A highly reusable genosensor for late-life depression diagnosis based on microRNA 184 attomolar detection in human plasma.

Late-Life Depression (LLD) is one of the most prevalent psychiatric disorders in elderly, causing significant functional impairments. MicroRNAs are small molecules involved in the post-transcriptional regulation of gene expression. Elderly individuals diagnosed with LLD present down regulation of miR-184 (hsa-miR-184) expression compared to healthy patients. Therefore, this miR-184 can be used as a biomarker to diagnose LLD. Current LLD diagnosis depends primarily on clinical subjective identification, based on symptoms and variable scales. This work introduces a novel and facile approach for the LLD diagnosis based on the development of an electrochemical genosensor for miR-184 detection in plasma, using differential pulse voltammetry (DPV) and electrochemical impedance spectroscopy (EIS). DPV results presented a 2-Fold increase in current value for healthy patients, compared to individuals with LLD when monitoring ethidium bromide oxidation peak. For EIS, a 1.5-fold increase in charge transfer resistance for healthy elderly subjects was observed in comparison with depressed patients. In addition, the analytical performance of the biosensor was evaluated using DPV, obtaining a linear response ranging from 10-9 mol L-1 to 10-17 mol L-1 of miR-184 in plasma and a detection limit of 10 atomoles L-1. The biosensor presented reusability, selectivity and stability, the current response remained 72% up to 50 days of storage. Thus, the genosensor proved to be efficient in the diagnosis of LLD, as well as the accurate quantification of miR-184 in real plasma samples of healthy and depressed patients.

Performance of glutamate dehydrogenase and triose phosphate isomerase genes in the analysis of genotypic variability of isolates of Giardia duodenalis from livestocks.

Giardia duodenalis is a small intestinal protozoan parasite of several terrestrial vertebrates. This work aims to assess the genotypic variability of Giardia duodenalis isolates from cattle, sheep and pigs in the Southeast of Brazil, by comparing the standard characterization between glutamate dehydrogenase (gdh) and triose phosphate isomerase (tpi) primers. Fecal samples from the three groups of animals were analyzed using the zinc sulphate centrifugal flotation technique. Out of 59 positive samples, 30 were from cattle, 26 from sheep and 3 from pigs. Cyst pellets were stored and submitted to PCR and nested-PCR reactions with gdh and tpi primers. Fragment amplification of gdh and tpi genes was observed in 25 (42.4%) and 36 (61.0%) samples, respectively. Regarding the sequencing, 24 sequences were obtained with gdh and 20 with tpi. For both genes, there was a prevalence of E specific species assemblage, although some isolates have been identified as A and B, by the tpi sequencing. This has also shown a larger number of heterogeneous sequences, which have been attribute to mixed infections between assemblages B and E. The largest variability of inter-assemblage associated to the frequency of heterogeneity provided by tpi sequencing reinforces the polymorphic nature of this gene and makes it an excellent target for studies on molecular epidemiology.

Evidence for reductive genome evolution and lateral acquisition of virulence functions in two Corynebacterium pseudotuberculosis strains.

BACKGROUND: Corynebacterium pseudotuberculosis, a gram-positive, facultative intracellular pathogen, is the etiologic agent of the disease known as caseous lymphadenitis (CL). CL mainly affects small ruminants, such as goats and sheep; it also causes infections in humans, though rarely. This species is distributed worldwide, but it has the most serious economic impact in Oceania, Africa and South America. Although C. pseudotuberculosis causes major health and productivity problems for livestock, little is known about the molecular basis of its pathogenicity. METHODOLOGY AND FINDINGS: We characterized two C. pseudotuberculosis genomes (Cp1002, isolated from goats; and CpC231, isolated from sheep). Analysis of the predicted genomes showed high similarity in genomic architecture, gene content and genetic order. When C. pseudotuberculosis was compared with other Corynebacterium species, it became evident that this pathogenic species has lost numerous genes, resulting in one of the smallest genomes in the genus. Other differences that could be part of the adaptation to pathogenicity include a lower GC content, of about 52%, and a reduced gene repertoire. The C. pseudotuberculosis genome also includes seven putative pathogenicity islands, which contain several classical virulence factors, including genes for fimbrial subunits, adhesion factors, iron uptake and secreted toxins. Additionally, all of the virulence factors in the islands have characteristics that indicate horizontal transfer. CONCLUSIONS: These particular genome characteristics of C. pseudotuberculosis, as well as its acquired virulence factors in pathogenicity islands, provide evidence of its lifestyle and of the pathogenicity pathways used by this pathogen in the infection process. All genomes cited in this study are available in the NCBI Genbank database (http://www.ncbi.nlm.nih.gov/genbank/) under accession numbers CP001809 and CP001829.

Biomarkers for serum diagnosis of infectious diseases and their potential application in novel sensor platforms.

Nanotechnological tools and biomarkers for diagnosis and prognosis, as well as strategies for disease control and monitoring populations at higher risk, are continuous worldwide challenges for infectious diseases. Phage display and monoclonal antibody combinatorial libraries are important sources for biomarker discovery and for improved diagnostic strategies. Mimetic peptides were selected against polyclonal antibodies from patients with dengue fever, leprosy, and leishmaniasis as model diseases, and from immunized chickens with total antigens from all three pathogens. Selected single or combined multi-epitope peptide biomarkers were further associated with four different sensor platforms, classified as affinity biosensors, that may be suitable as general protocols for field diagnosis. We have also developed two methods for nanoparticle agglutination assays (a particle gel agglutination test and a magnetic microparticle [MMP]-enzyme-linked immunosorbent assay [ELISA]) and two electrochemical biosensors (impedimetric and amperometric) for DNA and antibody detection. For the agglutination tests, micro- and nanoparticles were coupled with filamentous bacteriophages displaying the selected mimotopes on their surfaces, which has favored the formation of the antigen-antibody or peptide-protein complexes, amplifying the optical detection in ELISA assays or after the chromatographic separation of the microagglutinates. We have also demonstrated a proof-of-concept for the electrochemical biosensors by using electrodes modified with novel functionalized polymers. These electrochemical biosensors have proven to be fast, very sensitive, and specific for the detection of pathogen DNA and circulating antibodies of patients, which may become important in a wide range of diagnostic devices for many infectious agents.

Detection of all four human metapneumovirus subtypes in nasopharyngeal specimens from children with respiratory disease in Uberlândia, Brazil.

The human metapneumovirus (hMPV) is a pathogen of the respiratory tract identified first in the Netherlands in 2001 and since then it has been detected worldwide. The purpose of this study was to identify and characterize hMPV in samples collected from children <5 years presenting with acute respiratory disease (ARD) seen at a public hospital in Uberlândia, in Southeastern Brazil. One hundred fourteen nasopharyngeal aspirates (NPAs) samples that were negative for the presence of nine other respiratory viruses were tested by reverse transcription polymerase chain reaction (RT-PCR) for the presence of hMPV RNA. Fourteen out of 114 (12.3%) samples were positive for presence of hMPV RNA. PCR products, obtained by the amplification of partial nucleotide sequence of gene N, were sequenced and compared with sequences deposited in GenBank. Sequences from eight samples were obtained and all four subtypes were identified. Also, the recently proposed sublineages "a" and "b" of subtype A2 were found; mean age was 21 months old; upper respiratory tract infection (URTI) was the most common clinical symptom; the virus was detected in samples collected from March to November, a period that corresponds to late summer to mid-spring in Brazil. This is the first study to describe the circulation of all hMPV subtypes in Minas Gerais state.

Analysis of the intercaste transcriptional profile of Melipona scutellaris Latreille, 1811 (Hymenoptera, Apidae, Meliponini) by mRNA differential display.

In colonies of Melipona scutellaris Latreille, 1811 workers can be found with four ganglion nerve cells, a morphological characteristic of the queen. It is hypothesized that these workers, called intercastes, or phenocopies, are phenotypically-like workers, but genotypically identical to queens due to this specific trait. Workers with the same number of ganglion as queens seem to be intercastes between queens and workers. Our objective was to analyze the mRNA pro files of workers, queens, and intercastes of M. scutellaris through DDRT-PCR. Three hundred (300) pupae with white eyes were collected and externally identified according to the number of abdominal nerve ganglions: workers (5 ganglions), queens (4 ganglions) and intercastes (4 ganglions). The analysis identified differentially expressed transcripts that were present only in workers, but absent in intercastes and queens, confirming the hypothesis, by demonstrating the environmental effect on the queen genotype that generated phenotype-like workers.

