Inosine triphosphate pyrophosphatase from Trypanosoma brucei cleanses cytosolic pools from deaminated nucleotides.

Inosine triphosphate pyrophosphatases (ITPases) are ubiquitous house-cleaning enzymes that specifically recognize deaminated purine nucleotides and catalyze their hydrolytic cleavage. In this work, we have characterized the Trypanosoma brucei ITPase ortholog (TbITPA). Recombinant TbITPA efficiently hydrolyzes (deoxy)ITP and XTP nucleotides into their respective monophosphate form. Immunolocalization analysis performed in bloodstream forms suggests that the primary role of TbITPA is the exclusion of deaminated purines from the cytosolic nucleoside triphosphate pools. Even though ITPA-knockout bloodstream parasites are viable, they are more sensitive to inhibition of IMP dehydrogenase with mycophenolic acid, likely due to an expansion of IMP, the ITP precursor. On the other hand, TbITPA can also hydrolyze the activated form of the antiviral ribavirin although in this case, the absence of ITPase activity in the cell confers protection against this nucleoside analog. This unexpected phenotype is dependant on purine availability and can be explained by the fact that ribavirin monophosphate, the reaction product generated by TbITPA, is a potent inhibitor of trypanosomal IMP dehydrogenase and GMP reductase. In summary, the present study constitutes the first report on a protozoan inosine triphosphate pyrophosphatase involved in the removal of harmful deaminated nucleotides from the cytosolic pool.

Efectividad de las células madre mesenquimales en las elevaciones de seno maxilar. Revisión de la literatura / Effectiveness of mesenchymal stem cells in maxillary sinus lift. A literature review

Actualmente, la implantología está considerada como la terapia de elección para rehabilitar dientes ausentes. Sin embargo, debido a diversos factores, no siempre se consigue un enfoque inmediato para la colocación de los implantes. Se debe prestar una atención especial a los sectores maxilares posteriores, que debido al paso del tiempo y a la ausencia de dientes, sufren una gran atrofia alveolar que se encuentra asociada a una expansión del seno maxilar que dificulta, en ocasiones, la rehabilitación con implantes osteointegrados. Debido a esta pérdida ósea, se han desarrollado técnicas quirúrgicas como la elevación de seno maxilar para obtener una ganancia de hueso y poder favorecer la colocación y el pronóstico de los implantes. Sin embargo, existen situaciones en las que incluso esta técnica no aporta un volumen óseo suficiente, siendo preciso acompañarla de técnicas de regeneración ósea.En la actualidad, existen estudios que plantean el uso de células madre mesenquimales en procedimientos de regeneración ósea como alternativa a los procedimientos convencionales.El objetivo del presente trabajo es evaluar a través de la realización de una revisión sistemática de la literatura, la efectividad de las células madres mesenquimales en elevaciones de seno maxilar con diferentes tipos de matriz ósea. (AU)

Currently, implantology is considered the therapy of choice to rehabilitate missing teeth. However, due to various factors, we do not always get an immediate approach to the placement of our implants. Above all, we must pay special attention to the posterior maxillary sectors, which due to the passage of time and the absence of dental pieces suffer great alveolar atrophy associated with an expansion of the maxillary sinus that makes difficult and sometimes impossible the implant therapy. Due to this great bone loss, the professionals have been forced to practice surgical techniques such as maxillary sinus lift to gain bone and to be able to favour the placement and prognosis of the implants. However, there are clinical situations in which even this technique does not give us sufficient bone volume, being necessary a bone regeneration therapy.Thus, thanks to advances in the field of scientific research, various methods have been used to solve this type of problem, but one of the most innovative and current options is bone regeneration using mesenchymal stem cells.This work aims to evaluate, carrying out a systematic review of the literature, the effectiveness of mesenchymal stem cells in maxillary sinus elevations with several types of matrix. (AU)

Adquisición de conocimientos en cuidados paliativos mediante un plan de estudios transversal en enfermería / Acquisition of palliative care knowledge through a transversal approach in nursing studies

