[Diagnostic protocol for chronic manifestations of COVID-19]. / Protocolo diagnóstico de las manifestaciones crónicas de la COVID-19.

Many people who have had COVID-19 continue to have symptoms weeks and months after recovering from the acute disease. They present with a highly heterogeneous set of manifestations whose pathophysiology is unknown. There is no uniform nomenclature or agreed-upon diagnostic criteria. One of the main risk factors for its onset is having required intensive care unit admission. The most common manifestations are fatigue and respiratory symptoms, of note among which is dyspnea. The diagnostic approach is focused on ruling out other possible causes that could be responsible for said manifestations. Therefore, a comprehensive evaluation of the patient is conducted followed by a later approach aimed at the nonspecific symptoms.

A quantitative real-time PCR method for in planta monitoring of Phytophthora infestans growth.

AIMS: To establish a reliable and rapid protocol to simultaneously obtain high quality DNA from an infected host plant and the infecting pathogen. To develop an accurate and sensitive low-cost assay for the quantification and in planta monitoring of Phytophthora infestans growth. METHODS AND RESULTS: In this study, we describe a SYBR Green-based quantitative real-time PCR (qPCR) method for the quantification of P. infestans. The method is based on a simultaneous plant-pathogen DNA purification followed by a qPCR in which the relative quantification of pathogen and plant DNA is performed. Besides assuring an accurate quantification, the use of a plant gene provides a reliable indicator of sample quality, allowing the exclusion of inappropriate samples. By applying this methodology, we were able to detect P. infestans in potato leaf and tuber tissue before the first symptoms of the disease were observed and to monitor the in planta growth of the pathogen for 6 days. CONCLUSIONS: This is a reliable low-cost assay that provides rapid, accurate and sensitive quantification of the late blight pathogen, allowing the in planta monitoring of P. infestans growth. SIGNIFICANCE AND IMPACT OF THE STUDY: The quantitative nature of the assay described in this study may be useful in plant breeding programmes and basic research. The method is appropriate for the comparison of cultivars with different, and even subtle, degrees of pathogen resistance and in the screening of new anti-oomycete compounds. The method can be easily adapted to tomato and the model plant Nicotiana benthamiana.

Impact of erythropoietin on the reduction of blood transfusions and on survival of lung cancer patients receiving first-line chemotherapy.

INTRODUCTION: Anaemia is a common problem in patients with cancer who receive chemotherapy and is normally associated with a negative impact on patients' quality of life (QOL), poor cancer control and diminished survival. In clinical trials, recombinant human erythropoietin has been shown to correct and prevent anaemia, decrease the need for blood transfusions and improve cancer patients' QOL. METHODS: A retrospective study followed lung cancer patients who received first-line chemotherapy in our hospital in 1998 and in 2005. The incidence of anaemia was analysed, as was the impact of incorporating erythropoietin into the treatment. RESULTS: The incidence of anaemia was 68% (69% of which reported asthenia) in 1998 vs. 54% (60% with asthenia) in 2005. The comparison of anaemia rates (1998 vs. 2005) were grade 1 (16% vs. 32%), grade 2 (36% vs. 16%), grade 3 (16% vs. 5%) and grade 4 (none). Treatment for anaemia included transfusion 52%, intravenous iron 5% and epoetin 4% in 1998. In 2005 anaemia was treated with transfusion 9%, intravenous iron 41%, and epoetin 49%. Median survival (1998 vs. 2005) was 242 days [95% confidence interval (CI) 217-329) vs. 356 days (95% CI 322-382). CONCLUSIONS: Erythropoietin is a valid alternative for cancer patients with anaemia undergoing chemotherapy. It can possibly avoid the need for transfusions without negatively impacting survival.

[Patient safety]. / Seguridad del paciente.

Ensuring patient safety is essential for better heath care. Safety have gripped public attention ever since the release of the report "To Err is Human". To find strategies of promotion of patient safety has stimulated models that improve knowledge of adverse events. Adverse drug events are the most common cause of injury to hospitalized patients and are often preventable. Many tactics are available to make system changes to reduce errors and adverse events; they fall into five categories: Reduce complexity, optimise information processing, automate wisely, use constraints, and mitigate the unwanted side effects of change. These tactics can be deployed to support any of the three strategic components of error prevention, detection, and mitigation. Although progress has been slow, the pace of change is likely to accelerate, particularly in implementation of electronic health records and diffusion of safe practices.

