Molecular detection and identification of zygomycetes species from paraffin-embedded tissues in a murine model of disseminated zygomycosis: a collaborative European Society of Clinical Microbiology and Infectious Diseases (ESCMID) Fungal Infection Study Group (EFISG) evaluation.

The present study was performed to assess the interlaboratory reproducibility of the molecular detection and identification of species of Zygomycetes from formalin-fixed paraffin-embedded kidney and brain tissues obtained from experimentally infected mice. Animals were infected with one of five species (Rhizopus oryzae, Rhizopus microsporus, Lichtheimia corymbifera, Rhizomucor pusillus, and Mucor circinelloides). Samples with 1, 10, or 30 slide cuts of the tissues were prepared from each paraffin block, the sample identities were blinded for analysis, and the samples were mailed to each of seven laboratories for the assessment of sensitivity. A protocol describing the extraction method and the PCR amplification procedure was provided. The internal transcribed spacer 1 (ITS1) region was amplified by PCR with the fungal universal primers ITS1 and ITS2 and sequenced. As negative results were obtained for 93% of the tissue specimens infected by M. circinelloides, the data for this species were excluded from the analysis. Positive PCR results were obtained for 93% (52/56), 89% (50/56), and 27% (15/56) of the samples with 30, 10, and 1 slide cuts, respectively. There were minor differences, depending on the organ tissue, fungal species, and laboratory. Correct species identification was possible for 100% (30 cuts), 98% (10 cuts), and 93% (1 cut) of the cases. With the protocol used in the present study, the interlaboratory reproducibility of ITS sequencing for the identification of major Zygomycetes species from formalin-fixed paraffin-embedded tissues can reach 100%, when enough material is available.

Recent advances and prospective researches on molecular epidemiology of dengue viruses

The determination of amino acid changes in the envelop protein by direct sequencing of either genomic RNA or PCR-amplified cDNA fragments provides useful informations for assessing the genetic variability and the geographic distribution of the actually most widespread dengue-2 serotype. The possible link of variations in the envelope protein-gene and virus virulence is discussed.

Serum uric acid and gout: from the past to molecular biology.

Abstract Objectives: This review will briefly present the epidemiology and risk factors of gout, with a focus on recent advances. Methods: Key papers for inclusion were identified by a PubMed search, and articles were selected according to their relevance for the topic, according to authors' judgment. Results and conclusions: Gout therapy has remained very much unchanged for the last 50 years, but recently we have seen the approval of another gout treatment: the xanthine oxidase inhibitor febuxostat, and several new drugs are now in the late stages of clinical testing. Together with our enhanced level of understanding of the pathophysiology of the inflammatory process involved, we are entering a new era for the treatment of gout.

The fourth transmembrane segment of the Na,K-ATPase alpha subunit: a systematic mutagenesis study.

The Na,K-ATPase is a major ion-motive ATPase of the P-type family responsible for many aspects of cellular homeostasis. To determine the structure of the pathway for cations across the transmembrane portion of the Na,K-ATPase, we mutated 24 residues of the fourth transmembrane segment into cysteine and studied their function and accessibility by exposure to the sulfhydryl reagent 2-aminoethyl-methanethiosulfonate. Accessibility was also examined after treatment with palytoxin, which transforms the Na,K-pump into a cation channel. Of the 24 tested cysteine mutants, seven had no or a much reduced transport function. In particular cysteine mutants of the highly conserved "PEG" motif had a strongly reduced activity. However, most of the non-functional mutants could still be transformed by palytoxin as well as all of the functional mutants. Accessibility, determined as a 2-aminoethyl-methanethiosulfonate-induced reduction of the transport activity or as inhibition of the membrane conductance after palytoxin treatment, was observed for the following positions: Phe(323), Ile(322), Gly(326), Ala(330), Pro(333), Glu(334), and Gly(335). In accordance with a structural model of the Na,K-ATPase obtained by homology modeling with the two published structures of sarcoplasmic and endoplasmic reticulum calcium ATPase (Protein Data Bank codes 1EUL and 1IWO), the results suggest the presence of a cation pathway along the side of the fourth transmembrane segment that faces the space between transmembrane segments 5 and 6. The phenylalanine residue in position 323 has a critical position at the outer mouth of the cation pathway. The residues thought to form the cation binding site II ((333)PEGL) are also part of the accessible wall of the cation pathway opened by palytoxin through the Na,K-pump.

