[Immuno-oncological approaches in the perioperative therapy of muscle invasive bladder cancer]. / Immunonkologische Ansätze in der perioperativen Therapie des muskelinvasiven Urothelkarzinoms.

Perioperative chemotherapy has become a standard treatment for muscle invasive bladder cancer and is recommended by national and international guidelines. The treatment of metastatic urothelial cancer evolved by the use of immune-modulating therapies like checkpoint inhibitors. Many clinical trials have been initiated which try to evaluate the role of immune checkpoint inhibition in the neoadjuvant and adjuvant setting. These trials focus not only on monotherapy, but also on the combination of checkpoint inhibitors with classical chemotherapy or with local radiation therapy (radioimmunotherapy). In neoadjuvant radioimmunotherapy, the radiation is supposed to act as a sensitizer for the systemic effects of checkpoint inhibition, in addition to the local effects. This review presents and discusses current trials and published results for perioperative immunomodulating treatment-alone or in combination-in muscle invasive bladder cancer.

[Nutritional assessment of patients candidates for waiting list simultaneous kidney pancreas transplantation]. / Evaluación nutricional de pacientes candidatos a lista de espera de trasplante renopancreático.

BACKGROUND: Simultaneous Kidney Pancreas transplantation is currently the treatment of choice for patients with type 1 diabetes ERD advanced. Scientific studies on the therapeutic benefits of this guarantee acceptance of it as the most suitable for patients. The independence of dialysis and insulin therapy after the transplant represents a significant increase in the quality of life for these patients. In recent years, the importance of nutritional assessment in patients with chronic diseases has increased. There is increasing evidence that malnutrition is associated with an increased risk of morbidity and mortality after surgery. Given the therapeutic choice for these patients and the importance of nutritional status as an independent risk factor for post-transplant complications objective of our work is to describe the nutritional status of patients who are candidates for transplant through objective and subjective parameters of evaluation nutritional. MATERIALS AND METHODS: We included 45 patients with type 1 diabetes ERD interned in the Hospital Italiano of Buenos Aires for assessing pre Transplantation during the period June 2007-June 2008. They collected data through a sheet produced by themselves. As parameter anthropometric was calculated body mass index (BMI) by the formula Quetelet (post dialysis Weight (kg) / height 2 (m)) and was ranked as the ranges proposed by the committee of experts from WHO. As a subjective argument took place the Subjective Global Valuation (VGS), which ranked patients in well-nourished (A), mild malnourished (B), moderately malnourished (C) and severely malnourished (D). As biochemical parameters of the data was recorded serum albumin (g/dl) to hospitalizations and this data was obtained from the patient's medical history. RESULTS: The value of the average BMI of the total population was 21.83+/-2.65. According to the sex values were: 21.83+/-2.39 and 21.82+/-2.95 for male and female respectively. The nutritional status according to this indicator was anthropometric Normal in 89% of cases. The 6.66% of the cases submitted 13.33% pre malnutrition and obesity. We found no cases with obesity. Taking into account the albumin 47.45% of patients presented moderate risk of morbidity and mortality. According to GSV 62.22% were classified as well-nourished (Class A). It was the equitable proportion of women and men 33.33% were classified as mild malnourished (Class B). Only 2 cases (8.88%) had moderate malnutrition (Class C). None presented malnutrition Graves (Class C). CONCLUSION: In our study we can see that patients are candidates for waiting list while normal BMI have presented evidence of nutritional risk when one takes into account the VGS and the average value of albumin.

Evaluación nutricional de pacientes candidatos a lista de espera de trasplante renopancreático / Nutritional assessment of patients candidates for waiting list simultaneous kidney pancreas transplantation

