Emerging Microfluidic Devices for Sample Preparation of Undiluted Whole Blood to Enable the Detection of Biomarkers.

Blood testing allows for diagnosis and monitoring of numerous conditions and illnesses; it forms an essential pillar of the health industry that continues to grow in market value. Due to the complex physical and biological nature of blood, samples must be carefully collected and prepared to obtain accurate and reliable analysis results with minimal background signal. Examples of common sample preparation steps include dilutions, plasma separation, cell lysis, and nucleic acid extraction and isolation, which are time-consuming and can introduce risks of sample cross-contamination or pathogen exposure to laboratory staff. Moreover, the reagents and equipment needed can be costly and difficult to obtain in point-of-care or resource-limited settings. Microfluidic devices can perform sample preparation steps in a simpler, faster, and more affordable manner. Devices can be carried to areas that are difficult to access or that do not have the resources necessary. Although many microfluidic devices have been developed in the last 5 years, few were designed for the use of undiluted whole blood as a starting point, which eliminates the need for blood dilution and minimizes blood sample preparation. This review will first provide a short summary on blood properties and blood samples typically used for analysis, before delving into innovative advances in microfluidic devices over the last 5 years that address the hurdles of blood sample preparation. The devices will be categorized by application and the type of blood sample used. The final section focuses on devices for the detection of intracellular nucleic acids, because these require more extensive sample preparation steps, and the challenges involved in adapting this technology and potential improvements are discussed.

Complete functional analysis of type IV pilus components of a reemergent plant pathogen reveals neofunctionalization of paralog genes.

Type IV pilus (TFP) is a multifunctional bacterial structure involved in twitching motility, adhesion, biofilm formation, as well as natural competence. Here, by site-directed mutagenesis and functional analysis, we determined the phenotype conferred by each of the 38 genes known to be required for TFP biosynthesis and regulation in the reemergent plant pathogenic fastidious prokaryote Xylella fastidiosa. This pathogen infects > 650 plant species and causes devastating diseases worldwide in olives, grapes, blueberries, and almonds, among others. This xylem-limited, insect-transmitted pathogen lives constantly under flow conditions and therefore is highly dependent on TFP for host colonization. In addition, TFP-mediated natural transformation is a process that impacts genomic diversity and environmental fitness. Phenotypic characterization of the mutants showed that ten genes were essential for both movement and natural competence. Interestingly, seven sets of paralogs exist, and mutations showed opposing phenotypes, indicating evolutionary neofunctionalization of subunits within TFP. The minor pilin FimT3 was the only protein exclusively required for natural competence. By combining approaches of molecular microbiology, structural biology, and biochemistry, we determined that the minor pilin FimT3 (but not the other two FimT paralogs) is the DNA receptor in TFP of X. fastidiosa and constitutes an example of neofunctionalization. FimT3 is conserved among X. fastidiosa strains and binds DNA non-specifically via an electropositive surface identified by homolog modeling. This protein surface includes two arginine residues that were exchanged with alanine and shown to be involved in DNA binding. Among plant pathogens, fimT3 was found in ~ 10% of the available genomes of the plant associated Xanthomonadaceae family, which are yet to be assessed for natural competence (besides X. fastidiosa). Overall, we highlight here the complex regulation of TFP in X. fastidiosa, providing a blueprint to understand TFP in other bacteria living under flow conditions.

Changes in glycans on platelet microparticles released during storage of apheresis platelets are associated with phosphatidylserine externalization and phagocytosis.