Variability within the Seychelles cytoplasmic incompatibility system in Drosophila simulans.

In Drosophila simulans, we described a cytoplasmic incompatibility (CI) system (Seychelles) restricted to insular populations that harbor the mitochondrial type SiI. Since then, these populations have been shown to be heterogeneous, some being infected by one Wolbachia genetic variant only (wHa), while others are infected simultaneously by wHa and by another variant (wNo) always found in association with wHa. We have experimentally obtained two D. simulans strains only infected by the wNo variant. This variant determines its own cytoplasmic incompatibility type. In particular, the cross between wNo-bearing flies and wHa-bearing ones is bidirectionally incompatible. The Seychelles CI type, stricto sensu, is distinguished by being determined by the simultaneous presence of two Wolbachia variants that we found to be mutually incompatible. In addition, we observed incomplete maternal transmission of the Wolbachia.

Transcriptional regulation of the Saccharomyces cerevisiae DAL5 gene family and identification of the high affinity nicotinic acid permease TNA1 (YGR260w).

We have studied the transcript levels of YGR260w and YLR004c, two genes encoding members of the yeast Dal5p subfamily of the major facilitator family, and we show that they increase when extracellular nicotinic acid and thiamine, respectively, are absent. The deletion of YGR260w in a bna1 auxotrophic mutant for nicotinic acid prevents growth at low nicotinic acid concentration. This suggests that YGR260w is necessary for nicotinic acid import into the cell. The direct measurement of nicotinic acid uptake on whole cells demonstrates that YGR260w encodes the yeast high affinity nicotinic acid permease. Its apparent K(m) of 1.7 microM is low enough to allow the uptake of the low concentrations of nicotinic acid normally secreted by wild type cells.

Genomic exploration of the hemiascomycetous yeasts: 10. Kluyveromyces thermotolerans.

A genomic exploration of Kluyveromyces thermotolerans was performed by random sequence tag (RST) analysis. We sequenced 2653 RSTs corresponding to inserts sequenced from both ends. We performed a systematic comparison with a complete set of proteins from Saccharomyces cerevisiae, other completely sequenced genomes and SwissProt. We identified six mitochondrial genes and 1358-1496 nuclear genes by comparison with S. cerevisiae. In addition, 25 genes were identified by comparison with other organisms. This corresponds to about 24% of the estimated gene content of this organism. A lower level of conservation is observed with orthologues to genes of S. cerevisiae previously classified as orphans. Gene order was found to be conserved between S. cerevisiae and K. thermotolerans in 56.5% of studied cases.

Genomic exploration of the hemiascomycetous yeasts: 12. Kluyveromyces marxianus var. marxianus.

As part of the comparative genomics project 'GENOLEVURES', we studied the Kluyveromyces marxianus var. marxianus strain CBS712 using a partial random sequencing strategy. With a 0.2 x genome equivalent coverage, we identified ca. 1300 novel genes encoding proteins, some containing spliceosomal introns with consensus splice sites identical to those of Saccharomyces cerevisiae, 28 tRNA genes, the whole rDNA repeat, and retrotransposons of the Ty1/2 family of S. cerevisiae with diverged Long Terminal Repeats. Functional classification of the K. marxianus genes, as well as the analysis of the paralogous gene families revealed few differences with respect to S. cerevisiae. Only 42 K. marxianus identified genes are without detectable homolog in the baker's yeast. However, we identified several genetic rearrangements between these two yeast species.

Genomic exploration of the hemiascomycetous yeasts: 13. Pichia angusta.

As part of a comparative genomics project on 13 hemiascomycetous yeasts, the Pichia angusta type strain was studied using a partial random sequencing strategy. With coverage of 0.5 genome equivalents, about 2500 novel protein-coding genes were identified, probably corresponding to more than half of the P. angusta protein-coding genes, 6% of which do not have homologs in Saccharomyces cerevisiae. Some of them contain one or two introns, on average three times shorter than those in S. cerevisiae. We also identified 28 tRNA genes, a few retrotransposons similar to Ty5 of S. cerevisiae, solo long terminal repeats, the whole ribosomal DNA cluster, and segments of mitochondrial DNA. The P. angusta sequences were deposited in EMBL under the accession numbers AL430961 to AL436044.

