptional gene silencing in cells in culture is a laborious and expensive process. In this study, the effectiveness of two siRNA production strategies for the attenuation of abundant proteins for DNA repair were compared in human cells: (a) the in vitro production of siRNA mixtures by the Dicer enzyme (Diced siRNAs); and (b) the chemical synthesis of very specific and unique siRNA sequences (Stealth RNai (TM)). Materials, Methods & Results: For in vitro-produced siRNAs, two segments of the human Ku70 (167 bp in exon 5; and 249 bp in exon 13; NM001469) and Xrcc4 (172 bp in exon 2; and 108 bp in exon 6; NM003401) genes were chosen to generate dsRNA for subsequent "Dicing" to create mixtures of siRNAs. The Diced fragments of siRNA for each gene sequence were pooled and stored at -80 degrees C. Alternatively, chemically synthesized Stealth siRNAs were designed and generated to match two very specific gene sequence regions for each target gene of interest (Ku70 and Xrcc4). HCT116 cells were plated at 30% confluence in 24- or 6-well culture plates. The next day, cells were transfected by lipofection with either Diced or Stealth siRNAs for Ku70 or Xrcc4, in duplicate, at various doses, with blank and sham transfections used as controls. Cells were harvested at 0, 24, 48, 72 and 96 h post-transfection for protein determination. The knockdown of specific targeted gene products was quantified by Western blot using GAPDH as control. Transfection of gene-specific siRNA to either Ku70 or Xrcc4 with both Diced and Stealth siRNAs resulted in a down regulation of the targeted proteins to approximately 10 to 20% of control levels 48 h after transfection, with recovery to pre-treatment levels by 96 h. Discussion: By transfecting cells with Diced or chemically synthesized Stealth siRNAs, Ku70 and Xrcc4, two highly expressed proteins in cells, were effectively attenuated, demonstrating the great potential for the use of both siRNA production strategies as tools to perform loss of function experiments in mammalian cells. In fact, down-regulation of Ku70 and Xrcc4 has been shown to reduce the activity of the non-homologous end joining DNA pathway, a very desirable approach for the use of homologous recombination technology for gene targeting or knockout studies. Stealth RNAi (TM) was developed to achieve high specificity and greater stability when compared with mixtures of enzymatically-produced (Diced) siRNA fragments. In this study, both siRNA approaches inhibited the expression of Ku70 and Xrcc4 gene products, with no detectable toxic effects to the cells in culture. However, similar knockdown effects using Diced siRNAs were only attained at concentrations 10-fold higher than with Stealth siRNAs. The application of RNAi technology will expand and continue to provide new insights into gene regulation and as potential applications for new therapies, transgenic animal production and basic research.

Cyto and genotoxicity of gold nanoparticles in human hepatocellular carcinoma and peripheral blood mononuclear cells

Engineered nanomaterials have been extensively applied as active materials for technological applications. Since the impact of these nanomaterials on health and environment remains undefined, research on their possible toxic effects has attracted considerable attention. It is known that in humans, for example, the primary site of gold nanoparticles (AuNps) accumulation is the liver. The latter has motivated research regarding the use of AuNps for cancer therapy, since specific organs can be target upon appropriate functionalization of specific nanoparticles. In this study, we investigate the geno and cytotoxicity of two types of AuNps against human hepatocellular carcinoma cells (HepG2) and peripheral blood mononuclear cells (PBMC) from healthy human volunteers. The cells were incubated in the presence of different concentrations of AuNps capped with either sodium citrate or polyamidoamine dendrimers (PAMAM). Our results suggest that both types of AuNps interact with HepG2 cells and PBMC and may exhibit in vitro geno and cytotoxicity even at very low concentrations. In addition, the PBMC were less sensitive to DNA damage toxicity effects than cancer HepG2 cells upon exposure to AuNps. (C) 2012 Elsevier Ireland Ltd. All rights reserved.