Molecular pathology of colorectal cancer.

Colorectal cancer (CRC) is one of the most intensively studied cancer types, partly because of its high prevalence but also because of the existence of its precursor lesions, tubular or villous adenomas, and more recently (sessile) serrated adenomas, which can be detected endoscopically and removed. The morphological steps in the adenoma-carcinoma sequence have been elucidated at a molecular level, which has been facilitated by identification of the genes responsible for familial intestinal cancer. However, apart from early detection of familial forms of CRC and its use in genetic counseling, until recently such detailed molecular knowledge has had little impact on clinical management of the disease. This has dramatically changed in the last decade. With drugs specifically targeting the epidermal growth factor receptor (EGFR) having been shown effective in CRC, mechanisms responsible for resistance have been explored. The finding that KRAS mutated cancers do not respond to anti-EGFR treatment has had a profound impact on clinical management and on molecular diagnostics of CRC. Additional genetic tests for mutations in NRAS, BRAF and PIK3CA contribute to determining who to treat, and others will follow. New therapies effective in patients with advanced CRC are under investigation. Remaining burning questions for optimal management are which patients will relapse after resection of the primary tumor and which patients will respond to the standard 5FU-oxaliplatin adjuvant treatment regimen. Predictive tests to address these issues are eagerly awaited. New classifications of CRC, based on molecular parameters, are emerging, and we will be confronted with new subtypes of CRC, for which the definition is based on combinations of gene expression patterns, chromosomal alterations, gene mutations and epigenetic characteristics. This will be instrumental in designing new approaches for therapy but will also be translated into molecular diagnostics. Both will contribute to improved clinical management of CRC.

RNA-driven cyclin-dependent kinase regulation: when CDK9/cyclin T subunits of P-TEFb meet their ribonucleoprotein partners.

The positive transcription elongation factor (P-TEFb) consists of CDK9, a cyclin-dependent kinase and its cyclin T partner. It is required for transcription of most class II genes. Its activity is regulated by non-coding RNAs. The 7SK cellular RNA turns the HEXIM cellular protein into a P-TEFb inhibitor that binds its cyclin T subunit. Thus, P-TEFb activity responds to variations in global cellular transcriptional activity and to physiological conditions linked to cell differentiation, proliferation or cardiac hypertrophy. In contrast, the Tat activation region RNA plays an activating role. This feature at the 5' end of the human immunodeficiency (HIV) viral transcript associates with the viral protein Tat that in turn binds cyclin T1 and recruits active P-TEFb to the HIV promoter. This results in enhanced P-TEFb activity, which is critical for an efficient production of viral transcripts. Although discovered recently, the regulation of P-TEFb becomes a paradigm for non-coding RNAs that regulate transcription factors. It is also a unique example of RNA-driven regulation of a cyclindependent kinase.

Molecular characterization and phenotypic expression of mutations in genes for gonadotropins and their receptors in humans.

Gonadotropin hormones undergo important dynamic changes during life. Their rise during puberty stimulates gonadal steroid secretion, triggering the development of secondary sexual characteristics and the acquisition of fertility. The full spectrum of possible mutations and polymorphisms in the human gonadotropins and in their receptor genes has been described in recent years. Patients harboring these mutations display a very wide range of phenotypes affecting all aspects of the reproductive axis. An important insight provided by the careful study of these patients lies in the striking gender differences in the phenotypes associated with a given mutation. As a result, the careful study of these rare patients has allowed us to better define the respective roles of luteinizing hormone and follicle-stimulating hormone in normal human pubertal development and in the achievement of full fertility potential in either males or females. In this work, we describe briefly the known mutations in the genes for both gonadotropins and their receptors, and discuss their genotype/phenotype correlations in light of these important gender differences.

Use of molecular probes and PCR for detection and typing of Leishmania - a mini-review

The use of molecular tools to detect and type Leishmania species in humans, reservoirs or sandflies has been pursued using different approaches. The polymerase chain reaction provided sensitivity to case this task, since the use of hybridization procedures alone employing specifics probes is hampered due to the low detection limit. In this report, we describe the different molecular targets used in our laboratory, aiming at the detection and specific typing of these protozoa. Different kits based on hybridization assays and PCR amplification using kinetoplast and nuclear targets are described and the results obtained from their use are reported.