BACKGROUND: Platelets shed platelet microparticles (PMP) when activated or stored. As the removal of sialic acid (desialylation) promotes platelet uptake and clearance from the circulation, similar mechanisms for PMP uptake were hypothesized. The aim of the study was to investigate the role of surface glycans in the in vitro uptake of PMP from stored platelet components. STUDY DESIGN AND METHODS: Apheresis platelet components were stored in 40% plasma/60% SSP+ and sampled on day 1, 5, and 7 post-collection. PMP were characterized by staining with annexin-V (AnV) for phosphatidylserine (PS)-exposure, CD41 antibody, and fluorescently labeled glycan-binding lectins using flow cytometry. The procoagulant function of PMP following desialylation by neuraminidase treatment was assessed by AnV binding and a procoagulant phospholipid assay. PMP were isolated and stained with Deep Red, and phagocytosis by HepG2 cells was measured. Isolated PMP were deglycosylated with neuraminidase and galactosidase to assess the involvement of glycans in mediating phagocytosis. RESULTS: While the overall platelet surface glycan profile was unchanged during storage, PS+ platelets were sialylated, indicating different glycoproteins were changed. In contrast, sialic acid was removed from PS+ and CD41+ PMP, which specifically lost α-2,3-linked sialic acid during platelet storage. PMP were phagocytized by HepG2 cells, and PMP from platelets stored for 7 days were phagocytized to a lesser extent than on day 1. Desialylation by neuraminidase induced PS-exposure on PMP, decreased PPL clotting time, and increased PMP phagocytosis. CONCLUSION: PMP glycans change during platelet storage. Desialylation influences the procoagulant function of PMP and phagocytosis by HepG2 cells.

Host plant use of Helicoverpa spp. (Lepidoptera: Noctuidae) in the Brazilian agricultural landscape.

BACKGROUND: The Old-World bollworm, Helicoverpa armigera (Hübner), was recently documented attacking cotton and soybean plants in Brazil; however, restricted basic knowledge on host plant interactions and landscape use in Brazil have limited the effectiveness of control measures. In this study, we evaluated the suitability of different crops commonly cultivated in Brazil as hosts for H. armigera and H. zea, and examined their contribution to the establishment and size of H. armigera and H. zea field populations. We also estimated the proportions of H. armigera and H. zea moths that used cotton, noncotton C3 plants, and C4 plants as hosts in four regions in Brazil through the length of the cropping season. RESULTS: Viability of H. armigera larvae was highest on cotton (46.1%), followed by millet (39.5%), sorghum (31.2%), soybean (24.2%), and maize (21.1%). Noncotton C3 hosts served as the major source of H. armigera moths in all regions evaluated, and C4 hosts were a source of H. armigera mainly in regions where winter maize is typically cultivated. H. armigera moths that used cotton plants as natal hosts were observed during the reproductive stage of the crop mainly in the state of Bahia. Only C4 host plants were a consistent source of H. zea moths, primarily when maize was in the reproductive stage. H. armigera individuals were the main species infesting cotton and soybean fields while H. zea individuals were the main species infesting maize ears. CONCLUSIONS: Regional differences in the host use and population dynamics of H. armigera among the regions evaluated may be attributed to variation in alternative host utilization (crops, noncrops, and weeds) and the possible occurrence of facultative diapause and or migration.

Overexpression of Citrus reticulata SAMT in Nicotiana tabacum increases MeSA volatilization and decreases Xylella fastidiosa symptoms.

MAIN CONCLUSION: Nicotiana tabacum overexpressing CrSAMT from Citrus reticulata increased production of MeSA, which works as an airborne signal in neighboring wild-type plants, inducing PR1 and increasing resistance to the pathogen Xylella fastidiosa. Xylella fastidiosa is one of the major threats to plant health worldwide, affecting yield in many crops. Despite many efforts, the development of highly productive resistant varieties has been challenging. In studying host plant resistance, the S-adenosyl-L-methionine: salicylic acid carboxyl methyltransferase gene (SAMT) from Citrus reticulata, a X. fastidiosa resistant species, was upregulated in response to pathogen infection. SAMT is involved with the catalysis and production of methyl salicylate (MeSA), an airborne signal responsible for triggering systemic acquired resistance. Here we used tobacco as a model system and generated transgenic plants overexpressing C. reticulata SAMT (CrSAMT). We performed an in silico structural characterization of CrSAMT and investigated its biotechnological potential in modulating the immune system in transgenic plants. The increase of MeSA production in transgenic lines was confirmed by gas chromatography (GC-MS). The transgenic lines showed upregulation of PR1, and their incubation with neighboring wild-type plants activated PR1 expression, indicating that MeSA worked as an airborne signal. In addition, transgenic plants showed significantly fewer symptoms when challenged with X. fastidiosa. Altogether, these data suggest that CrSAMT plays a role in host defense response and can be used in biotechnology approaches to confer resistance against X. fastidiosa.