Production of human factor VIII-FL in 293T cells using the bicistronic MGMT(P140K)-retroviral vector

Hemophilia A is the most common X-linked bleeding disorder; it is caused by deficiency of coagulation factor VIII (FVIII). Replacement therapy with rFVIII produced from human cell line is a major goal for treating hemophilia patients. We prepared a full-length recombinant FVIII (FVIII-FL), using the pMFG-P140K retroviral vector. The IRES DNA fragment was cloned upstream to the P140K gene, providing a 9.34-kb bicistronic vector. FVIII-FL cDNA was then cloned upstream to IRES, resulting in a 16.6-kb construct. In parallel, an eGFP control vector was generated, resulting in a 10.1-kb construct. The 293T cells were transfected with these constructs, generating the 293T-FVIII-FL/P140K and 293T-eGFP/P140K cell lines. In 293T-FVIII-FL/P140K cells, FVIII and P140K mRNAs levels were 4,410 (+/- 931.7)- and 295,400 (+/- 75,769)-fold higher than in virgin cells. In 293T-eGFP/P140K cells, the eGFP and P140K mRNAs levels were 1,501,000 (+/- 493,700)- and 308,000 (+/- 139,300)-fold higher than in virgin cells. The amount of FVIII-FL was 0.2 IU/mL and 45 ng/mL FVIII cells or 4.4 IU/mu g protein. These data demonstrate the efficacy of the bicistronic retroviral vector expressing FVIII-FL and MGMT(P140K), showing that it could be used for producing the FVIII-FL protein in a human cell line.

