Beta-adaptin: key molecule for microglial scavenger receptor function under oxidative stress

Scavenger receptors internalize chemically modified low density lipoprotein particles (ac-LDL) and other ligands through the process of receptor-mediated endocytosis. During this investigation using amyloid-beta as a natural ligand for the SR, we studied under a ligand-induced oxidative stress condition, changes in protein expression of several adaptor proteins important in the organization of the endocytic machinery in microglia and macrophages. Differential expression experiments of beta-adaptin, alpha-adaptin, SR-AI, and SR-BI in RAW (macrophages) and EOC (microglia) cells were performed according to dosage and exposure time to amyloid-beta. Our results show that according to dosage, amyloid-beta produces an oxidative stress state that importantly affects the availability of beta-adaptin. Under these conditions, RT-PCR assays show that beta-adaptin mRNA is normally synthesized, reason why protein translation or protein structure of beta-adaptin might be altered. These observations might have impact in the understanding of the mechanisms microglia employ to process amyloid-beta in the brain.     

Simultaneous detection of the release of glutamate and nitric oxide from adherently growing cells using an array of glutamate and nitric oxide selective electrodes

The simultaneous detection of nitric oxide and glutamate using an array of individually addressable electrodes, in which the individual electrodes in the array were suitably modified with a highly sensitive nitric oxide sensing chemistry or a glutamate oxidase/redox hydrogel-based glutamate biosensor is presented. In a sequence of modification steps one of the electrodes was covered first with a positively charged Ni porphyrin entrapped into a negatively charged electrodeposition paint followed by the manual modification of the second working electrode by a bienzyme sensor architecture based on crosslinked redox hydrogels with entrapped peroxidase and glutamate oxidase. Adherently growing C6-glioma cells were grown on membrane inserts and placed in close distance to the modified sensor surfaces. The current responses recorded at each electrode after stimulation of glutamate and NO release by means of K+ and bradykinin clearly demonstrate the ability of the individual electrode in the array to detect the analyte towards which its sensitivity and selectivity was targeted without interference from the neighbouring electrode or other analytes present in the test mixture.     

Selection on a behaviour‐related gene during the first stages of the biological invasion pathway

Human‐induced biological invasions are common worldwide and often have negative impacts on wildlife and human societies. Several studies have shown evidence for selection on invaders after introduction to the new range. However, selective processes already acting prior to introduction have been largely neglected. Here, we tested whether such early selection acts on known behaviour‐related gene variants in the yellow‐crowned bishop (Euplectes afer), a pet‐traded African songbird. We tested for nonrandom allele frequency changes after trapping, acclimation and survival in captivity. We also compared the native source population with two independent invasive populations. Allele frequencies of two SNPs in the dopamine receptor D4 (DRD4) gene—known to be linked to behavioural activity in response to novelty in this species—significantly changed over all early invasion stages. They also differed between the African native population and the two invading European populations. The two‐locus genotype associated with reduced activity declined consistently, but strongest at the trapping stage. Overall genetic diversity did not substantially decrease, and there is little evidence for new alleles in the introduced populations, indicating that selection at the DRD4 gene predominantly worked on the standing genetic variation already present in the native population. Our study demonstrates selection on a behaviour‐related gene during the first stages of a biological invasion. Thus, pre‐establishment stages of a biological invasion do not only determine the number of propagules that are introduced (their quantity), but also their phenotypic and genetic characteristics (their quality).     

Characteristics of a Dengue Outbreak in a Remote Pacific Island Chain – Republic of the Marshall Islands, 2011–2012

Dengue is a potentially fatal acute febrile illness caused by four mosquito-transmitted dengue viruses (DENV-1–4). Although dengue outbreaks regularly occur in many regions of the Pacific, little is known about dengue in the Republic of the Marshall Islands (RMI). To better understand dengue in RMI, we investigated an explosive outbreak that began in October 2011. Suspected cases were reported to the Ministry of Health, serum specimens were tested with a dengue rapid diagnostic test (RDT), and confirmatory testing was performed using RT-PCR and IgM ELISA. Laboratory-positive cases were defined by detection of DENV nonstructural protein 1 by RDT, DENV nucleic acid by RT-PCR, or anti-DENV IgM antibody by RDT or ELISA. Secondary infection was defined by detection of anti-DENV IgG antibody by ELISA in a laboratory-positive acute specimen. During the four months of the outbreak, 1,603 suspected dengue cases (3% of the RMI population) were reported. Of 867 (54%) laboratory-positive cases, 209 (24%) had dengue with warning signs, six (0.7%) had severe dengue, and none died. Dengue incidence was highest in residents of Majuro and individuals aged 10–29 years, and ∼95% of dengue cases were experiencing secondary infection. Only DENV-4 was detected by RT-PCR, which phylogenetic analysis demonstrated was most closely related to a virus previously identified in Southeast Asia. Cases of vertical DENV transmission, and DENV/Salmonella Typhi and DENV/Mycobacterium leprae co-infection were identified. Entomological surveys implicated water storage containers and discarded tires as the most important development sites for Aedes aegypti and Ae. albopictus, respectively. Although this is the first documented dengue outbreak in RMI, the age groups of cases and high prevalence of secondary infection demonstrate prior DENV circulation. Dengue surveillance should continue to be strengthened in RMI and throughout the Pacific to identify and rapidly respond to future outbreaks.     