La implementación de los cuidados paliativos en las universidades ha sido paulatina. Su reciente implantación requiere de su evaluación para la mejora en la formación de nuestros futuros profesionales. Por tanto, el objetivo de este trabajo es valorar el conocimiento en paliativos, en estudiantes de enfermería de la Universidad Jaume I, tras la implementación de un plan de estudios de enseñanza transversal y evaluación mediante una asignatura de carácter obligatorio. MÉTODO: Estudio observacional, de corte transversal, durante el curso académico 2017/2018 mediante la escala Palliative Care Quiz for Nursing. Análisis descriptivo, comparativo de las variables cuantitativas y cualitativas global y según aciertos/errores, significación estadística p-value <0,05. RESULTADOS: La muestra de n=155, edad 22,21±6,61 años, con predominio del sexo femenino en el 80% (n=124). La esfera con mayor número de aciertos fue la relacionada con el control de dolor y los síntomas 69,1% y la esfera con más fallos la relacionada con la filosofía y los principios 46,9%. El conocimiento en paliativos viene explicado en un 16% por el curso académico y con la experiencia previa en paliativos. CONCLUSIONES: Mediante nuestros resultados observamos, que la educación en paliativos desarrollada de forma transversal con la culminación de una asignatura obligatoria en cuarto curso, resulta ser efectiva en estudiantes de enfermería, donde el 63% ostentó una buena tasa de aciertos en conocimientos de paliativos

The inclusion of palliative care content in university curricula has been gradual. Due to the recent incorporation of palliative care as a subject, an evaluation is necessary in order improve the education of our future health professionals. The objective of this study was thus to assess palliative care knowledge in nursing students at Jaume I University after the transversal inclusion of palliative care content throughout the program, culminating in a subject of an obligatory nature. METHOD: This observational cross-sectional study was undertaken over the 2017/2018 academic year using the Palliative Care Quiz for Nursing. A descriptive and comparative analysis of the quantitative and qualitative global variables was performed with respect to correct/incorrect responses. Statistical significance was set at p <0.05. RESULTS: The sample included 155 participants, with a mean age of 22.21±6.61 years. Females (n=124) were predominant in the sample (80%). The domain which received the highest number of correct responses was related to the control of pain and symptoms (69.1%) and the domain which received the highest number of incorrect responses was related to philosophy and principles (46.9%). Academic year and prior palliative care experience explained 16% of the variance in knowledge. CONCLUSIONS: Through our results, we observed that palliative care education, implemented in a transversal manner and with the completion of compulsory subject in the fourth year, was effective in nursing students, with 63% achieving good scores with respect to palliative care knowledge

DCTPP1 prevents a mutator phenotype through the modulation of dCTP, dTTP and dUTP pools.

To maintain dNTP pool homeostasis and preserve genetic integrity of nuclear and mitochondrial genomes, the synthesis and degradation of DNA precursors must be precisely regulated. Human all-alpha dCTP pyrophosphatase 1 (DCTPP1) is a dNTP pyrophosphatase with high affinity for dCTP and 5'-modified dCTP derivatives, but its contribution to overall nucleotide metabolism is controversial. Here, we identify a central role for DCTPP1 in the homeostasis of dCTP, dTTP and dUTP. Nucleotide pools and the dUTP/dTTP ratio are severely altered in DCTPP1-deficient cells, which exhibit an accumulation of uracil in genomic DNA, the activation of the DNA damage response and both a mitochondrial and nuclear hypermutator phenotype. Notably, DNA damage can be reverted by incubation with thymidine, dUTPase overexpression or uracil-DNA glycosylase suppression. Moreover, DCTPP1-deficient cells are highly sensitive to down-regulation of nucleoside salvage. Our data indicate that DCTPP1 is crucially involved in the provision of dCMP for thymidylate biosynthesis, introducing a new player in the regulation of pyrimidine dNTP levels and the maintenance of genomic integrity.

Overview of the role of kinetoplastid surface carbohydrates in infection and host cell invasion: prospects for therapeutic intervention.

Kinetoplastid parasites are responsible for serious diseases in humans and livestock such as Chagas disease and sleeping sickness (caused by Trypanosoma cruzi and Trypanosoma brucei, respectively), and the different forms of cutaneous, mucocutaneous and visceral leishmaniasis (produced by Leishmania spp). The limited number of antiparasitic drugs available together with the emergence of resistance underscores the need for new therapeutic agents with novel mechanisms of action. The use of agents binding to surface glycans has been recently suggested as a new approach to antitrypanosomal design and a series of peptidic and non-peptidic carbohydrate-binding agents have been identified as antiparasitics showing efficacy in animal models of sleeping sickness. Here we provide an overview of the nature of surface glycans in three kinetoplastid parasites, T. cruzi, T. brucei and Leishmania. Their role in virulence and host cell invasion is highlighted with the aim of identifying specific glycan-lectin interactions and carbohydrate functions that may be the target of novel carbohydrate-binding agents with therapeutic applications.

Base excision repair plays an important role in the protection against nitric oxide- and in vivo-induced DNA damage in Trypanosoma brucei.

Uracil-DNA glycosylase (UNG) initiates the base excision repair pathway by excising uracil from DNA. We have previously shown that Trypanosoma brucei cells defective in UNG exhibit reduced infectivity thus demonstrating the relevance of this glycosylase for survival within the mammalian host. In the early steps of the immune response, nitric oxide (NO) is released by phagocytes, which in combination with oxygen radicals produce reactive nitrogen species (RNS). These species can react with DNA generating strand breaks and base modifications including deaminations. Since deaminated cytosines are the main substrate for UNG, we hypothesized that the glycosylase might confer protection towards nitrosative stress. Our work establishes the occurrence of genotoxic damage in Trypanosoma brucei upon exposure to NO in vitro and shows that deficient base excision repair results in increased levels of damage in DNA and a hypermutator phenotype. We also evaluate the incidence of DNA damage during infection in vivo and show that parasites recovered from mice exhibit higher levels of DNA strand breaks, base deamination and repair foci compared to cells cultured in vitro. Notably, the absence of UNG leads to reduced infectivity and enhanced DNA damage also in animal infections. By analysing mRNA and protein levels, we found that surviving UNG-KO trypanosomes highly express tryparedoxin peroxidase involved in trypanothione/tryparedoxin metabolism. These observations suggest that the immune response developed by the host enhances the activation of genes required to counteract oxidative stress and emphasize the importance of DNA repair pathways in the protection to genotoxic and oxidative stress in trypanosomes.

Targeting Kinetoplastid and Apicomplexan Thymidylate Biosynthesis as an Antiprotozoal Strategy.

Kinetoplastid and apicomplexan parasites comprise a group of protozoans responsible for human diseases, with a serious impact on human health and the socioeconomic growth of developing countries. Chemotherapy is the main option to control these pathogenic organisms and nucleotide metabolism is considered a promising area for the provision of antimicrobial therapeutic targets. Impairment of thymidylate (dTMP) biosynthesis severely diminishes the viability of parasitic protozoa and the absence of enzymatic activities specifically involved in the formation of dTMP (e.g. dUTPase, thymidylate synthase, dihydrofolate reductase or thymidine kinase) results in decreased deoxythymidine triphosphate (dTTP) levels and the so-called thymineless death. In this process, the ratio of deoxyuridine triphosphate (dUTP) versus dTTP in the cellular nucleotide pool has a crucial role. A high dUTP/dTTP ratio leads to uracil misincorporation into DNA, the activation of DNA repair pathways, DNA fragmentation and eventually cell death. The essential character of dTMP synthesis has stimulated interest in the identification and development of drugs that specifically block the biochemical steps involved in thymine nucleotide formation. Here, we review the available literature in relation to drug discovery studies targeting thymidylate biosynthesis in kinetoplastid (genera Trypanosoma and Leishmania) and apicomplexan (Plasmodium spp and Toxoplasma gondii) protozoans. The most relevant findings concerning novel inhibitory molecules with antiparasitic activity against these human pathogens are presented herein.

Characterization of Mycosphaerellaceae species associated with citrus greasy spot in Panama and Spain.

