Collagenase mRNA Overexpression and Decreased Extracellular Matrix Components Are Early Events in the Pathogenesis of Emphysema

To describe the progression of parenchymal remodeling and metalloproteinases gene expression in earlier stages of emphysema, mice received porcine pancreatic elastase (PPE) instillation and Control groups received saline solution. After PPE instillation (1, 3, 6 hours, 3 and 21 days) we measured the mean linear intercept, the volume proportion of types I and III collagen, elastin, fibrillin and the MMP-1, -8, -12 and -13 gene expression. We observed an initial decrease in type I (at the 3rd day) and type III collagen (from the 6th hour until the 3rd day), in posterior time points in which we detected increased gene expression for MMP-8 and -13 in PPE groups. After 21 days, the type III collagen fibers increased and the type I collagen values returned to similar values compared to control groups. The MMP-12 gene expression was increased in earlier times (3 and 6 hours) to which we detected a reduced proportion of elastin (3 days) in PPE groups, reinforcing the already established importance of MMP-12 in the breakdown of ECM. Such findings will be useful to better elucidate the alterations in ECM components and the importance of not only metalloelastase but also collagenases in earlier emphysema stages, providing new clues to novel therapeutic targets.     

Surface Plasmon Resonance Imaging Sensors: A Review

Surface plasmon resonance (SPR) imaging sensors realize label-free, real-time, highly sensitive, quantitative, high-throughput biological interaction monitoring and the binding profiles from multi-analytes further provide the binding kinetic parameters between different biomolecules. In the past two decades, SPR imaging sensors found rapid increasing applications in fundamental biological studies, medical diagnostics, drug discovery, food safety, precision measurement, and environmental monitoring. In this paper, we review the recent advances of SPR imaging sensor technology towards high-throughput multi-analyte screening. Finally, we describe our multiplex spectral-phase SPR imaging biosensor for high-throughput biosensing applications.     

A systematic bioinformatics approach for selection of epitope-based vaccine targets

Epitope-based vaccines provide a new strategy for prophylactic and therapeutic application of pathogen-specific immunity. A critical requirement of this strategy is the identification and selection of T-cell epitopes that act as vaccine targets. This study describes current methodologies for the selection process, with dengue virus as a model system. A combination of publicly available bioinformatics algorithms and computational tools are used to screen and select antigen sequences as potential T-cell epitopes of supertype human leukocyte antigen (HLA) alleles. The selected sequences are tested for biological function by their activation of T-cells of HLA transgenic mice and of pathogen infected subjects. This approach provides an experimental basis for the design of pathogen specific, T-cell epitope-based vaccines that are targeted to majority of the genetic variants of the pathogen, and are effective for a broad range of differences in human leukocyte antigens among the global human population.     

Spectroscopic characterization and photobleaching kinetics of hypericin-N-methyl pyrrolidone formulations.

Hypericin (HY) is a promising photosensitizer in photodynamic therapy (PDT). It was recently reported that appropriate use of N-methyl pyrrolidone (NMP) enhanced in vivo PDT efficacy of HY and enhanced in vivo delivery of HY. This present study further investigates the use of NMP and other known non-toxic pharmaceutical additives, polyvinylpyrrolidone (PVP, K29/32) and copolyvidonum (S630), for formulating HY to enhance its delivery with photodynamic activity as a goal in mind. Hence, the first objective of this study was to characterize the solubilization of HY by NMP, K29/32 and S630. Thermodynamic considerations were used to explain the solvation process. Photobleaching is another important property of photosensitizers. There is no report on the photostability of HY in pharmaceutical formulations used for PDT. Therefore, the second objective of this study was to investigate the photobleaching of HY in these formulations. The fluorescence of HY was found to increase significantly in higher concentrations of NMP or when 5% of polymer was co-mixed with 5% of NMP solution. The photobleaching of HY in these formulations followed first-order kinetics. The loss of fluorescence paralleled to the loss of absorption of HY. The formulation of HY with 40% NMP was found to be the most stable.     

Insights into amebiasis using a human 3D‐intestinal model

Entamoeba histolytica is the causative agent of amebiasis, an infectious disease targeting the intestine and the liver in humans. Two types of intestinal infection are caused by this parasite: silent infection, which occurs in the majority of cases, and invasive disease, which affects 10% of infected persons. To understand the intestinal pathogenic process, several in vitro models, such as cell cultures, human tissue explants or human intestine xenografts in mice, have been employed. Nevertheless, our knowledge on the early steps of amebic intestinal infection and the molecules involved during human–parasite interaction is scarce, in part due to limitations in the experimental settings. In the present work, we took advantage of tissue engineering approaches to build a three‐dimensional (3D)‐intestinal model that is able to replicate the general characteristics of the human colon. This system consists of an epithelial layer that develops tight and adherens junctions, a mucus layer and a lamina propria‐like compartment made up of collagen containing macrophages and fibroblast. By means of microscopy imaging, omics assays and the evaluation of immune responses, we show a very dynamic interaction between E. histolytica and the 3D‐intestinal model. Our data highlight the importance of several virulence markers occurring in patients or in experimental models, but they also demonstrate the involvement of under described molecules and regulatory factors in the amoebic invasive process.     

