Jaime E. Castellanos

Dengue-yellow fever sera cross-reactivity; challenges for diagnosis

OBJECTIVE The Flavivirus genera share epitopes inducing cross-reactive antibodies leading to great difficulty in differentially diagnosing flaviviral infections. This work was aimed at evaluating the complexity of dengue and yellow fever serological differential diagnosis. MATERIAL AND METHODS Dengue antibody capture ELISA and a yellow fever neutralisation test were carried out on 13 serum samples obtained from yellow fever patients, 20 acute serum samples from dengue patients and 19 voluntary serum samples pre- and post-vaccination with YF vaccine. RESULTS Dengue ELISA revealed IgM reactivity in 46,2 % of yellow fever patients and 42 % of vaccinees. Sixteen out of 20 dengue patients (80 %) had high YF virus neutralisation titres. CONCLUSIONS Such very high cross-reactivity data challenged differential laboratory diagnosis of dengue and yellow fever in areas where both flaviviruses co-circulate. New laboratory strategies are thus needed for improving the tests and providing a specific laboratory diagnosis. Cross-reactivity between Flaviviruses represents a great difficulty for epidemiological surveillance and preventing dengue, both of which demand urgent attention.

Easy and inexpensive molecular detection of dengue, chikungunya and zika viruses in febrile patients.

Dengue (DENV), chikungunya (CHIKV) and zika (ZIKV) are arthropod-borne viruses (arboviruses) sharing a common vector, the mosquito Aedes aegypti. At initial stages, patients infected with these viruses have similar clinical manifestations, however, the outcomes and clinical management of these diseases are different, for this reason early and accurate identification of the causative virus is necessary. This paper reports the development of a rapid and specific nested-PCR for detection of DENV, CHIKV and ZIKV infection in the same sample. A set of six outer primers targeting the C-preM, E1, and E gene respectively was used in a multiplex one-step RT-PCR assay, followed by the second round of amplification with specific inner primers for each virus. The specificity of the present assay was validated with positive and negative serum samples for viruses and supernatants of infected cells. The assay was tested using clinical samples from febrile patients. In these samples, we detected mono and dual infections and a case of triple co-infection DENV-CHIKV-ZIKV. This assay might be a useful and an inexpensive tool for detection of these infections in regions where these arboviruses co-circulate.

Identification of proteins from human permanent erupted enamel

Proteins from the extracellular matrix of enamel are highly specific and necessary for proper enamel formation. Most proteins are removed from the matrix by enamel proteases before complete mineralization is achieved; however, some residual protein fragments persist in the mineralized matrix of erupted enamel. So far, only amelogenin peptides obtained by traditional bottom-up proteomics have been recovered and identified in human permanent erupted enamel. In this study, we hypothesize that other enamel-specific proteins are also found in human permanent enamel, by analysing human erupted third molars. Pulverized enamel was used to extract proteins, and the protein extract was subjected directly to liquid-chromatography coupled to tandem mass spectrometry (LC-MS/MS) without a previous trypsin-digestion step. Amelogenin and non-amelogenin proteins (ameloblastin and enamelin) were succesfully identified. The sequences of the naturally occurring peptides of these proteins are reported, finding in particular that most of the peptides from the amelogenin X-isoform come from the tyrosine-rich amelogenin peptide (TRAP) and that some were identified in all specimens. In conclusion, our LC-MS/MS method without trypsin digestion increased the coverage of identification of the enamel proteome from a few amelogenin peptides to a higher number of peptides from three enamel-specific proteins.

Virus del dengue: estructura y ciclo viral

Dengue virus (DENV) is responsible for the clinical entity known as dengue that is a great concern for economy and public health of tropical countries. This flavivirus is a single strand RNA virus that after their translation and replication in host cells produces three structural and seven non-structural proteins with specific function in replication or cell binding process that we will describe here. Intracellular viral cycle has begun to be described and this knowledge will impact the rational design of new antiviral drugs. Patients suffering dengue can have an undifferentiated fever or in the severe cases, show an aberrant immunological activation process that lead to soluble inflammatory mediators secretion, affecting tissue function, mainly endothelium. This organ dysfunction is associated with plasma leakage and coagulatory imbalance. Despite it has been recently described some host factors associated with severity of infection, it remains unknown some aspects of viral biology or the role of DENV proteins in disease pathogenesis. This article pretends to make an updated revision about DENV structure, cell viral cycle and introduce some concepts about dengue immunopathogenesis.

Study of interferon-β antiviral activity against Herpes simplex virus type 1 in neuron-enriched trigeminal ganglia cultures.

Herpes simplex virus type 1 (HSV-1) causes a lytic infection in epithelial cells before being captured and moved via retrograde axonal transport to the nuclei of the sensory neurons of the trigeminal ganglion or dorsal root, where it establishes a latent infection. HSV-1 infection induces an antiviral response through the production of Beta Interferon (IFN-β) in infected trigeminal ganglia. The aim of this work was to characterize the response induced by IFN-β in neuron-enriched trigeminal ganglia primary cultures infected with HSV-1. An antiviral effect of IFN-β in these cultures was observed, including reduced viral production and increased cell survival. In contrast, viral infection significantly decreased both double stranded RNA dependent protein kinase (Pkr) transcription and Jak-1 and Stat-1 phosphorylation, suggesting a possible HSV-1 immune evasion mechanism in trigeminal cells. Additionally, HSV-1 infection upregulated Suppressor of Cytokine Signaling-3 (Socs3) mRNA; upregulation of socs3 was inhibited in IFN-β treated cultures. HSV-1 infection increased the number of Socs3 positive cells and modified the intracellular distribution of Socs3 protein, in infected cells. This neuron-enriched trigeminal ganglia culture model could be used to elucidate the HSV-1 viral cycle in sensory neurons and to study cellular antiviral responses and possible viral evasion mechanisms that underlie the choice between viral replication and latency.

Higher Fluorosis Severity Makes Enamel Less Resistant to Demineralization

Fluorotic teeth could either be more resistant or more susceptible to the caries process than sound ones due to their higher enamel fluoride concentration and higher porosity (subsurface hypomineralization), respectively; however, there is no consensus on this subject. In this study, a total of 49 human unerupted third molars presenting Thylstrup and Fejerskov (TF) fluorosis scores 0-4 were used. Two enamel slabs were obtained from each tooth. The rest of the tooth crown was powdered, and the enamel was separated from the dentine. In purified powdered enamel, the calcium (Ca), inorganic phosphate (Pi), and fluoride (F) concentrations were determined. The F concentration gradient throughout the enamel and in the enamel volume was determined in one slab. The other enamel slab was isolated with acid-resistant varnish, subjecting the exposed enamel surface half to a pH-cycling model to evaluate its demineralization resistance and to calculate the demineralization area. The nonexposed surface was used to determine the natural hypomineralization area found in fluorotic enamel and normalize the demineralization data. The hypomineralization and demineralization areas were assessed by cross-sectional microhardness. For statistical analyses, the data for TF1 and 2, and for TF3 and 4 were pooled. Concentrations of powered enamel Ca and Pi were not significantly different (p > 0.05) among groups TF0, TF1-2 and TF3-4, but a higher F concentration was found in fluorotic enamel (p < 0.05). Highly fluorotic teeth (TF3-4) presented a greater hypomineralization subsurface area and demonstrated lower demineralization resistance than sound enamel (p < 0.05). The findings suggest that a higher severity of fluorosis makes enamel less resistant to the caries process due to its greater subsurface mineral area exposed to demineralization and deeper acid diffusion through the enamel.

Genomic Epidemiology of NDM-1-Encoding Plasmids in Latin American Clinical Isolates Reveals Insights into the Evolution of Multidrug Resistance

Abstract Bacteria that produce the broad-spectrum Carbapenem antibiotic New Delhi Metallo-β-lactamase (NDM) place a burden on health care systems worldwide, due to the limited treatment options for infections caused by them and the rapid global spread of this antibiotic resistance mechanism. Although it is believed that the associated resistance gene blaNDM-1 originated in Acinetobacter spp., the role of Enterobacteriaceae in its dissemination remains unclear. In this study, we used whole genome sequencing to investigate the dissemination dynamics of blaNDM-1-positive plasmids in a set of 21 clinical NDM-1-positive isolates from Colombia and Mexico (Providencia rettgeri, Klebsiella pneumoniae, and Acinetobacter baumannii) as well as six representative NDM-1-positive Escherichia coli transconjugants. Additionally, the plasmids from three representative P. rettgeri isolates were sequenced by PacBio sequencing and finished. Our results demonstrate the presence of previously reported plasmids from K. pneumoniae and A. baumannii in different genetic backgrounds and geographically distant locations in Colombia. Three new previously unclassified plasmids were also identified in P. rettgeri from Colombia and Mexico, plus an interesting genetic link between NDM-1-positive P. rettgeri from distant geographic locations (Canada, Mexico, Colombia, and Israel) without any reported epidemiological links was discovered. Finally, we detected a relationship between plasmids present in P. rettgeri and plasmids from A. baumannii and K. pneumoniae. Overall, our findings suggest a Russian doll model for the dissemination of blaNDM-1 in Latin America, with P. rettgeri playing a central role in this process, and reveal new insights into the evolution and dissemination of plasmids carrying such antibiotic resistance genes.

Fractured reservoirs in the Eastern Foothills, Colombia, and their relationship with fold kinematics

Analysis of fracture systems in subsurface structures is limited by the amount and uncertainty of available data. With the aim of analyzing the distribution of fracture systems, we studied surface structures as analogs for oil fields in the fractured reservoirs of the Llanos foothills of Colombia. Here, we document the presence of four widespread fracture systems whose distribution is related to fold geometry and folding mechanism. At surface, in the Tierranegra and Silbadero anticlines, the principal fracture systems are symmetrical with respect to northeast- and northwest-trending fold axes, showing higher fracture intensities in the forelimbs of the structures. In the Guavio anticline, higher fracture intensities are located in the backlimb, with principal east–west and northwest–southeast directions. In contrast, we document northeast–southwest fractures near the hinge zones in the adjacent synclines. This distribution suggests that in the Guavio anticline, fractures respond to movement of the hanging-wall above a ramp, consistent with a fault-bend-fold model. Whereas, in the Tierranegra and Silbadero anticlines, fractures respond to limb rotation and hinge migration consistent with detachment fold models. Comparing these with subsurface structures, we identified that El Morro anticline has fracture distributions like those in the Tierranegra and Silbadero anticlines, but have higher fracture intensities. In the case of the Cusiana Structure, fracture intensities are higher in the crest but not in the limbs, and intensities differ from the ones found in the Guavio anticline, showing that these structures are not appropriate analogs. The results show how fracture distribution depends on structural position and fold evolution, and is controlled in part by folding mechanism. This suggests that models based on Holocene fold geometry cannot accurately predict the observed fracture distributions and should not be used to construct discrete fracture network models. Instead, the patterns we describe can be used as a guide for similar structures. Our work illustrates the possibility of having different fracture patterns and fracture abundances in adjacent folds in the same fold-thrust belt.

Odontoblast-Like Cells Differentiated from Dental Pulp Stem Cells Retain Their Phenotype after Subcultivation

Odontoblasts, the main cell type in teeth pulp tissue, are not cultivable and they are responsible for the first line of response after dental restauration. Studies on dental materials cytotoxicity and odontoblast cells physiology require large quantity of homogenous cells retaining most of the phenotype characteristics. Odontoblast-like cells (OLC) were differentiated from human dental pulp stem cells using differentiation medium (containing TGF-β1), and OLC expanded after trypsinization (EXP-21) were evaluated and compared. Despite a slower cell growth curve, EXP-21 cells express similarly the odontoblast markers dentinal sialophosphoprotein and dentin matrix protein-1 concomitantly with RUNX2 transcripts and low alkaline phosphatase activity as expected. Both OLC and EXP-21 cells showed similar mineral deposition activity evidenced by alizarin red and von Kossa staining. These results pointed out minor changes in phenotype of subcultured EXP-21 regarding the primarily differentiated OLC, making the subcultivation of these cells a useful strategy to obtain odontoblasts for biocompatibility or cell physiology studies in dentistry.