Hybridization and introgression between Helicoverpa armigera and H. zea: an adaptational bridge.

BACKGROUND: Invasion of organisms into new ecosystems is increasingly common, due to the global trade in commodities. One of the most complex post-invasion scenarios occurs when an invasive species is related to a native pest, and even more so when they can hybridize and produce fertile progeny. The global pest Helicoverpa armigera was first detected in Brazil in 2013 and generated a wave of speculations about the possibility of hybridization with the native sister taxon Helicoverpa zea. In the present study, we used genome-wide single nucleotide polymorphisms from field-collected individuals to estimate hybridization between H. armigera and H. zea in different Brazilian agricultural landscapes. RESULTS: The frequency of hybridization varied from 15 to 30% depending on the statistical analyses. These methods showed more congruence in estimating that hybrids contained approximately 10% mixed ancestry (i.e. introgression) from either species. Hybridization also varied considerably depending on the geographic locations where the sample was collected, forming a 'mosaic' hybrid zone where introgression may be facilitated by environmental and landscape variables. Both landscape composition and bioclimatic variables indicated that maize and soybean cropland are the main factors responsible for high levels of introgression in agricultural landscapes. The impact of multiple H. armigera incursions is reflected in the structured and inbred pattern of genetic diversity. CONCLUSIONS: Our data showed that the landscape composition and bioclimatic variables influence the introgression rate between H. armigera and H. zea in agricultural areas. Continuous monitoring of the hybridization process in the field is necessary, since agricultural expansion, climatic fluctuations, changing composition of crop species and varieties, and dynamic planting seasons are some factors in South America that could cause a sudden alteration in the introgression rate between Helicoverpa species. Introgression between invasive and native pests can dramatically impact the evolution of host ranges and resistance management.

Common Origin of Brazilian and Colombian Populations of the Neotropical Coffee Leaf Miner, Leucoptera coffeella (Lepidoptera: Lyonetiidae).

The coffee leaf miner, Leucoptera coffeella (Guérin-Mèneville & Perrottet, 1842), probably infested coffee plants in Neotropical America during the 19th century. The species subsequently became a key pest of coffee plants in Brazil, but not in Colombia, the two main coffee producers in the region. The contrasting importance of the coffee leaf miner in Brazil and Colombia may be the result of the evolutionary and demographic history of this species. Therefore, our goal was to test two alternative hypotheses regarding the possible genetic origins of this species: 1) leaf miners in both countries share the same origin and 2) the leaf miner arrived in both countries independently from distinct sources and subsequently diversified without genetic exchange between countries. Thus, DNA sequence data of 21 populations were collected (Brazil, 16; Colombia, 5), and partial sequences of their cytochrome oxidase subunit I (COI), cytochrome b (Cytb), and the nuclear internal transcribed spacer (ITS) region were obtained to test these hypotheses. Both nuclear and mitochondrial molecular markers showed low nucleotide diversity. Analyses of molecular variance indicated higher variability within population in both concatenated mitochondrial genes and ITS region (70.57 and 84.01%, respectively). Finally, geno/haplotype networks showed each central geno/haplotypes that displayed high frequency and were distributed widely in both countries. Low-frequency geno/haplotypes were at tip positions connected to the central geno/haplotypes through single mutation steps, suggesting that the Neotropical coffee leaf miner in both Brazil and Colombia consists of a single species and exhibits a common and recent genetic origin.