Transcriptional activation of the utrophin promoter B by a constitutively active Ets-transcription factor.

Duchenne muscular dystrophy is an X-linked genetic disease caused by the absence of functional dystrophin. Pharmacological upregulation of utrophin, the autosomal homologue of dystrophin, offers a potential therapeutic approach to treat Duchenne patients. Full-length utrophin mRNA is transcribed from two alternative promoters, called A and B. In contrast to the utrophin promoter A, little is known about the factors regulating the activity of the utrophin promoter B. Computer analysis of this second promoter revealed the presence of several conserved binding motives for Ets-transcription factors. Using electrotransfer of cDNA into mouse muscles, we demonstrate that a genetically modified beta-subunit of the Ets-transcription factor GA-binding protein potently activates a utrophin promoter B reporter construct in innervated muscle fibers in vivo. These results make the GA-binding protein and the signaling cascade regulating its activity in muscle cells, potential targets for the pharmacological modulation of utrophin expression in Duchenne patients.

Molecular characterization of chromosome termini of the arbuscular mycorrhizal fungus Glomus intraradices (Glomeromycota).

The minimum chromosome number of Glomus intraradices was assessed through cloning and sequencing of the highly divergent telomere-associated sequences (TAS) and by pulsed field gel electrophoresis (PFGE). The telomere of G. intraradices, as in other filamentous fungi, consists of TTAGGG repeats, this was confirmed using Bal31 nuclease time course reactions. Telomere length was estimated to be roughly 0.9 kb by Southern blots on genomic DNA and a telomere probe. We have identified six classes of cloned chromosomal termini based on the TAS. An unusually high genetic variation was observed within two of the six TAS classes. To further assess the total number of chromosome termini, we used telomere fingerprinting. Surprisingly, all hybridization patterns showed smears, which demonstrate that TAS are remarkably variable in the G. intraradices genome. These analyses predict the presence of at least three chromosomes in G. intraradices while PFGE showed a pattern of four bands ranging from 1.2 to 1.5 Mb. Taken together, our results indicate that there are at least four chromosomes in G. intraradices but there are probably more. The information on TAS and telomeres in the G. intradicies will be essential for making a physical map of the G. intraradices genome and could provide molecular markers for future studies of genetic variation among nuclei in these multigenomic fungi.

Functional effects of Na+,K+-ATPase gene mutations linked to familial hemiplegic migraine.

Familial hemiplegic migraine type 2, an autosomal dominant form of migraine with aura, has been associated with four distinct mutations in the alpha2-subunit of the Na+,K+-ATPase. We have introduced these mutations in the alpha2-subunit of the human Na+,K+-ATPase and the corresponding mutations in the Bufo marinus alpha1-subunit and studied these mutants by expression in Xenopus oocyte. Metabolic labeling studies showed that the mutants were synthesized and associated with the beta-subunit, except for the alpha2HW887R mutant, which was poorly synthesized, and the alpha1BW890R, which was partially retained in the endoplasmic reticulum. [3H]ouabain binding showed the presence of the alpha2HR689Q and alpha2HM731T at the membrane, whereas the alpha2HL764P and alpha2HW887R could not be detected. Functional studies with the mutants of the B. marinus Na+,K+-ATPase showed a reduced or abolished electrogenic activity and a low K+ affinity for the alpha1BW890R mutant. Through different mechanisms, all these mutations result in a strong decrease of the functional expression of the Na+,K+-pump. The decreased activity in alpha2 isoform of the Na+,K+-pump expressed in astrocytes seems an essential component of hemiplegic migraine pathogenesis and may be responsible for the cortical spreading depression, which is one of the first events in migraine attacks.

Molecular aspects of Schistosoma mansoni female maturation

Incubation of total protein extracts of Schistosoma mansoni with 3H 17-beta-estradiol and 20-hydroxyecdysone, revealed steroid binding proteins in both, male and female worms. The interaction of nuclear proteins with restriction fragments of the gender and stage-specific gene F-10 was investigated using the "Band-Shift" technique. Distinct male and female nuclear proteins bound to the fragments of this gene. Among the nuclear proteins, only those rich in cysteine residues bound to DNA. In vitro incubation of live worms with the estrogen antagonist Tamoxifen, altered the pattern of the DNA binding proteins, producing in females, a band profile similar to that obtained with male worm protein extracts. When Tamoxifen was injected into schistosome infected mice, the eggs produced by females presented an abnormal morphology, compatible with non-viable eggs. These results suggest that the regulation of transcription of the F-10 gene might involve steroid receptors.