Introducción: El trasplante renopancreático es en la actualidad el tratamiento elegido para los pacientes diabéticos tipo 1 con ERD avanzada. Estudios científicos sobre los beneficios de esta terapéutica avalan la aceptación de la misma como lo más adecuado para los pacientes. La independencia de diálisis y de tratamiento insulínico luego del trasplante representa un importante incremento en la calidad de vida de esos pacientes. En los últimos años, la importancia de la evaluación nutricional en pacientes con enfermedades crónicas ha aumentado. Existe cada vez más evidencia científica que la malnutrición esta asociada a un riesgo mayor de morbilidad y mortalidad post intervención quirúrgica. Teniendo en cuenta la terapéutica elegida para estos pacientes y la importancia del estado nutricional como factor predictor independiente de riesgo de complicaciones post trasplante es objetivo de nuestro trabajo describir el estado nutricional de los pacientes candidatos a trasplante renopancreatico a través de parámetros objetivos y subjetivos de valoración nutricional. Materiales y métodos: Se incluyeron 45 pacientes Diabéticos tipo 1 con ERD internados en el Hospital Italiano de Buenos Aires para evaluación pre trasplante renopancreático durante el período Junio 2007-Junio 2008. Se recolectaron los datos mediante una ficha de elaboración propia. Como parámetro antropométrico se calculó el Índice de Masa corporal (IMC) mediante la fórmula de Quetelet (Peso post diálisis (kg)/talla 2 (mts)) y se clasificó según los rangos propuestos por el comité de Expertos de la OMS. Como parámetro subjetivo se realizo la Valoración Global Subjetiva (VGS) que clasificó a los pacientes en Bien nutrido (A), desnutrido leve (B), desnutrido moderado (C) y desnutrido severo (D). Como parámetros bioquímicos se registró el dato de albúmina sérica (g/dl) al ingreso hospitalario y este dato se obtuvo de la historia clínica del paciente. Resultados: El valor de la media para IMC del total de la población fue de 21,83 ± 2,65. Según el sexo los valores fueron: 21,83 ± 2,39 y de 21,82 ± 2,95 para el sexo mascureprelino y femenino respectivamente. El estado nutricional según este indicador antropométrico fue Normal en el 89% de los casos. El 6,66% de los casos presentó desnutrición y 13,33% pre obesidad. No se encontraron casos con Obesidad. Teniendo en cuenta la albúmina el 47,45% de los pacientes presenta riesgo moderado de morbimortalidad. Según la VGS el 62,22% se clasificaron como bien nutridos (Clase A). Fue equitativa la proporción de mujeres y de hombres. 33,33% se clasificaron como desnutridos leve (Clase B). Sólo 2 casos (8,88%) presentaron desnutrición moderada (Clase C). Ninguno presentó desnutrición Graves (Clase C). Conclusión: En nuestro estudio se puede observar que los pacientes candidatos a lista de espera si bien presentan valores de IMC dentro de la clasificación normal presentan indicios de riesgo nutricional cuando se tienen en cuenta la VGS y el valor medio de albúmina (AU)

Background: Simultaneous Kidney Pancreas transplantation is currently the treatment of choice for patients with type 1 diabetes ERD advanced. Scientific studies on the therapeutic benefits of this guarantee acceptance of it as the most suitable for patients. The independence of dialysis and insulin therapy after the transplant represents a significant increase in the quality of life for these patients. In recent years, the importance of nutritional assessment in patients with chronic diseases has increased. There is increasing evidence that malnutrition is associated with an increased risk of morbidity and mortality after surgery. Given the therapeutic choice for these patients and the importance of nutritional status as an independent risk factor for post-transplant complications objective of our work is to describe the nutritional status of patients who are candidates for transplant through objective and subjective parameters of evaluation nutritional. Materials and methods: We included 45 patients with type 1 diabetes ERD interned in the Hospital Italiano of Buenos Aires for assessing pre Transplantation during the period June 2007-June 2008.Se collected data through a sheet produced by themselves. As parameter anthropometric was calculated body mass index (BMI) by the formula Quetelet (post dialysis Weight (kg) / height 2 (m)) and was ranked as the ranges proposed by the committee of experts from WHO. As a subjective argument took place the Subjective Global Valuation (VGS), which ranked patients in well-nourished (A), mild malnourished (B), moderately malnourished (C) and severely malnourished (D). As biochemical parameters of the data was recorded serum albumin (g / dl) to hospitalizations and this data was obtained from the patient's medical history. Results: The value of the average BMI of the total population was 21.83 ± 2.65. According to the sex values were: 21.83 ± 2.39 and 21.82 ± 2.95 for male and female respectively. The nutritional status according to this indicator was anthropometric Normal in 89% of cases. The 6.66% of the cases submitted 13.33% pre malnutrition and obesity. We found no cases with obesity. Taking into account the albumin 47.45% of patients presented moderate risk of morbidity and mortality. According to GSV 62.22% were classified as well-nourished (Class A). It was the equitable proportion of women and hombres.33, 33% were classified as mild malnourished (Class B). Only 2 cases (8.88%) had moderate malnutrition (Class C). None presented malnutrition Graves (Class C). Conclussion: In our study we can see that patients are candidates for waiting list while normal BMI have presented evidence of nutritional risk when one takes into account the VGS and the average value of albumin (AU)