RNA Interference and cyclooxygenase-2 (COX-2) regulation in colon cancer cells

Despite new methods and combined strategies, conventional cancer chemotherapy still lacks specificity and induces drug resistance. Gene therapy can offer the potential to obtain the success in the clinical treatment of cancer and this can be achieved by replacing mutated tumour suppressor genes, inhibiting gene transcription, introducing new genes encoding for therapeutic products, or specifically silencing any given target gene. Concerning gene silencing, attention has recently shifted onto the RNA interference (RNAi) phenomenon. Gene silencing mediated by RNAi machinery is based on short RNA molecules, small interfering RNAs (siRNAs) and microRNAs (miRNAs), that are fully o partially homologous to the mRNA of the genes being silenced, respectively. On one hand, synthetic siRNAs appear as an important research tool to understand the function of a gene and the prospect of using siRNAs as potent and specific inhibitors of any target gene provides a new therapeutical approach for many untreatable diseases, particularly cancer. On the other hand, the discovery of the gene regulatory pathways mediated by miRNAs, offered to the research community new important perspectives for the comprehension of the physiological and, above all, the pathological mechanisms underlying the gene regulation. Indeed, changes in miRNAs expression have been identified in several types of neoplasia and it has also been proposed that the overexpression of genes in cancer cells may be due to the disruption of a control network in which relevant miRNA are implicated. For these reasons, I focused my research on a possible link between RNAi and the enzyme cyclooxygenase-2 (COX-2) in the field of colorectal cancer (CRC), since it has been established that the transition adenoma-adenocarcinoma and the progression of CRC depend on aberrant constitutive expression of COX-2 gene. In fact, overexpressed COX-2 is involved in the block of apoptosis, the stimulation of tumor-angiogenesis and promotes cell invasion, tumour growth and metastatization. On the basis of data reported in the literature, the first aim of my research was to develop an innovative and effective tool, based on the RNAi mechanism, able to silence strongly and specifically COX-2 expression in human colorectal cancer cell lines. In this study, I firstly show that an siRNA sequence directed against COX-2 mRNA (siCOX-2), potently downregulated COX-2 gene expression in human umbilical vein endothelial cells (HUVEC) and inhibited PMA-induced angiogenesis in vitro in a specific, non-toxic manner. Moreover, I found that the insertion of a specific cassette carrying anti-COX-2 shRNA sequence (shCOX-2, the precursor of siCOX-2 previously tested) into a viral vector (pSUPER.retro) greatly increased silencing potency in a colon cancer cell line (HT-29) without activating any interferon response. Phenotypically, COX-2 deficient HT-29 cells showed a significant impairment of their in vitro malignant behaviour. Thus, results reported here indicate an easy-to-use, powerful and high selective virus-based method to knockdown COX-2 gene in a stable and long-lasting manner, in colon cancer cells. Furthermore, they open up the possibility of an in vivo application of this anti-COX-2 retroviral vector, as therapeutic agent for human cancers overexpressing COX-2. In order to improve the tumour selectivity, pSUPER.retro vector was modified for the shCOX-2 expression cassette. The aim was to obtain a strong, specific transcription of shCOX-2 followed by COX-2 silencing mediated by siCOX-2 only in cancer cells. For this reason, H1 promoter in basic pSUPER.retro vector [pS(H1)] was substituted with the human Cox-2 promoter [pS(COX2)] and with a promoter containing repeated copies of the TCF binding element (TBE) [pS(TBE)]. These promoters were choosen because they are partculary activated in colon cancer cells. COX-2 was effectively silenced in HT-29 and HCA-7 colon cancer cells by using enhanced pS(COX2) and pS(TBE) vectors. In particular, an higher siCOX-2 production followed by a stronger inhibition of Cox-2 gene were achieved by using pS(TBE) vector, that represents not only the most effective, but also the most specific system to downregulate COX-2 in colon cancer cells. Because of the many limits that a retroviral therapy could have in a possible in vivo treatment of CRC, the next goal was to render the enhanced RNAi-mediate COX-2 silencing more suitable for this kind of application. Xiang and et al. (2006) demonstrated that it is possible to induce RNAi in mammalian cells after infection with engineered E. Coli strains expressing Inv and HlyA genes, which encode for two bacterial factors needed for successful transfer of shRNA in mammalian cells. This system, called “trans-kingdom” RNAi (tkRNAi) could represent an optimal approach for the treatment of colorectal cancer, since E. Coli in normally resident in human intestinal flora and could easily vehicled to the tumor tissue. For this reason, I tested the improved COX-2 silencing mediated by pS(COX2) and pS(TBE) vectors by using tkRNAi system. Results obtained in HT-29 and HCA-7 cell lines were in high agreement with data previously collected after the transfection of pS(COX2) and pS(TBE) vectors in the same cell lines. These findings suggest that tkRNAi system for COX-2 silencing, in particular mediated by pS(TBE) vector, could represent a promising tool for the treatment of colorectal cancer. Flanking the studies addressed to the setting-up of a RNAi-mediated therapeutical strategy, I proposed to get ahead with the comprehension of new molecular basis of human colorectal cancer. In particular, it is known that components of the miRNA/RNAi pathway may be altered during the progressive development of colorectal cancer (CRC), and it has been already demonstrated that some miRNAs work as tumor suppressors or oncomiRs in colon cancer. Thus, my hypothesis was that overexpressed COX-2 protein in colon cancer could be the result of decreased levels of one or more tumor suppressor miRNAs. In this thesis, I clearly show an inverse correlation between COX-2 expression and the human miR- 101(1) levels in colon cancer cell lines, tissues and metastases. I also demonstrate that the in vitro modulating of miR-101(1) expression in colon cancer cell lines leads to significant variations in COX-2 expression, and this phenomenon is based on a direct interaction between miR-101(1) and COX-2 mRNA. Moreover, I started to investigate miR-101(1) regulation in the hypoxic environment since adaptation to hypoxia is critical for tumor cell growth and survival and it is known that COX-2 can be induced directly by hypoxia-inducible factor 1 (HIF-1). Surprisingly, I observed that COX-2 overexpression induced by hypoxia is always coupled to a significant decrease of miR-101(1) levels in colon cancer cell lines, suggesting that miR-101(1) regulation could be involved in the adaption of cancer cells to the hypoxic environment that strongly characterize CRC tissues.