Greasy spot of citrus, caused by Zasmidium citri-griseum (= Mycosphaerella citri), is widely distributed in the Caribbean Basin, inducing leaf spots, premature defoliation, and yield loss. Greasy spot-like symptoms were frequently observed in humid citrus-growing regions in Panama as well as in semi-arid areas in Spain, but disease aetiology was unknown. Citrus-growing areas in Panama and Spain were surveyed and isolates of Mycosphaerellaceae were obtained from citrus greasy spot lesions. A selection of isolates from Panama (n = 22) and Spain (n = 16) was assembled based on their geographical origin, citrus species, and affected tissue. The isolates were characterized based on multi-locus DNA (ITS and EF-1α) sequence analyses, morphology, growth at different temperatures, and independent pathogenicity tests on the citrus species most affected in each country. Reference isolates and sequences were also included in the analysis. Isolates from Panama were identified as Z. citri-griseum complex, and others from Spain attributed to Amycosphaerella africana. Isolates of the Z. citri-griseum complex had a significantly higher optimal growth temperature (26.8°C) than those of A. africana (19.3°C), which corresponded well with their actual biogeographical range. The isolates of the Z. citri-griseum complex from Panama induced typical greasy spot symptoms in 'Valencia' sweet orange plants and the inoculated fungi were reisolated. No symptoms were observed in plants of the 'Ortanique' tangor inoculated with A. africana. These results demonstrate the presence of citrus greasy spot, caused by Z. citri-griseum complex, in Panama whereas A. africana was associated with greasy spot-like symptoms in Spain.

Insights into the role of endonuclease V in RNA metabolism in Trypanosoma brucei.

Inosine may arise in DNA as a result of oxidative deamination of adenine or misincorporation of deoxyinosine triphosphate during replication. On the other hand, the occurrence of inosine in RNA is considered a normal and essential modification induced by specific adenosine deaminases acting on mRNA and tRNA. In prokaryotes, endonuclease V (EndoV) can recognize and cleave inosine-containing DNA. In contrast, mammalian EndoVs preferentially cleave inosine-containing RNA, suggesting a role in RNA metabolism for the eukaryotic members of this protein family. We have performed a biochemical characterization of EndoV from the protozoan parasite Trypanosoma brucei. In vitro, TbEndoV efficiently processes single-stranded RNA oligonucleotides with inosine, including A to I-edited tRNA-like substrates but exhibits weak activity over DNA, except when a ribonucleotide is placed 3' to the inosine. Immunolocalization studies performed in procyclic forms indicate that TbEndoV is mainly cytosolic yet upon nutritional stress it redistributes and accumulates in stress granules colocalizing with the DEAD-box helicase TbDhh1. RNAi-mediated depletion of TbEndoV results in moderate growth defects in procyclic cells while the two EndoV alleles could be readily knocked out in bloodstream forms. Taken together, these observations suggest an important role of TbEndoV in RNA metabolism in procyclic forms of the parasite.

The nucleotidohydrolases DCTPP1 and dUTPase are involved in the cellular response to decitabine.

Decitabine (5-aza-2'-deoxycytidine, aza-dCyd) is an anti-cancer drug used clinically for the treatment of myelodysplastic syndromes and acute myeloid leukaemia that can act as a DNA-demethylating or genotoxic agent in a dose-dependent manner. On the other hand, DCTPP1 (dCTP pyrophosphatase 1) and dUTPase are two 'house-cleaning' nucleotidohydrolases involved in the elimination of non-canonical nucleotides. In the present study, we show that exposure of HeLa cells to decitabine up-regulates the expression of several pyrimidine metabolic enzymes including DCTPP1, dUTPase, dCMP deaminase and thymidylate synthase, thus suggesting their contribution to the cellular response to this anti-cancer nucleoside. We present several lines of evidence supporting that, in addition to the formation of aza-dCTP (5-aza-2'-deoxycytidine-5'-triphosphate), an alternative cytotoxic mechanism for decitabine may involve the formation of aza-dUMP, a potential thymidylate synthase inhibitor. Indeed, dUTPase or DCTPP1 down-regulation enhanced the cytotoxic effect of decitabine producing an accumulation of nucleoside triphosphates containing uracil as well as uracil misincorporation and double-strand breaks in genomic DNA. Moreover, DCTPP1 hydrolyses the triphosphate form of decitabine with similar kinetic efficiency to its natural substrate dCTP and prevents decitabine-induced global DNA demethylation. The data suggest that the nucleotidohydrolases DCTPP1 and dUTPase are factors involved in the mode of action of decitabine with potential value as enzymatic targets to improve decitabine-based chemotherapy.