Methicillin-resistant Staphylococcus aureus infection after lung transplantation: 5-year review of clinical and molecular epidemiology.

BACKGROUND: Data on the epidemiology of MRSA infection in lung transplantation is limited. METHODS: We performed a 5-year retrospective study to assess the incidence and microbiologic and clinical characteristics of methicillin-resistant Staphylococcus aureus (MRSA) infection in a cohort of 163 lung transplant recipients. RESULTS: Seventeen patients with MRSA colonization and/or infection were identified, for a calculated incidence rate of 76.1 cases per 1,000 transplanted-years. Pulsed-field gel electrophoresis identified 3 different distinct MRSA profiles, all of them consistent with hospital-associated MRSA infection. CONCLUSION: Despite negative polymerase chain reaction (PCR) for the virulence factor Panton-Valentine leukocidin, MRSA infections resulted in significant disease and morbidity.

A molecular genetic study of the variations in metabolic function during schistosome development

During their complex life cycle schistosomes alternate between the use of stored glycogen and reliance on host glucose to provide for their energy needs. In addition, there is dramatic variation between the relative contribution of aerobic versus anaerobic glucose metabolism during development. We have cloned a set of representative cDNAs that encode proteins involved in glucose uptake, glycolysis, Kreb's cycle and oxidative phosphorylation. The different cDNAs were used as probes to examine the expression of glucose metabolism genes during the schistosome life cycle. Steady state mRNA levels from whole cercariae, isolated cercarial tails, schistosomula and adult worms were analysed on Northern blots and dot blots which were quantified using storage phosphor technology. These studies reveal: (1) Transcripts encoding glycogen metabolic enzymes are expressed to much higher levels in cercarial tails than whole cercariae or schistosomula while the opposite pattern is found for glucose transporters and hexokinase transcripts; (2) Schistosomula contain low levels of transcripts encoding respiratory enzymes but regain the capacity for aerobic glucose metabolism as they mature to adulthood; (3) Male and female adults contain similar levels of the different transcripts involved in glucose metabolism.

Anàlisi genètica i molecular de les malalties Niemann-Pick tipus A/B i tipus C. Estratègies terapèutiques.

Aquest treball es basa en l’estudi de dues malalties lisosòmiques: la malaltia de Niemann-Pick A/B (NPAB) i la malaltia de Niemann-Pick tipus C (NPC). En relació a la malaltia de NPAB, s’ha realitzat l’expressió in vitro d’algunes de les mutacions de canvi d’aminoàcid trobades en pacients espanyols per tal de detectar les activitats enzimàtiques residuals. Totes les mutacions presenten una activitat molt baixa, gairebé nul•la, excepte la p.L225P i la R608del que tenen un 11% i 20% d’activitat respectivament. Els resultats obtinguts són coherents amb la severitat del fenotip que presenten els pacients. D’altra banda, s’ha caracteritzat un al•lel amb una mutació que afecta a una posició poc conservada d’un donador de splicing i que produeix la generació de trànscrits aberrants corresponents a trànscrits minoritaris de SMPD1, prèviament descrits, que no codifiquen per proteïna funcional. Respecte a malaltia de NPC, s’ha realitzat una anàlisi molecular de pacients espanyols prèviament estudiats identificant, en la majoria dels casos, la segona mutació responsable de la patologia. S’ha descrit per primer cop per aquesta malaltia una gran deleció que inclou el gen NPC1 i altres gens flanquejants i s’ha estudiat l’efecte que tenen les mutacions de splicing trobades a nivell de RNA. Per una d’aquestes mutacions, c.1554-1009G&A, s’ha assajat amb èxit una estratègia terapèutica basada en la utilització d’oligonuclèotids antisentit. D’altra banda, s’està desenvolupant un model cel•lular neuronal de la malaltia de Niemann-Pick tipus C, basat en la utilització de RNAs d’interferència, sobre el qual es podran assajar possibles estratègies terapèutiques en un futur.