Rapid fusion and syncytium formation of heterologous cells upon expression of the FGFRL1 receptor

The fusion of mammalian cells into syncytia is a developmental process that is tightly restricted to a limited subset of cells. Besides gamete and placental trophoblast fusion, only macrophages and myogenic stem cells fuse into multinucleated syncytia. In contrast to viral cell fusion, which is mediated by fusogenic glycoproteins that actively merge membranes, mammalian cell fusion is poorly understood at the molecular level. A variety of mammalian transmembrane proteins, among them many of the immunoglobulin superfamily, have been implicated in cell-cell fusion, but none has been shown to actively fuse cells in vitro. Here we report that the FGFRL1 receptor, which is up-regulated during the differentiation of myoblasts into myotubes, fuses cultured cells into large, multinucleated syncytia. We used luciferase and GFP-based reporter assays to confirm cytoplasmic mixing and to identify the fusion inducing domain of FGFRL1. These assays revealed that Ig-like domain III and the transmembrane domain are both necessary and sufficient to rapidly fuse CHO cells into multinucleated syncytia comprising several hundred nuclei. Moreover, FGFRL1 also fused HEK293 and HeLa cells with untransfected CHO cells. Our data show that FGFRL1 is the first mammalian protein that is capable of inducing syncytium formation of heterologous cells in vitro.

Exocyst subunits are involved in isoproterenol-induced amylase release from rat parotid acinar cells

Exocytosis of secretory granules in parotid acinar cells requires multiple events: tethering, docking, priming, and fusion with a luminal plasma membrane. The exocyst complex, which is composed of eight subunits (Sec3, Sec5, Sec6, Sec8, Sec10, Sec15, Exo70, and Exo84) that are conserved in yeast and mammalian cells, is thought to participate in the exocytotic pathway. However, to date, no exocyst subunit has been identified in salivary glands. In the present study, we investigated the expression and function of exocyst subunits in rat parotid acinar cells. The expression of mRNA for all eight exocyst subunits was detected in parotid acinar cells by RT-PCR, and Sec6 and Sec8 proteins were localized on the luminal plasma membrane. Sec6 interacted with Sec8 after 5 min of stimulation with isoproterenol. In addition, antibodies to-Sec6 and Sec8 inhibited isoproterenol-induced amylase release from streptolysin O-permeabilized parotid acinar cells. These results suggest that an exocyst complex of eight subunits is required for amylase release from parotid acinar cells.

Radioprotection of cultured cells by preincubation in medium containing deuterium oxide

Pretreatment with deuterium oxide (D2O) has been shown to protect mice against lethal effects of X-rays. In contrast, X-irradiation of cultured mammalian cells in D2O-containing medium has previously been reported to result in increased cell killing. Therefore, the effects of preincubation in medium containing 20% D2O on radiosensitivity were tested, using cells of a heat-sensitive cell-cycle mutant (21-Tb) of the murine mastocytoma P 815-X2. The mutant cells proliferate at 33 degrees C and are arrested in G1 phase in a state of reversible proliferative quiescence at 39.5 degrees C. Prior to irradiation with single X-ray doses of 0-10 Gy, the cells were cultured in normal or D2O-containing medium, either for 96 h at 33 degrees C ('proliferating cells'), or for 72 h at 33 degrees C followed by 24 h at 39.5 degrees C ('arrested cells'). After X-irradiation the cells were resuspended in normal medium, and cell survival was determined by the capacity of cells to form colonies in fibrin gels. Preincubation in medium containing 20% D2O resulted in a radioprotective effect on both proliferating and arrested cells, particularly at the higher X-ray doses. This radioprotection was manifested as a decreased slope of the semilogarithmic survival curves, whereas pretreatment with D2O had no significant effect on postirradiation repair as judged from Dq values. These results support the interpretation that the increase in postirradiation survival may be attributed to incorporation of deuterium into cellular metabolites during the period of preincubation.