Adrenohepatic fusion: Adhesion or invasion in primary virilizant giant adrenal carcinoma? Implications for surgical resection. Two case report and review of the literature.

INTRODUCTION: Adrenohepatic fusion means union between the adrenal gland and the liver, intermingling its parenchymas. It is not possible to identify this condition by image tests. Its presence implies radical and multidisciplinar approach. PRESENTATION OF CASES: We report two female cases of 45 and 50 years old with clinical virilization and palpable mass on the abdominal right upper quadrant corresponding to adrenocortical carcinoma with hepatic fusion. The contrast-enhanced tomography showed an indistinguishable mass involving the liver and the right adrenal gland. In the first case, the patient had a two-time operation, the former removing only the adrenal carcinoma, and the second performing a radical surgery after an early relapse. In the second case, a radical right en bloc adrenohepatectomy was performed. Both cases were pathologically reported as liver-infiltrating adrenal carcinoma. Only in the second case the surgery was radical effective as first intention to treat, with 3 years of disease-free survival. DISCUSSION: ACC is a rare entity with poor prognosis. The major indicators of malignancy are tumour diameter over 6cm, local invasion or metastasis, secretion of corticosteroids, virilization and hypertension and hypokalaemia. The parenchymal fusion of the adrenal cortical layer can be misdiagnosed as hepatocellular carcinoma with adhesion with the Glisson capsule. AHF in such cases may be misinterpreted during surgery, what may impair its resectability, and therefore the survival. The surgical treatment must be performed en bloc, often using liver vascular control. Postoperative treatment must be offered immediately after surgery. CONCLUSION: We report two consecutive rare cases of adrenohepatic fusion in giant right adrenocortical carcinoma, not detectable by imaging, what has important implications for the surgical decision-making. As radical surgery is the best choice to offer a curative treatment, it has to be performed by a multidisciplinary well-assembled team, counting with endocrine and liver surgeons, and transplant surgeons in case of vena cava involvement, in order to maximize the disease-free survival.

Post-bariatric surgery body contouring treatment in the public health system: cost study and perception by patients.

BACKGROUND: Post-bariatric, body contouring surgery to treat the sequelae of massive weight loss is an undervalued topic by patients and in most of the literature. The objective of this study was to determine the mean cost per patient of this treatment in a public morbid obesity unit, and compare it with the perception by the patients. METHODS: Costs were estimated using a specific Diagnosis-Related Group-based method and a questionnaire in a sample of 100 patients who had completed body contouring treatment. RESULTS: This study included 23 men and 77 women with a mean age of 48.5 years, a mean reduction of body mass index of 20.77 kg/m, and a median follow-up of 58 months. These patients had undergone surgery, as needed, as follows: on the lower part of the trunk (109 operations; mean cost, &OV0556;6348.6), cruroplasty (43 operations; mean cost, &OV0556;3490), brachioplasty (28 operations; mean cost &OV0556;3150), and the upper part of the trunk (10 operations; mean cost, &OV0556;4290). The rate of complications has been high (up to 50 percent) and, although the more severe complications are rare (10.5 percent Clavien grade IIIb), these represent high costs (mean, &OV0556;24462.6). Forty-five patients answered the questionnaire. Although they think that this surgery improves their quality of life, they have undervalued its total cost (17.58 percent; &OV0556;2034) (p = 0.16). CONCLUSIONS: The average cost of post-bariatric surgery body contouring treatment in this unit is &OV0556;8263.95 (1.66 operations per patient). The severe complications increase by 2.96 times the average cost per patient.

The NTP pyrophosphatase DCTPP1 contributes to the homoeostasis and cleansing of the dNTP pool in human cells.