Synergistic and additive interactions among components of food-based baits underlie female fruit fly attraction

Attraction of tephritid fruit flies to some food sources can be enhanced by the presence of ammonia derivatives, compounds that are perceived as volatile cues for protein-rich food sources. Using a comparative approach, we (1) evaluated the behavioral responses of females of three invasive fruit fly species, Bactrocera (Zeugodacus) cucurbitae (Coquillett), Bactrocera dorsalis (Hendel), and Ceratitis capitata (Wiedemann) (all Diptera: Tephritidae) to trub (a protein-rich waste brewer’s yeast product generated during the production of beer), Concord grape juice (a protein-deficient material), and ammonium acetate, and (2) identified synergistic and additive interactions between low- and high-attractiveness materials and ammonium acetate. We established the attractiveness of fresh trub, grape juice, and ammonium acetate when tested singly to females of all three fly species. Although ammonium acetate did not enhance significantly the response of females of any species to fresh trub, the most attractive material, ammonium acetate, did significantly enhance females’ level of response to aged trub (a comparatively less attractive material) and to grape juice. Our research found a synergistic interaction between diluted grape juice and ammonium acetate for B. cucurbitae, as well as between aged trub and ammonium acetate for B. dorsalis. For C. capitata, additive effects among food attractants and ammonium acetate were identified. Our findings increase our understanding of fruit fly female olfactory-driven behavior in response to food-based materials and the extent to which ammonium acetate modulates female response to protein-rich and protein-deficient materials.     

Culture-free genome-wide locus sequence typing (GLST) provides new perspectives on Trypanosoma cruzi dispersal and infection complexity

Analysis of genetic polymorphism is a powerful tool for epidemiological surveillance and research. Powerful inference from pathogen genetic variation, however, is often restrained by limited access to representative target DNA, especially in the study of obligate parasitic species for which ex vivo culture is resource-intensive or bias-prone. Modern sequence capture methods enable pathogen genetic variation to be analyzed directly from host/vector material but are often too complex and expensive for resource-poor settings where infectious diseases prevail. This study proposes a simple, cost-effective ‘genome-wide locus sequence typing’ (GLST) tool based on massive parallel amplification of information hotspots throughout the target pathogen genome. The multiplexed polymerase chain reaction amplifies hundreds of different, user-defined genetic targets in a single reaction tube, and subsequent agarose gel-based clean-up and barcoding completes library preparation at under 4 USD per sample. Our study generates a flexible GLST primer panel design workflow for Trypanosoma cruzi, the parasitic agent of Chagas disease. We successfully apply our 203-target GLST panel to direct, culture-free metagenomic extracts from triatomine vectors containing a minimum of 3.69 pg/μl T. cruzi DNA and further elaborate on method performance by sequencing GLST libraries from T. cruzi reference clones representing discrete typing units (DTUs) TcI, TcIII, TcIV, TcV and TcVI. The 780 SNP sites we identify in the sample set repeatably distinguish parasites infecting sympatric vectors and detect correlations between genetic and geographic distances at regional (< 150 km) as well as continental scales. The markers also clearly separate TcI, TcIII, TcIV and TcV + TcVI and appear to distinguish multiclonal infections within TcI. We discuss the advantages, limitations and prospects of our method across a spectrum of epidemiological research.     

Secondary metabolites from two species of Pulicaria and their cytotoxic activity

Two new compounds, the sesquiterpene (1E,5E)-8β-acetoxy-4α-hydroxy-7βH-germacra-1(10),5-dien-14-oic acid (2), and a nor-sesquiterpene, (5E)-8β-acetoxy-4α-hydroxy-7βH-germacr-5-en-10-one (3), were isolated from Pulicaria canariensis ssp. lanata, along with ten known compounds, including the flavonoid 5,3'-dihydroxy-3,7,4'-trimethoxyflavone (4). From Pulicaria burchardii, we isolated seven known compounds; the physical and spectroscopic data of the triterpenoid 3β-hydroxytaraxaster-20-en-30-al (1) are reported. The structures of compounds 1-3 were determined on the basis of HR-MS, and 1D- and 2D-NMR studies. The structure of 2 was corroborated by X-ray crystal diffraction. Cell viability experiments revealed that the semisynthetic flavonoid 4b was the most cytotoxic compound against human leukemia cells, and the cytotoxicity was caused by induction of apoptosis, as determined by microscopy of nuclear changes.     

Towards the production of fungal biocontrol candidates using inert supports: a case of study of Trichoderma asperellum in a pilot fixed bed fermenter

The production of fungal biocontrol agents by solid-state fermentation (SSF) processes in inert supports demands deeper studies in SSF-modelling and SSF-optimisation to cope with scale-up issues. Here, we report the systematic application of fractional factorial and central composite designs to optimise the conidia productivity and maximum specific growth rate of the biological control candidate Trichoderma asperellum strain Th204 using two inert supports: polyurethane foam (PUF) and rice husk (RH), in a pilot 16 L fixed bed fermenter. By using response surface methodology, 2D contour graphs and Spearman’s correlation coefficients, axial temperature, conidia concentration, bed moisture and pressure drop gradients were modelled. C:N ratio and airflow rate were identified as significant factors. Optimal conditions using PUF a C:N ratio of 18.1 and airflow of 0.8?m³?h^?1 were found, with the highest productivity of 3.09?×?10⁷conidia g^?1 initial dry matter h¹. Polynomial models and response surfaces found in this study are advantageous to design strategies to scale-up the SSF process in fixed bed fermenters for fungal biological control candidates.     

Mycobacterium tuberculosis alters the differentiation of monocytes into macrophages in vitro

This paper shows that in vitro infection of human monocytes by Mycobacterium tuberculosis affected monocyte to macrophage differentiation. Despite the low bacterial load used, M. tuberculosis-infected monocytes had fewer granules, displayed a reduced number of cytoplasmic projections and decreased HLA class II, CD68, CD86 and CD36 expression compared to cells differentiated in the absence of mycobacteria. