A new approach to dengue fatal cases diagnosis: NS1 antigen capture in tissues

Abstract/Background

Dengue is the most important arthropod borne viral disease worldwide in terms of morbidity and mortality and is caused by any of the four serotypes of dengue virus (DENV-1 to 4). Brazil is responsible for approximately 80% of dengue cases in the Americas, and since the introduction of dengue in 1986, a total of 5,944,270 cases have been reported including 21,596 dengue hemorrhagic fever and 874 fatal cases. DENV can infect many cell types and cause diverse clinical and pathological effects. The goal of the study was to investigate the usefulness of NS1 capture tests as an alternative tool to detect DENV in tissue specimens from previously confirmed dengue fatal cases (n = 23) that occurred in 2002 in Brazil.

Methodology/Principal Findings

A total of 74 tissue specimens were available: liver (n = 23), lung (n = 14), kidney (n = 04), brain (n = 10), heart (n = 02), skin (n = 01), spleen (n = 15), thymus (n = 03) and lymph nodes (n = 02). We evaluated three tests for NS1 antigen capture: first generation Dengue Early ELISA (PanBio Diagnostics), Platelia NS1 (BioRad Laboratories) and the rapid test NS1 Ag Strip (BioRad Laboratories). The overall dengue fatal case diagnosis based on the tissues analyzed by Dengue Early ELISA, Platelia NS1 and the NS1 Ag Strip was 34.7% (08/23), 60.8% (14/23) and 91.3% (21/23), respectively. The Dengue Early ELISA detected NS1 in 22.9% (17/74) of the specimens analyzed and the Platelia NS1 in 45.9% (34/74). The highest sensitivity (78.3%; 58/74) was achieved by the NS1 Ag Strip, and the differences in the sensitivities were statistically significant (p<0.05). The NS1 Ag Strip was the most sensitive in liver (91.3%; 21/23), lung (71.4%; 10/14), kidney (100%; 4/4), brain (80%; 8/10), spleen (66.6%, 10/15) and thymus (100%, 3/3) when compared to the other two ELISA assays.

Conclusions/Significance

This study shows the DENV NS1 capture assay as a rapid and valuable approach to postmortem dengue confirmation. With an increasing number of DHF and fatal cases, the availability of new approaches useful for cases confirmation plays an important tool for the disease surveillance.     

Omega-3 Eicosapentaenoic Acid Decreases CD133 Colon Cancer Stem-Like Cell Marker Expression While Increasing Sensitivity to Chemotherapy

Colorectal cancer is the third leading cause of cancer-related death in the western world. In vitro and in vivo experiments showed that omega-3 polyunsaturated fatty acids (n-3 PUFAs) can attenuate the proliferation of cancer cells, including colon cancer, and increase the efficacy of various anticancer drugs. However, these studies address the effects of n-3 PUFAs on the bulk of the tumor cells and not on the undifferentiated colon cancer stem-like cells (CSLCs) that are responsible for tumor formation and maintenance. CSLCs have also been linked to the acquisition of chemotherapy resistance and to tumor relapse. Colon CSLCs have been immunophenotyped using several antibodies against cellular markers including CD133, CD44, EpCAM, and ALDH. Anti-CD133 has been used to isolate a population of colon cancer cells that retains stem cells properties (CSLCs) from both established cell lines and primary cell cultures. We demonstrated that the n-3 PUFA, eicosapentaenoic acid (EPA), was actively incorporated into the membrane lipids of COLO 320 DM cells. 25 uM EPA decreased the cell number of the overall population of cancer cells, but not of the CD133 (+) CSLCs. Also, we observed that EPA induced down-regulation of CD133 expression and up-regulation of colonic epithelium differentiation markers, Cytokeratin 20 (CK20) and Mucin 2 (MUC2). Finally, we demonstrated that EPA increased the sensitivity of COLO 320 DM cells (total population) to both standard-of-care chemotherapies (5-Fluorouracil and oxaliplatin), whereas EPA increased the sensitivity of the CD133 (+) CSLCs to only 5-Fluorouracil.     

Recent Findings in the Chemistry of Odorants from Four Baccharis Species and Their Impact as Chemical Markers

Baccharis is a widespread genus belonging to the Asteraceae family that includes almost 400 species exclusively from the Americas. Even when studied in detail, the taxonomic classification among species from this genus is not yet fully defined. Within the framework of our study of the volatile composition of the Baccharis genus, four species (B. trimera, B. milleflora, B. tridentata, and B. uncinella) were collected from the ‘Campos de Cima da Serra’ highlands of the Brazilian state of Rio Grande do Sul. The aerial parts were dried and extracted by the simultaneous distillation extraction (SDE) procedure. This is the first time that SDE has been applied to obtain and compare the volatile‐extract composition in the Baccharis genus. Characterization of the volatile extracts allowed the identification of 180 peaks with many coeluting components; these latter being detailed for the first time for this genus. The multivariate statistical analyses allowed separating the volatile extracts of the four populations of Baccharis into two separate groups. The first one included the B. milleflora, B. trimera, and B. uncinella volatile extracts. The three species showed a high degree of similarity in their volatile composition, which was characterized by the presence of high contents of sesquiterpene compounds, in particular of spathulenol. The second group comprised the extract of B. tridentata, which contained α‐pinene, β‐pinene, limonene, and (E)‐β‐ocimene in high amounts.     

Genome Editing by CRISPR/Cas9: A Game Change in the Genetic Manipulation of Protists

Genome editing by CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR‐associated gene 9) system has been transformative in biology. Originally discovered as an adaptive prokaryotic immune system, CRISPR/Cas9 has been repurposed for genome editing in a broad range of model organisms, from yeast to mammalian cells. Protist parasites are unicellular organisms producing important human diseases that affect millions of people around the world. For many of these diseases, such as malaria, Chagas disease, leishmaniasis and cryptosporidiosis, there are no effective treatments or vaccines available. The recent adaptation of the CRISPR/Cas9 technology to several protist models will be playing a key role in the functional study of their proteins, in the characterization of their metabolic pathways, and in the understanding of their biology, and will facilitate the search for new chemotherapeutic targets. In this work we review recent studies where the CRISPR/Cas9 system was adapted to protist parasites, particularly to Apicomplexans and trypanosomatids, emphasizing the different molecular strategies used for genome editing of each organism, as well as their advantages. We also discuss the potential usefulness of this technology in the green alga Chlamydomonas reinhardtii.     

Comparative spermatology in Plecoptera (Insecta): an ultrastructural investigation on four species

The ultrastructure of spermatozoa of four Plecoptera species, Leuctra fusca Linnaeus, Brachyptera risi (Morton), Taeniopteryx stankovitchi Ikonomov, T. kuehtreiberi (Aubert), was investigated by light microscopy, scanning and transmission electron microscopy, and immunofluorescence microscopy. The spermatozoa of all the species have a complex acrosome, are filiform and flagellate, with a '9+9+2' axoneme flanked by two mitochondrial derivatives. However, the structure shows a certain interspecific heterogeneity. L. fusca has a short conical nucleus in the apical position and an axoneme flanked by two accessory bodies. In the sperm of the Taeniopterygidae, the nucleus is a long cylindrical body which lies between the two mitochondrial derivatives flanking the axoneme for most of its length. Our results suggest a close phylogenetic affinity between Plecoptera and the other orders of Polyneoptera, although the proposed sister-group relationship between Plecoptera and Embioptera cannot be confirmed.     

DNA-protein flow cytometric analysis in non-Hodgkin lymphoma

A flow cytometric study of DNA and protein contents was performed on cell suspensions obtained from 73 adult patients with non-Hodgkin lymphoma. Bivariate analysis identified a second subpopulation, not revealed by DNA determination, in 25% of the tumors. Protein heterogeneity was more frequently observed in diffuse than in nodular histology according the Rappaport classification and in high-grade than in low-grade malignancy tumors by the Kiel classification and the Working Formulation, but it was not related to ploidy or cell proliferative rate. The presence of an additional subpopulation, detected by protein analysis, defined as monoclonal by DNA analysis, could adversely affect clinical outcome in terms of response to treatment and overall survival.     

Ultrastructure of the seminal vesicle of Phlebotomus perniciosus Newstead (Diptera, Psychodidae)

The seminal vesicles of Phlebotomus perniciosus were investigated by light microscopy, confocal scanning laser microscopy and by scanning and transmission electron microscopy. They have a complex structure, and three different morphological compartments called A, B and C are distinguished on the basis of their position and fine structure. Compartment A is continuous with the vasa deferentia and consists of a cylindrical wall limiting a lumen in which the spermatozoa are stored. Compartment B is hemispherical and surrounds compartment A like a muff. Compartment C constitutes an external coat surrounding A and B. The epithelial cells of each compartment are characterized by morphologically different secretory granules. The ultrastructural features of these cells are described and their role in sandfly reproductive biology is discussed.     

Periods of organogenesis in shoots of Nothofagus dombeyi (Mirb.) oersted (Nothofagaceae)

The organogenetic cycle of main-branch shoots of Nothofagus dombeyi (Nothofagaceae) was studied. Twelve samples of 52-59 parent shoots were collected from a roadside population between September 1999 and October 2000. Variations over time in the number of nodes of terminal and axillary buds, and the length, diameter and number of leaves of shoots derived from these buds (sibling shoots) were analysed. The number of nodes of buds developed by parent shoots was compared with the number of nodes of buds developed, I year later, by sibling shoots. The length, diameter and number of leaves of sibling shoots increased from October 1999 to February 2000 in those shoots with a terminal bud. However, extension of most sibling shoots, including the first five most distal leaf primordia, ceased before February due to abscission of the shoot apex. Axillary buds located most distally on a shoot had more nodes than both terminal buds and more proximal axillary buds. The longest shoots included a preformed part and a neoformed part. The organogenetic event which initiated the neoformed organs continued until early autumn, giving rise to the following year's preformation. The absence of cataphylls in terminal buds could indicate a low intensity of shoot rest. The naked terminal bud of Nothofagus spp. could be interpreted as a structure less specialized than the scaled bud found in genera of Fagaceae and Betulaceae.