Genomic exploration of the hemiascomycetous yeasts: 3. Methods and strategies used for sequence analysis and annotation.

The primary analysis of the sequences for our Hemiascomycete random sequence tag (RST) project was performed using a combination of classical methods for sequence comparison and contig assembly, and of specifically written scripts and computer visualization routines. Comparisons were performed first against DNA and protein sequences from Saccharomyces cerevisiae, then against protein sequences from other completely sequenced organisms and, finally, against protein sequences from all other organisms. Blast alignments were individually inspected to help recognize genes within our random genomic sequences despite the fact that only parts of them were available. For each yeast species, validated alignments were used to infer the proper genetic code, to determine codon usage preferences and to calculate their degree of sequence divergence with S. cerevisiae. The quality of each genomic library was monitored from contig analysis of the DNA sequences. Annotated sequences were submitted to the EMBL database, and the general annotation tables produced served as a basis for our comparative description of the evolution, redundancy and function of the Hemiascomycete genomes described in other articles of this issue.

Genomic exploration of the hemiascomycetous yeasts: 1. A set of yeast species for molecular evolution studies.

The identification of molecular evolutionary mechanisms in eukaryotes is approached by a comparative genomics study of a homogeneous group of species classified as Hemiascomycetes. This group includes Saccharomyces cerevisiae, the first eukaryotic genome entirely sequenced, back in 1996. A random sequencing analysis has been performed on 13 different species sharing a small genome size and a low frequency of introns. Detailed information is provided in the 20 following papers. Additional tables available on websites describe the ca. 20000 newly identified genes. This wealth of data, so far unique among eukaryotes, allowed us to examine the conservation of chromosome maps, to identify the 'yeast-specific' genes, and to review the distribution of gene families into functional classes. This project conducted by a network of seven French laboratories has been designated 'Génolevures'.

Genomic exploration of the hemiascomycetous yeasts: 4. The genome of Saccharomyces cerevisiae revisited.

Since its completion more than 4 years ago, the sequence of Saccharomyces cerevisiae has been extensively used and studied. The original sequence has received a few corrections, and the identification of genes has been completed, thanks in particular to transcriptome analyses and to specialized studies on introns, tRNA genes, transposons or multigene families. In order to undertake the extensive comparative sequence analysis of this program, we have entirely revisited the S. cerevisiae sequence using the same criteria for all 16 chromosomes and taking into account publicly available annotations for genes and elements that cannot be predicted. Comparison with the other yeast species of this program indicates the existence of 50 novel genes in segments previously considered as 'intergenic' and suggests extensions for 26 of the previously annotated genes.

Genomic exploration of the hemiascomycetous yeasts: 18. Comparative analysis of chromosome maps and synteny with Saccharomyces cerevisiae.

We have analyzed the evolution of chromosome maps of Hemiascomycetes by comparing gene order and orientation of the 13 yeast species partially sequenced in this program with the genome map of Saccharomyces cerevisiae. From the analysis of nearly 8000 situations in which two distinct genes having homologs in S. cerevisiae could be identified on the sequenced inserts of another yeast species, we have quantified the loss of synteny, the frequency of single gene deletion and the occurrence of gene inversion. Traces of ancestral duplications in the genome of S. cerevisiae could be identified from the comparison with the other species that do not entirely coincide with those identified from the comparison of S. cerevisiae with itself. From such duplications and from the correlation observed between gene inversion and loss of synteny, a model is proposed for the molecular evolution of Hemiascomycetes. This model, which can possibly be extended to other eukaryotes, is based on the reiteration of events of duplication of chromosome segments, creating transient merodiploids that are subsequently resolved by single gene deletion events.

Genomic exploration of the hemiascomycetous yeasts: 19. Ascomycetes-specific genes.

Comparisons of the 6213 predicted Saccharomyces cerevisiae open reading frame (ORF) products with sequences from organisms of other biological phyla differentiate genes commonly conserved in evolution from 'maverick' genes which have no homologue in phyla other than the Ascomycetes. We show that a majority of the 'maverick' genes have homologues among other yeast species and thus define a set of 1892 genes that, from sequence comparisons, appear 'Ascomycetes-specific'. We estimate, retrospectively, that the S. cerevisiae genome contains 5651 actual protein-coding genes, 50 of which were identified for the first time in this work, and that the present public databases contain 612 predicted ORFs that are not real genes. Interestingly, the sequences of the 'Ascomycetes-specific' genes tend to diverge more rapidly in evolution than that of other genes. Half of the 'Ascomycetes-specific' genes are functionally characterized in S. cerevisiae, and a few functional categories are over-represented in them.