Bug22p, a conserved centrosomal/ciliary protein also present in higher plants, is required for an effective ciliary stroke in Paramecium.

Centrioles, cilia, and flagella are ancestral conserved organelles of eukaryotic cells. Among the proteins identified in the proteomics of ciliary proteins in Paramecium, we focus here on a protein, Bug22p, previously detected by cilia and basal-body high-throughput studies but never analyzed per se. Remarkably, this protein is also present in plants, which lack centrioles and cilia. Bug22p sequence alignments revealed consensus positions that distinguish species with centrioles/cilia from plants. In Paramecium, antibody and green fluorescent protein (GFP) fusion labeling localized Bug22p in basal bodies and cilia, and electron microscopy immunolabeling refined the localization to the terminal plate of the basal bodies, the transition zone, and spots along the axoneme, preferentially between the membrane and the microtubules. RNA interference (RNAi) depletion of Bug22p provoked a strong decrease in swimming speed, followed by cell death after a few days. High-speed video microscopy and morphological analysis of Bug22p-depleted cells showed that the protein plays an important role in the efficiency of ciliary movement by participating in the stroke shape and rigidity of cilia. The defects in cell swimming and growth provoked by RNAi can be complemented by expression of human Bug22p. This is the first reported case of complementation by a human gene in a ciliate.

Genetic evidence for interaction between eta- and beta-tubulins.

The thermosensitive allelic mutations sm19-1 and sm19-2 of Paramecium tetraurelia cause defective basal body duplication: growth at the nonpermissive temperature yields smaller and smaller cells with fewer and fewer basal bodies. Complementation cloning of the SM19 gene identified a new tubulin, eta-tubulin, showing low homology with each of the other five tubulins, alpha to epsilon, characterized in P. tetraurelia. In order to analyze eta-tubulin functions, we used a genetic approach to identify interacting molecules. Among a series of extragenic suppressors of the sm19-1 mutation, the su3-1 mutation was characterized as an E288K substitution in the beta-PT2 gene coding for a beta-tubulin, while the mutation nocr1 conferring nocodazole resistance and localized in another beta-tubulin gene, beta-PT3, was shown to enhance the mutant phenotype. The interaction between eta-tubulin and microtubules, revealed by genetic data, is supported by two further types of evidence: first, the mutant phenotype is rescued by taxol, which stabilizes microtubules; second, molecular modeling suggests that eta-tubulin, like gamma- and delta-tubulins, might be a microtubule minus-end capping molecule. The likely function of eta-tubulin as part of a complex specifically involved in basal body biogenesis is discussed.

Mitochondrial membrane potential and ageing in Podospora anserina.

Some filamentous fungi exhibit a limited vegetative growth with modifications in the mitochondria, suggesting the involvement of mitochondria in the process of ageing. Nevertheless, the relationship between the ability to grow or the fate of these cells relative to their mitochondrial membrane potential (Psi(mt)) level has not been investigated. Using flow cytometric analysis, we have assessed Psi(mt) in young and senescent cultures of wild type strains and mitochondrial or nuclear mutant strains of Podospora anserina that present very long or brief life span. When we compared two distinct populations of cells obtained from the same strain, we can show a correlation not only between Psi(mt) and ageing, but also between Psi(mt) and the frequency of regeneration and/or the life span. However, this relationship is not observed when we compared the cells obtained from different physiological states or mutants strains. These results allow us to suggest that the Psi(mt) modifications during senescence could be only one of the possible consequences of the process and are not the factor driving towards death. We also show that the driving force of Psi(mt) is principally maintained by the alternative pathway during ageing, suggesting a role of the alternative oxidase pathway.