Mechanistic aspects of NMD in human cells

Eukaryotic mRNAs with premature translation termination codons (PTCs) are recognized and degraded through a process termed nonsense-mediated mRNA decay (NMD). To get more insight into the recruitment of the central NMD factor UPF1 to target mRNAs, we mapped transcriptome-wide UPF1-binding sites by individual-nucleotide-resolution UV cross-linking and immunoprecipitation (iCLIP) in human cells and found that UPF1 preferentially associated with 3′ UTRs in translationally active cells but underwent significant redistribution toward coding regions (CDS) upon translation inhibition. This indicates that UPF1 binds RNA before translation and gets displaced from the CDS by translating ribosomes. Corroborated by RNA immunoprecipitation and by UPF1 cross-linking to long noncoding RNAs, our evidence for translation-independent UPF1-RNA interaction suggests that the triggering of NMD occurs after UPF1 binding to mRNA, presumably through activation of RNA-bound UPF1 by aberrant translation termination. Unlike in yeast, in mammalian cells NMD has been reported to be restricted to cap-binding complex (CBC)–bound mRNAs during the pioneer round of translation. However, we compared decay kinetics of two NMD reporter genes in mRNA fractions bound to either CBC or the eukaryotic initiation factor 4E (eIF4E) in human cells and show that NMD destabilizes eIF4E-bound transcripts as efficiently as those associated with CBC. These results corroborate an emerging unified model for NMD substrate recognition, according to which NMD can ensue at every aberrant translation termination event.

Post-transcriptional Regulation of Mammalian Gene Expression in Non-coding Region of Target RNA

Tumor Suppressor Candidate 2 (TUSC2) is a novel tumor suppressor gene located in the human chromosome 3p21.3 region. TUSC2 mRNA transcripts could be detected on Northern blots in both normal lung and some lung cancer cell lines, but no endogenous TUSC2 protein could be detected in a majority of lung cancer cell lines. Mechanisms regulating TUSC2 protein expression and its inactivation in primary lung cancer cells are largely unknown. We investigated the role of the 5’- and 3’-untranslated regions (UTRs) of the TUSC2 gene in the regulation of TUSC2 protein expression. We found that two small upstream open-reading frames (uORFs) in the 5’UTR of TUSC2 could markedly inhibit the translational initiation of TUSC2 protein by interfering with the “scanning” of the ribosome initiation complexes. Site-specific stem-loop array reverse transcription-polymerase chain reaction (SLA-RT-PCR) verified several micoRNAs (miRNAs) targeted at 3’UTR and directed TUSC2 cleavage and degradation. In addition, we used the established let-7-targeted high mobility group A2 (Hmga2) mRNA as a model system to study the mechanism of regulation of target mRNA by miRNAs in mammalian cells under physiological conditions. There have been no evidence of direct link between mRNA downregulation and mRNA cleavages mediated by miRNAs. Here we showed that the endonucleolytic cleavages on mRNAs were initiated by mammalian miRNA in seed pairing style. Let-7 directed cleavage activities among the eight predicted potential target sites have varied efficiency, which are influenced by the positional and the structural contexts in the UTR. The 5’ cleaved RNA fragments were mostly oligouridylated at their 3’-termini and accumulated for delayed 5’–3’ degradation. RNA fragment oligouridylation played important roles in marking RNA fragments for delayed bulk degradation and in converting RNA degradation mode from 3’–5’ to 5’–3’ with cooperative efforts from both endonucleolytic and non-catalytic miRNA-induced silencing complex (miRISC). Our findings point to a mammalian miRNA-mediated mechanism for the regulation of mRNA that miRNA can decrease target mRNA through target mRNA cleavage and uridine addition