Genomic exploration of the hemiascomycetous yeasts: 20. Evolution of gene redundancy compared to Saccharomyces cerevisiae.

We have evaluated the degree of gene redundancy in the nuclear genomes of 13 hemiascomycetous yeast species. Saccharomyces cerevisiae singletons and gene families appear generally conserved in these species as singletons and families of similar size, respectively. Variations of the number of homologues with respect to that expected affect from 7 to less than 24% of each genome. Since S. cerevisiae homologues represent the majority of the genes identified in the genomes studied, the overall degree of gene redundancy seems conserved across all species. This is best explained by a dynamic equilibrium resulting from numerous events of gene duplication and deletion rather than by a massive duplication event occurring in some lineages and not in others.

Genomic exploration of the hemiascomycetous yeasts: 21. Comparative functional classification of genes.

We explored the biological diversity of hemiascomycetous yeasts using a set of 22000 newly identified genes in 13 species through BLASTX searches. Genes without clear homologue in Saccharomyces cerevisiae appeared to be conserved in several species, suggesting that they were recently lost by S. cerevisiae. They often identified well-known species-specific traits. Cases of gene acquisition through horizontal transfer appeared to occur very rarely if at all. All identified genes were ascribed to functional classes. Functional classes were differently represented among species. Species classification by functional clustering roughly paralleled rDNA phylogeny. Unequal distribution of rapidly evolving, ascomycete-specific, genes among species and functions was shown to contribute strongly to this clustering. A few cases of gene family amplification were documented, but no general correlation could be observed between functional differentiation of yeast species and variations of gene family sizes. Yeast biological diversity seems thus to result from limited species-specific gene losses or duplications, and for a large part from rapid evolution of genes and regulatory factors dedicated to specific functions.

Comparative toxicity of sulfur mustard and nitrogen mustard on tracheal epithelial cells in primary culture.

Sulfur mustard (SM) and mechlorethamine (HN2) are two alkylating agents. SM represents a potential chemical warfare agent and HN2 is used in cancer chemotherapy. Based on the similarities of their action, although few comparative studies of their effects have been performed on the same model, many compounds effective against HN2 side-effects have been proposed, unsuccessfully, against SM-induced lesions. We performed this study to compare the toxic effects of these two alkylating agents on rabbit tracheal epithelium in primary culture. Using neutral red uptake, we evidenced that for a time of contact of 24hr, HN2 LC(50) was significantly lower than SM LC(50) (0.034+/-0.009 and 0.132+/-0.023mm, respectively; P<0.001). On the other hand, for exposure at 10(-3)m, the time necessary to decrease the cell viability rate to 50% was shorter with SM than with HN2 (11+/-1min and 54+/-2min, respectively; P<0.0001). These two alkylating agents induced apoptosis which was evidenced by DNA ladder and by 4',6-diamidino-2-phenylindole (DAPI) DAPI staining. The apoptosis rates were time and dose dependent for the two toxics: mild doses induced apoptosis, while higher doses induced necrosis.

[Impact of arterial hypertension on the central nervous system]. / Repercusión de la hipertensión arterial sobre el sistema nervioso central.

Cerebrovascular diseases (CVD) entail high costs associated to therapies, hospitalizations and disabilities. Arterial hypertension (AHT) is the major modifiable risk factor for the development of all types of CVDs (cerebrovascular accidents, vascular dementias, etc.). The increase of arterial pressure causes functional and anatomical changes in the cerebral circulation which facilitate the development of CVDs. The time of evolution and the severity of the AHT, as well as the associated vascular risk factors, will determine its impact on the central nervous system.

[Cardiovascular complications due to cocaine]. / Complicaciones cardiovasculares por cocaína.

The increase of cocaine consumption is a common new item in the medical literature and in the national press and, hence, it does not surprises us any more. However, we are seeing every day the appearance of unknown or forgotten complications. In this paper, we review the cardiovascular complications due to the consumption of this drug, stressing the physiopathological mechanisms and the therapeutical aspects. In addition, we describe our experience with the management of these patients.