The size and composition of dNTP (deoxyribonucleoside triphosphate) pools influence the accuracy of DNA synthesis and consequently the genetic stability of nuclear and mitochondrial genomes. In order to keep the dNTP pool in balance, the synthesis and degradation of DNA precursors must be precisely regulated. One such mechanism involves catabolic activities that convert deoxynucleoside triphosphates into their monophosphate form. Human cells possess an all-α NTP (nucleoside triphosphate) pyrophosphatase named DCTPP1 [dCTP pyrophosphatase 1; also known as XTP3-TPA (XTP3-transactivated protein A)]. In the present study, we provide an extensive characterization of this enzyme which is ubiquitously distributed in the nucleus, cytosol and mitochondria. Interestingly, we found that in addition to dCTP, methyl-dCTP and 5-halogenated nucleotides, DCTPP1 hydrolyses 5-formyl-dCTP very efficiently and with the lowest Km value described so far. Because the biological function of mammalian all-α NTP pyrophosphatases remains uncertain, we examined the role of DCTPP1 in the maintenance of pyrimidine nucleotide pools and cellular sensitivity to pyrimidine analogues. DCTPP1-deficient cells accumulate high levels of dCTP and are hypersensitive to exposure to the nucleoside analogues 5-iodo-2'-deoxycytidine and 5-methyl-2'-deoxycytidine. The results of the present study indicate that DCTPP1 has a central role in the balance of dCTP and the metabolism of deoxycytidine analogues, thus contributing to the preservation of genome integrity.

La Disfasia desde el enfoque de la Comunicación / Dysphasia from the communication perspective

La disfasia es un término que conceptualiza una entidad gnoseológica relacionada con la organización del lenguaje en su evolución. Es un trastorno grave y de prolongada duración que afecta a niños desde el inicio del desarrollo del lenguaje, se extiende a toda la infancia y la adolescencia y puede dejar secuelas en el estado adulto. En esta revisión bibliográfica se propone un acercamiento a la comprensión de sus dimensiones, concepto, síntomas y pautas para el diagnóstico e intervención desde el enfoque de la Comunicación (AU)

Dysphasia is a term that conceptualizes a gnoseologic entity related to the organization of language in its evolution. It is a severe and prolonged duration disorder that affects children since the beginning of the language development and extends to all children and adolescents; it can also leave consequences in the adult stage. This review proposes an approach to the understanding of its dimensions, concept, symptoms and guidelines for the diagnosis and involvement from a communication perspective (AU)

Carbohydrate-binding agents act as potent trypanocidals that elicit modifications in VSG glycosylation and reduced virulence in Trypanosoma brucei.

The surface of Trypanosoma brucei is covered by a dense coat of glycosylphosphatidylinositol-anchored glycoproteins. The major component is the variant surface glycoprotein (VSG) which is glycosylated by both paucimannose and oligomannose N-glycans. Surface glycans are poorly accessible and killing mediated by peptide lectin-VSG complexes is hindered by active endocytosis. However, contrary to previous observations, here we show that high-affinity carbohydrate binding agents bind to surface glycoproteins and abrogate growth of T. brucei bloodstream forms. Specifically, binding of the mannose-specific Hippeastrum hybrid agglutinin (HHA) resulted in profound perturbations in endocytosis and parasite lysis. Prolonged exposure to HHA led to the loss of triantennary oligomannose structures in surface glycoproteins as a result of genetic rearrangements that abolished expression of the oligosaccharyltransferase TbSTT3B gene and yielded novel chimeric enzymes. Mutant parasites exhibited markedly reduced infectivity thus demonstrating the importance of specific glycosylation patterns in parasite virulence.

La Disfasia desde el enfoque de la Comunicación / Dysphasia from the communication perspective

La disfasia es un término que conceptualiza una entidad gnoseológica relacionada con la organización del lenguaje en su evolución. Es un trastorno grave y de prolongada duración que afecta a niños desde el inicio del desarrollo del lenguaje, se extiende a toda la infancia y la adolescencia y puede dejar secuelas en el estado adulto. En esta revisión bibliográfica se propone un acercamiento a la comprensión de sus dimensiones, concepto, síntomas y pautas para el diagnóstico e intervención desde el enfoque de la Comunicación.

