Genetic variation in the ribosomal DNA (rDNA) internal transcribed spacer (ITS) region has been studied among fungi. However, the numbers of ITS sequence polymorphisms in the various Candida species ...and their associations with sources of invasive fungal infections remain poorly investigated. Here, we characterized the intraspecific and interspecific ITS diversity of Candida spp. strains collected from patients with bloodstream or oroesophageal candidiasis.
We selected cultures of representative medically important species of Candida as well as some rare and emerging pathogens. Identification was performed by micromorphology and by biochemical testing using an ID32C system, as well as by the sequencing of rDNA ITS. The presence of intraspecific ITS polymorphisms was characterized based on haplotype networks, and interspecific diversity was characterized based on Bayesian phylogenetic analysis.
Among 300 Candida strains, we identified 76 C. albicans, 14 C. dubliniensis, 40 C. tropicalis, 47 C. glabrata, 34 C. parapsilosis (sensu stricto), 31 C. orthopsilosis, 3 C. metapsilosis, 21 Meyerozyma guilliermondii (C. guilliermondii), 12 Pichia kudriavzevii (C. krusei), 6 Clavispora lusitaniae (C. lusitaniae), 3 C. intermedia, 6 Wickerhamomyces anomalus (C. pelliculosa), and 2 C. haemulonii strains, and 1 C. duobushaemulonii, 1 Kluyveromyces marxianus (C. kefyr), 1 Meyerozyma caribbica (C. fermentati), 1 Pichia norvegensis (C. norvegensis), and 1 Lodderomyces elongisporus strain. Out of a total of seven isolates with inconsistent ID32C profiles, ITS sequencing identified one C. lusitaniae strain, three C. intermedia strains, two C. haemulonii strains and one C. duobushaemulonii strain. Analysis of ITS variability revealed a greater number of haplotypes among C. albicans, C. tropicalis, C. glabrata and C. lusitaniae, which are predominantly related to endogenous sources of acquisition. Bayesian analysis confirmed the major phylogenetic relationships among the isolates and the molecular identification of the different Candida spp.
Molecular studies based on ITS sequencing are necessary to identify closely related and emerging species. Polymorphism analysis of the ITS rDNA region demonstrated its utility as a genetic marker for species identification and phylogenetic relationships as well as for drawing inferences concerning the natural history of hematogenous infections caused by medically important and emerging Candida species.
We present the sequencing and annotation of the Leishmania (Leishmania) amazonensis genome, an etiological agent of human cutaneous leishmaniasis in the Amazon region of Brazil. L. (L.) amazonensis ...shares features with Leishmania (L.) mexicana but also exhibits unique characteristics regarding geographical distribution and clinical manifestations of cutaneous lesions (e.g. borderline disseminated cutaneous leishmaniasis). Predicted genes were scored for orthologous gene families and conserved domains in comparison with other human pathogenic Leishmania spp. Carboxypeptidase, aminotransferase, and 3'-nucleotidase genes and ATPase, thioredoxin, and chaperone-related domains were represented more abundantly in L. (L.) amazonensis and L. (L.) mexicana species. Phylogenetic analysis revealed that these two species share groups of amastin surface proteins unique to the genus that could be related to specific features of disease outcomes and host cell interactions. Additionally, we describe a hypothetical hybrid interactome of potentially secreted L. (L.) amazonensis proteins and host proteins under the assumption that parasite factors mimic their mammalian counterparts. The model predicts an interaction between an L. (L.) amazonensis heat-shock protein and mammalian Toll-like receptor 9, which is implicated in important immune responses such as cytokine and nitric oxide production. The analysis presented here represents valuable information for future studies of leishmaniasis pathogenicity and treatment.
Trypanosoma cruzi is highly diverse genetically and has been partitioned into six discrete typing units (DTUs), recently re-named
T. cruzi I–VI. Although
T. cruzi reproduces predominantly by binary ...division, accumulating evidence indicates that particular DTUs are the result of hybridization events. Two major scenarios for the origin of the hybrid lineages have been proposed. It is accepted widely that the most heterozygous TcV and TcVI DTUs are the result of genetic exchange between TcII and TcIII strains. On the other hand, the participation of a TcI parental in the current genome structure of these hybrid strains is a matter of debate. Here, sequences of the
T. cruzi-specific 195-bp satellite DNA of TcI, TcII, TcIII, TcV, and TcVI strains have been used for inferring network genealogies. The resulting genealogy showed a high degree of reticulation, which is consistent with more than one event of hybridization between the Tc DTUs. The data also strongly suggest that TcIII is a hybrid with two distinct sets of satellite sequences, and that genetic exchange between TcI and TcII parentals occurred within the pedigree of the TcV and TcVI DTUs. Although satellite DNAs belong to the fast-evolving portion of eukaryotic genomes, in >100 satellite units of nine
T. cruzi strains we found regions that display 100% identity. No DTU-specific consensus motifs were identified, inferring species-wide conservation.