Characterization of within-host Plasmodium falciparum diversity using next-generation sequence data

Our understanding of the composition of multi-clonal malarial infections and the epidemiological factors which shape their diversity remain poorly understood. Traditionally within-host diversity has been defined in terms of the multiplicity of infection (MOI) derived by PCR-based genotyping. Massively parallel, single molecule sequencing technologies now enable individual read counts to be derived on genome-wide datasets facilitating the development of new statistical approaches to describe within-host diversity. In this class of measures the F_WS metric characterizes within-host diversity and its relationship to population level diversity. Utilizing P. falciparum field isolates from patients in West Africa we here explore the relationship between the traditional MOI and F_WS approaches. F_WS statistics were derived from read count data at 86,158 SNPs in 64 samples sequenced on the Illumina GA platform. MOI estimates were derived by PCR at the msp-1 and -2 loci. Significant correlations were observed between the two measures, particularly with the msp-1 locus (P = 5.92×10⁻⁵). The F_WS metric should be more robust than the PCR-based approach owing to reduced sensitivity to potential locus-specific artifacts. Furthermore the F_WS metric captures information on a range of parameters which influence out-crossing risk including the number of clones (MOI), their relative proportions and genetic divergence. This approach should provide novel insights into the factors which correlate with, and shape within-host diversity.     

West Nile virus T-cell ligand sequences shared with other flaviviruses: a multitude of variant sequences as potential altered peptide ligands

Phylogenetic relatedness and cocirculation of several major human pathogen flaviviruses are recognized as a possible cause of deleterious immune responses to mixed infection or immunization and call for a greater understanding of the inter-Flavivirus protein homologies. This study focused on the identification of human leukocyte antigen (HLA)-restricted West Nile virus (WNV) T-cell ligands and characterization of their distribution in reported sequence data of WNV and other flaviviruses. H-2-deficient mice transgenic for either A2, A24, B7, DR2, DR3, or DR4 HLA alleles were immunized with overlapping peptides of the WNV proteome, and peptide-specific T-cell activation was measured by gamma interferon (IFN-γ) enzyme-linked immunosorbent spot (ELISpot) assays. Approximately 30% (137) of the WNV proteome peptides were identified as HLA-restricted T-cell ligands. The majority of these ligands were conserved in ～≥88% of analyzed WNV sequences. Notably, only 51 were WNV specific, and the remaining 86, chiefly of E, NS3, and NS5, shared an identity of nine or more consecutive amino acids with sequences of 64 other flaviviruses, including several major human pathogens. Many of the shared ligands had an incidence of >50% in the analyzed sequences of one or more of six major flaviviruses. The multitude of WNV sequences shared with other flaviviruses as interspecies variants highlights the possible hazard of defective T-cell activation by altered peptide ligands in the event of dual exposure to WNV and other flaviviruses, by either infection or immunization. The data suggest the possible preferred use of sequences that are pathogen specific with minimum interspecies sequence homology for the design of Flavivirus vaccines.     

Analysis of viral diversity for vaccine target discovery

Background

Viral vaccine target discovery requires understanding the diversity of both the virus and the human immune system. The readily available and rapidly growing pool of viral sequence data in the public domain enable the identification and characterization of immune targets relevant to adaptive immunity. A systematic bioinformatics approach is necessary to facilitate the analysis of such large datasets for selection of potential candidate vaccine targets.

Results

This work describes a computational methodology to achieve this analysis, with data of dengue, West Nile, hepatitis A, HIV-1, and influenza A viruses as examples. Our methodology has been implemented as an analytical pipeline that brings significant advancement to the field of reverse vaccinology, enabling systematic screening of known sequence data in nature for identification of vaccine targets. This includes key steps (i) comprehensive and extensive collection of sequence data of viral proteomes (the virome), (ii) data cleaning, (iii) large-scale sequence alignments, (iv) peptide entropy analysis, (v) intra- and inter-species variation analysis of conserved sequences, including human homology analysis, and (vi) functional and immunological relevance analysis.

Conclusion

These steps are combined into the pipeline ensuring that a more refined process, as compared to a simple evolutionary conservation analysis, will facilitate a better selection of vaccine targets and their prioritization for subsequent experimental validation.

     

Surface‐enhanced Raman scattering‐active photonic crystal fiber probe: Towards next generation liquid biopsy sensor with ultra high sensitivity

The tremendous enhancement factors that surface‐enhanced Raman scattering (SERS) possesses coupled with the flexibility of photonic crystal fibers (PCFs) pave the way to a new generation of ultrasensitive biosensors. Thanks to the unique structure of PCFs, which allows direct incorporation of an analyte into the axially aligned air channels, interaction between the analyte and excitation light could be increased many folds leading to flexible, reliable and sensitive probes that can be used in preclinical or clinical biosensing. SERS‐active PCF probes provide unique opportunity to develop an opto‐fluidic liquid biopsy needle sensor that enables one‐step integrated sample collection and testing for disease diagnosis. Specificity being a key parameter to biosensors, the PCF inside the biopsy needle could be functionalized with targeting moieties to detect specific biomarkers. In this review article, we present some of the most promising recent biosensors based on PCFs including hollow‐core PCFs, suspended‐core PCFs and side‐channel PCFs. We provide a wide range of applications of such platform using Raman spectroscopy, label free SERS or labeled SERS detection and analyze some of the main challenges to be addressed for translating it to a clinically viable next generation sensitive biopsy needle sensing probe.