Analysis of the intercaste transcriptional profile of Melipona scutellaris Latreille, 1811 (Hymenoptera, Apidae, Meliponini) by mRNA differential display

In colonies of Melipona scutellaris Latreille, 1811 workers can be found with four ganglion nerve cells, a morphological characteristic of the queen. It is hypothesized that these workers, called intercastes, or phenocopies, are phenotypically-like workers, but genotypically identical to queens due to this specific trait. Workers with the same number of ganglion as queens seem to be intercastes between queens and workers. Our objective was to analyze the mRNA pro files of workers, queens, and intercastes of M. scutellaris through DDRT-PCR. Three hundred (300) pupae with white eyes were collected and externally identified according to the number of abdominal nerve ganglions: workers (5 ganglions), queens (4 ganglions) and intercastes (4 ganglions). The analysis identified differentially expressed transcripts that were present only in workers, but absent in intercastes and queens, confirming the hypothesis, by demonstrating the environmental effect on the queen genotype that generated phenotype-like workers.

Farnesoic acid O-methyl transferase (FAMeT) isoforms: conserved traits and gene expression patterns related to caste differentiation in the stingless bee, Melipona scutellaris.

Farnesoic acid O-methyl transferase (FAMeT) is the enzyme that catalyzes the formation of methyl farnesoate (MF) from farnesoic acid (FA) in the biosynthetic pathway of juvenile hormone (JH). This work reports the cloning, sequencing, and expression of FAMeT gene from the stingless bee Melipona scutellaris (MsFAMeT). The MsFAMeT in silico analysis showed that greatest sequence similarity is found in Apis mellifera and other insects, while relatively less similarity is shown in crustaceans. Evidence of alternative splicing of a 27 nucleotide (nt) microexon explains the presence of the detected isoforms, 1 and 2. The expression analysis of the two isoforms showed a marked difference when castes were compared, suggesting that they could be involved differently in the JH metabolism in M. scutellaris, providing new insights for the comprehension of female plasticity.

Differentially displayed expressed sequence tags in Melipona scutellaris (Hymenoptera, Apidae, Meliponini) development.

We have compared gene expression, using the Differential Display Reverse Transcriptase-Polymerase Chain Reaction (DDRT-PCR) technique, by means of mRNA profile in Melipona scutellaris during ontogenetic postembryonic development, in adult worker, and in both Natural and Juvenile Hormone III-induced adult queen. Six, out of the nine ESTs described here, presented differentially expressed in the phases L1 or L2, or even in both of them, suggesting that key mechanisms to the development of Melipona scutellaris are regulated in these stages. The combination HT11G-AP05 revealed in L1 and L2 a product which matches to thioredoxin reductase protein domain in the Clostridium sporogenes, an important protein during cellular oxidoreduction processes. This study represents the first molecular evidence of differential gene expression profiles toward a description of the genetic developmental traits in the genus Melipona.

Differentially displayed expressed sequence tags in Melipona scutellaris (Hymenoptera, Apidae, Meliponini) development

Nesse estudo nós usamos a técnica de Differential Display Reverse Transcriptase - Polymerase Chain Reaction (DDRT-PCR) para comparamos o perfil de mRNA em Melipona scutellaris durante o desenvolvimento ontogenético pós-embrionário e em operárias adultas, rainha natural e induzida pelo Hormônio Juvenil III. Fragmentos diferencialmente expressos foram detectados usando as seguintes combinações de primers: HT11G-AP05; HT11C-AP05; HT11G-OPF12; HT11G-OPA16. Dos 9 ESTs descrito nesse trabalho, 6 tiveram expressão diferencial nas fases de larva L1 e L2, sugerindo serem mecanismos chave no regulação do desenvolvimento larval em Melipona. A combinação HT11G-AP05 revelou em L1 e L2 um produto com similaridade à proteína tioredoxina redutase de Clostridium sporogenes, uma proteína importante durante os processos de oxidoredução. Esse estudo representa as primeiras evidências moleculares do perfil de expressão durante o desenvolvimento ontogenético em abelhas do gênero Melipona.