The SM19 gene, required for duplication of basal bodies in Paramecium, encodes a novel tubulin, eta-tubulin.

The discovery of delta-tubulin, the fourth member of the tubulin superfamily, in Chlamydomonas [1] has led to the identification in the genomes of vertebrates and protozoa of putative delta homologues and of additional tubulins, epsilon and zeta [2-4]. These discoveries raise questions concerning the functions of these novel tubulins, their interactions with microtubule arrays and microtubule-organising centres, and their evolutionary status. The sm19-1 mutation of Paramecium specifically inhibits basal body duplication [5] and causes delocalisation of gamma-tubulin, which is also required for basal body duplication [6]. We have cloned the SM19 gene by functional complementation and found that it encodes another new member of the tubulin superfamily. SM19p, provisionally called eta-tubulin (eta-tubulin), shows low sequence identity with the tubulins previously identified in Paramecium, namely, alpha [7], beta [8], gamma [6], delta (this work) and epsilon (P. Dupuis-Williams, personal communication). Phylogenetic analysis indicated that SM19p is not consistently grouped with any phylogenetic entity.

Intronic GIY-YIG endonuclease gene in the mitochondrial genome of Podospora curvicolla: evidence for mobility.

Endonuclease genes encoded in invasive introns are themselves supposed to be mobile elements which, during evolution, have colonized pre-existing introns converting them into invasive elements. This hypothesis is supported by numerous data concerning the LAGLI-DADG subclass of intronic endonucleases. Less is known about the GIY-YIG ORFs which constitute another family of endonucleases. In this paper we describe the presence of one optional GIY-YIG ORF in the second intron of the mitochondrial cytochrome b gene in the fungus Podospora curvicolla. We show that this GIY-YIG ORF is efficiently transferred from an ORF-containing intron to an ORF-less allele. We also show that the products of both the GIY-YIG ORF and the non-canonical LAGLI-DADG-GIY-YIG ORF, which is generated by its integration, have endonuclease activities which recognize and cut the insertion site of the optional sequence. This constitutes the first direct evidence for potential mobility of an intronic GIY-YIG endonuclease. We discuss the role that such a mobile sequence could have played during evolution.

Plasticity of the mitochondrial genome in Podospora. Polymorphism for 15 optional sequences: group-I, group-II introns, intronic ORFs and an intergenic region.

The mitochondrial chromosome of 15 Podospora anserina and one Podospora comata wild-type strains have been extensively examined for the presence of optional elements and for sequence divergence. Among the P. anserina strains, nine optional sequences were found. By comparing P. anserina with the closely related and weakly interfertile P. comata species, six additional optional sequences were detected. These optional elements correspond to mitochondrial introns belonging to different groups and subgroups (11 cases), intronic open reading frames (two cases), a complex insert and an intergenic region. Although difficult to explain, the distribution of optional mitochondrial sequences among the 15 wild-type isolates of P. anserina is far from random.

Cytosolic ribosomal mutations that abolish accumulation of circular intron in the mitochondria without preventing senescence of Podospora anserina.

The filamentous fungus Podospora anserina presents a degeneration syndrome called Senescence associated with mitochondrial DNA modifications. We show that mutations affecting the two different and interacting cytosolic ribosomal proteins (S7 and S19) systematically and specifically prevent the accumulation of senDNA alpha (a circular double-stranded DNA plasmid derived from the first intron of the mitochondrial cox1 gene or intron alpha) without abolishing Senescence nor affecting the accumulation of other usually observed mitochondrial DNA rearrangements. One of the mutant proteins is homologous to the Escherichia coli S4 and Saccharomyces cerevisiae S13 ribosomal proteins, known to be involved in accuracy control of cytosolic translation. The lack of accumulation of senDNA alpha seems to result from a nontrivial ribosomal alteration unrelated to accuracy control, indicating that S7 and S19 proteins have an additional function. The results strongly suggest that modified expression of nucleus-encoded proteins contributes to Senescence in P. anserina. These data do not fit well with some current models, which propose that intron alpha plays the role of the cytoplasmic and infectious Determinant of Senescence that was defined in early studies.