Dysphasia is a term that conceptualizes a gnoseologic entity related to the organization of language in its evolution. It is a severe and prolonged duration disorder that affects children since the beginning of the language development and extends to all children and adolescents; it can also leave consequences in the adult stage. This review proposes an approach to the understanding of its dimensions, concept, symptoms and guidelines for the diagnosis and involvement from a communication perspective.

Pyrimidine requirements in deoxyuridine triphosphate nucleotidohydrolase deficient Trypanosoma brucei mutants.

Trypanosomal all-alpha dUTPases are homodimeric enzymes that catalyze the hydrolysis of dUTP and dUDP to dUMP and PPi. Trypanosomes lack dCTP/dCMP deaminase and therefore strongly depend on dUDP/dUTP hydrolysis for dUMP production. Here we have addressed by gene replacement the consequences of elimination of dUTPase activity in bloodstream forms of Trypanosoma brucei. We first generated conditional DUT-knockout strains that allowed an effective decrease of dUTPase resulting in proliferation arrest, although gene repression could not be sustained long enough to cause lethality. Alternatively, DUT null mutants could be isolated in the presence of high levels of thymidine while exogenous supplementation with uracil, uridine or deoxyuridine could not complement metabolically the dUTPase deficiency. Upon thymidine removal, trypanosomes exhibited impaired proliferation and eventually died. These data establish a strict requirement for dUTPase in T. brucei viability and support a major role of the enzyme in the provision of pyrimidine nucleotides in kinetoplastids.

Ubiquitin mediates the physical and functional interaction between human DNA polymerases η and ι.

Human DNA polymerases η and ι are best characterized for their ability to facilitate translesion DNA synthesis (TLS). Both polymerases (pols) co-localize in 'replication factories' in vivo after cells are exposed to ultraviolet light and this co-localization is mediated through a physical interaction between the two TLS pols. We have mapped the polη-ι interacting region to their respective ubiquitin-binding domains (UBZ in polη and UBM1 and UBM2 in polι), and demonstrate that ubiquitination of either TLS polymerase is a prerequisite for their physical and functional interaction. Importantly, while monoubiquitination of polη precludes its ability to interact with proliferating cell nuclear antigen (PCNA), it enhances its interaction with polι. Furthermore, a polι-ubiquitin chimera interacts avidly with both polη and PCNA. Thus, the ubiquitination status of polη, or polι plays a key regulatory function in controlling the protein partners with which each polymerase interacts, and in doing so, determines the efficiency of targeting the respective polymerase to stalled replication forks where they facilitate TLS.

Increased uracil insertion in DNA is cytotoxic and increases the frequency of mutation, double strand break formation and VSG switching in Trypanosoma brucei.

Deoxyuridine 5'-triphosphate pyrophosphatase (dUTPase) and uracil-DNA glycosylase (UNG) are key enzymes involved in the control of the presence of uracil in DNA. While dUTPase prevents uracil misincorporation by removing dUTP from the deoxynucleotide pool, UNG excises uracil from DNA as a first step of the base excision repair pathway (BER). Here, we report that strong down-regulation of dUTPase in UNG-deficient Trypanosoma brucei cells greatly impairs cell viability in both bloodstream and procyclic forms, underscoring the extreme sensitivity of trypanosomes to uracil in DNA. Depletion of dUTPase activity in the absence of UNG provoked cell cycle alterations, massive dUTP misincorporation into DNA and chromosomal fragmentation. Overall, trypanosomatid cells that lack dUTPase and UNG activities exhibited greater proliferation defects and DNA damage than cells deficient in only one of these activities. To determine the mutagenic consequences of uracil in DNA, mutation rates and spectra were analyzed in dUTPase-depleted cells in the presence of UNG activity. These cells displayed a spontaneous mutation rate 9-fold higher than the parental cell line. Base substitutions at A:T base pairs and deletion frequencies were both significantly enhanced which is consistent with the generation of mutagenic AP sites and DNA strand breaks. The increase in strand breaks conveyed a concomitant increase in VSG switching in vitro. The low tolerance of T. brucei to uracil in DNA emphasizes the importance of uracil removal and regulation of intracellular dUTP pool levels in cell viability and genetic stability and suggests potential strategies to compromise parasite survival.