Human and animal fungal pathogens are a growing threat worldwide leading to emerging infections and creating new risks for established ones. There is a growing need for a rapid and accurate ...identification of pathogens to enable early diagnosis and targeted antifungal therapy. Morphological and biochemical identification methods are time-consuming and require trained experts. Alternatively, molecular methods, such as DNA barcoding, a powerful and easy tool for rapid monophasic identification, offer a practical approach for species identification and less demanding in terms of taxonomical expertise. However, its wide-spread use is still limited by a lack of quality-controlled reference databases and the evolving recognition and definition of new fungal species/complexes. An international consortium of medical mycology laboratories was formed aiming to establish a quality controlled ITS database under the umbrella of the ISHAM working group on “DNA barcoding of human and animal pathogenic fungi.” A new database, containing 2800 ITS sequences representing 421 fungal species, providing the medical community with a freely accessible tool at http://www.isham.org/ and http://its.mycologylab.org/ to rapidly and reliably identify most agents of mycoses, was established. The generated sequences included in the new database were used to evaluate the variation and overall utility of the ITS region for the identification of pathogenic fungi at intra-and interspecies level. The average intraspecies variation ranged from 0 to 2.25%. This highlighted selected pathogenic fungal species, such as the dermatophytes and emerging yeast, for which additional molecular methods/genetic markers are required for their reliable identification from clinical and veterinary specimens.
Acute kidney injury is associated with many conditions, and no interventions to improve the outcomes of established acute kidney injury have been developed. We performed this study to determine ...whether goal-directed therapy conducted during the early stages of acute kidney injury could change the course of the disease.
This was a multicenter prospective randomized controlled study. Patients with early acute kidney injury in the critical care unit were randomly allocated to a standard care (control) group or a goal-directed therapy group with 8h of intensive treatment to maximize oxygen delivery, and all patients were evaluated during a period of 72h. ClinicalTrials.gov: NCT02414906.
A total of 143 patients were eligible for the study, and 99 patients were randomized. Central venous oxygen saturation was significantly increased and the serum lactate level significantly was decreased from baseline levels in the goal-directed therapy group (p=0.001) compared to the control group (p=0.572). No significant differences in the change in serum creatinine level (p=0.96), persistence of acute kidney injury beyond 72h (p=0.064) or the need for renal replacement therapy (p=0.82) were observed between the two groups. In-hospital mortality was significantly lower in the goal-directed therapy group than in the control group (33% vs. 51%; RR: 0.61, 95% CI: 0.37-1.00, p=0.048, number needed to treat=5).
Goal-directed therapy for patients in the early stages of acute kidney injury did not change the disease course.
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Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)
Conselho Nacional de ...Desenvolvimento Científico e Tecnológico (CNPq)
Howard Hughes Medical Institute (HHMI)
Os índices que medem adaptação dos códons são amplamente utilizados para;
predizer o nível de expressão de um gene. Para verificar se: (i) o índice de adaptação;
dos códons (CAI) pode ser utilizado para ordenar e caracterizar genes hipotéticos em;
seqüências de genomas completos; (ii) a variação estocástica da expressão gênica;
entre as células (ruído) pode ser utilizada como um marcador quantitativo para;
diferenciar genes essenciais de não essenciais e (iii) qual a correlação existente entre;
o ruído e o CAI, subdividiu-se esse trabalho em duas partes. Na primeira parte analisei;
os índices de padrão de uso dos códons como preditores do nível de expressão de;
genes hipotéticos da levedura Candida albicans. Foram selecionados 744 genes;
hipotéticos que satisfazem três características simultaneamente sendo elas: (i) ORFs;
maiores que 500bp; (ii) fases abertas de leitura sem sobreposição com outras ORFs e;
(iii) ORFs com similaridade ≥ 60% com genes de Saccharomyces cerevisiae e;
Schizosaccharomyces pombe. O padrão de uso dos códons para os genes hipotéticos;
apresenta uma grande variedade nos diferentes genes selecionados, porém nota-se;
uma tendência a um baixo e médio nível de expressão independente do índice;
analisado. Os valores de CAI variam entre 0,044 e 0,540 com média de 0,170 (±;
0,051). ORFs com baixo nível de expressão e potencialmente essenciais são ótimas;
candidatas para serem alvos para drogas potenciais. Essas características são;
importantes uma vez que um baixo nível de expressão implicaria numa menor;
dosagem da droga, e o gene de ser potencialmente essencial uma vez que se deseja a;
morte do fungo causando a infecção. Baseando-se nessas duas características;
selecionou-se duas ORFs hipotéticas para estudo de função: CaYdr187c (CAI = 0,084);
e CaYlr339c (CAI = 0,088). A ORF CaYdr187c é uma possível proteína do envelope;
celular, com ontologia de resposta imune; e apresenta tanto peptídeo sinal quanto;