Contribution of ultra-short invasive elements to the evolution of the mitochondrial genome in the genus Podospora.

In the filamentous fungus Podospora anserina, senescence is associated with major rearrangements of the mitochondrial DNA. The undecamer GGCGCAAGCTC has been described as a preferential site for these recombination events. We show that: (i) copies of this short sequence GGCGCAAGCTC are present in unexpectedly high numbers in the mitochondrial genome of this fungus; (ii) a short cluster of this sequence, localised in a group II intronic ORF, encodes amino acids that disrupt a protein domain that is otherwise highly conserved between various species; (iii) most of the polymorphisms observed between three related species, P.anserina, P.curvicolla and P.comata, are associated with the presence/absence of this sequence; (iv) this element lies at the boundaries of major rearrangements of the mitochondrial genomes; (v) at least two other short elements in the Podospora mitochondrial genomes display similar features. We suggest that these short elements, called MUSEs (mitochondrial ultra-short elements), could be mobile and that they contribute to evolution of the mitochondrial genome in the genus Podospora. A model for mobility involving a target DNA-primed reverse transcription step is discussed.

Site-specific deletions of the mitochondrial genome in the Pearson marrow-pancreas syndrome.

The Pearson marrow-pancreas syndrome is a fatal disorder involving the hematopoietic system and the exocrine pancreas in early infancy. We have previously shown that this disease results from a widespread defect of oxidative phosphorylation. Here, we describe deletions of the mitochondrial (mt) genome between repeated 8- to 13-bp sequences as consistent features of the disease. Studying a series of nine unrelated children, including the patient originally reported by H. Pearson, we found five different types of direct repeats at the boundaries of the mtDNA deletions and we provided evidence for conservation of the 3'-repeated sequence in the deletions. In addition, we found a certain degree of homology between the nucleotide composition of the direct repeats and several structures normally involved in mtDNA replication and mtRNA processing. These results are consistent either with the recognition and cleavage of a particular DNA sequence with a factor of still unknown origin or with a homologous recombination between direct-repeat mtDNA sequences in the Pearson syndrome.

Insertion of short poly d(A) d(T) sequences at recombination junctions in mitochondrial DNA of Podospora.

We have characterized the DNA sequences at recombination points in the mitochondrial DNA of two independent mitochondrial mutants of Podospora anserina. These sequences reveal the presence of foreign DNA at each recombination border, consisting of short stretches of A and T residues. We discuss the possible origin of this DNA and suggest the involvement of a reverse transcriptase activity.

A 1100-bp sequence of mitochondrial DNA is involved in senescence process in Podospora: study of senescent and mutant cultures.

In Podospora, senescence is assumed to be caused by the amplification of short sequences of the mitochondrial genome (sen-DNAs). We have characterized a 1100-bp-long mitochondrial DNA sequence which could be directly involved in the phenomenon. Indeed, by hybridization experiments, we show that this sequence is both present in all the sen-DNA molecules which originate from the beta region of the mitochondrial chromosome and rearranged in the mitochondrial genome of two mitochondrial mutants selected as resistant to senescence.

Ethidium bromide rejuvenation of senescent cultures of Podospora anserina : Loss of senescence-specific DNA and recovery of normal mitochondrial DNA.

The effect of ethidium bromide (EB) which is known to be able to "rejuvenate" senescent mycelia in Podospora anserina, has been investigated at the level of the mitochondrial DNA (mtDNA) by restriction analysis and molecular hybridization. While senescent mycelia display a very low growth ability and gross mtDNA modifications (tandem amplification of short sequences and disorganization of the mitochondrial chromosome: deletion of large sequences), the rejuvenated mycelia display a normal life span and contain a mtDNA in all respects identical to that of wild type mycelium (neither circular molecules nor amplified fragments could be detected). These results demonstrate a strict correlation between the senescent state and the presence of amplified mtDNA and suggest that EB rejuvenation could proceed by an efficient selection of intact mitochondrial chromosomes still present in senescent cultures.