regiões transmembrana. De acordo com o RT-PCR esse gene é transcrito apenas na;
fase de levedura do fungo. A ORF CaYlr339c é uma possível proteína relacionada com;
a tradução, com ontologia de resposta imune; e não apresenta nem peptídeo sinal e;
nem regiões transmembrana. De acordo com o RT-PCR esse gene é transcrito tanto;
na fase de levedura quanto na de hifa do fungo. Na segunda parte, analisei a;
distribuição do ruído transcricional e sua possível utilização como classificador para a;
essencialidade dos genes. Para se estudar as distribuições estatísticas do ruído;
temporal no sistema eucariótico modelo Saccharomyces cerevisiae, nós analisamos;
dados de microarray correspondendo à um ciclo celular para 6.200 genes. Nós;
descobrimos que o ruído temporal segue uma distribuição log-normal com invariância;
de escala nos níveis genômico, cromossômico e sub-cromossômico. A correlação do;
ruído temporal com o índice de adaptação dos códons sugere que pelo menos 70%;
dos genes codificadores de proteínas são o centro de minimização de ruído do;
genoma. Nós propusemos um modelo matemático da dinâmica da expressão de um;
único gene, utilizando a teoria de operadores, o qual revela condições rígidas para a;
variabilidade do ruído e uma estratégia possível para a minimização / otimização do;
ruído em nível genômico. Nosso modelo e dados mostram que o ruído mínimo não;
corresponde a genes obedecendo a uma dinâmica estritamente determinística. A;
estratégia natural de minimização do ruído consiste em igualar o ruído (η) com a média;
do valor absoluto da variação relativa do nível de expressão (α). Nós hipotetizamos;
que o padrão do ruído temporal é uma propriedade emergente do genoma e mostra;
como a dinâmica da expressão gênica pode estar relacionada com a organização;
cromossômica. Os índices utilizados neste estudo foram validados como preditores do;
nível de expressão, caso do CAI para a Candida albicans, e de essencialidade, caso;
do ruído da expressão gênica. O índice de adaptação dos códons (CAI) é válido como;
preditor do nível de ruído bem como da dinâmica da expressão gênica.
The indices that measure the codon adaptation are widely used for predicting;
the expression level of a gene. To verify if: (i) the codon adaptation index (CAI) can be;
used to characterize hypothetical genes in complete genomes; (ii) the variation of gene;
expression within cells (noise) can be used as a quantitative marker for essentiality and;
(iii) what’s the correlation between noise and CAI, this work is divided in two parts. In;
first section we analyzed the indices that measure the codon adaptation as predictors;
of the expression level in hypothetical genes in the yeast Candida albicans. We;
selected 744 hypothetical ORFs that satisfied three characteristic: (i) ORFs longer than;
500bp (bp); (ii) open reading frames not superposed with other ORFs and (iii) ORFs;
with similarity ≥ 60% with Saccharomyces cerevisiae and Schizosaccharomyces;
pombe genes. The codon usage for the hypothetical genes vary a lot between the;
selected ORFs, regardless the index. The CAI vary between 0,044 and 0,540 with;
average 0,170 (±0,051). ORFs with low expression level and that are possibly essential;
are good candidates to be new drug targets. Based on this two characteristics we;
selected two hypothetical ORFs for function studies: CaYdr187c (CAI=0,084) and;
CaYlr339c (CAI=0,088). The ORF CaYdr187c is a possible cell envelope protein, with;
gene ontology of immune response, and prediction of both signal peptide and;
transmembrane regions. Based on the RT-PCR this gene is expressed only in the yeast;
fase. The ORF CaYlr339c is a possible protein involved in translation, with gene;
ontology of immune response, and no prediction of both signal peptide and;
transmembrane regions. Based on the RT-PCR this gene is expressed both in the;
yeast and hypha fases. In the second section, I analyzed the transcription noise and its;
possible use as a classifier of gene essentiality. The analysis of transcriptional temporal;
noise could be an interesting means to study gene expression dynamics and;
stochasticity in eukaryotes. To study the statistical distributions of temporal noise in the;
eukaryotic model system Saccharomyces cerevisiae, we analyzed microarray data;
corresponding to one cell cycle for 6,200 genes. We found that the temporal noise;
follows a lognormal distribution with scale invariance at the genome, chromosomal and;
sub-chromosomal levels. Correlation of temporal noise with the codon adaptation index;
suggests that at least 70% of all protein-coding genes are a noise minimization core of;
the genome. Accordingly, a mathematical model of individual gene expression;
dynamics was proposed, using an operator theoretical approach, which reveals strict;
conditions for noise variability and a possible global noise minimization/optimization;
strategy at the genome level. Our model and data show that minimal noise does not;
correspond to genes obeying a strictly deterministic dynamics. The natural strategy of;
minimization consists in equating the mean of the absolute value of the relative;
variation of the expression level (α) with noise (η). We hypothesize that the temporal;
noise pattern is an emergent property of the genome and shows how the dynamics of;
gene expression could be related to chromosomal organization.
BV UNIFESP: Teses e dissertações
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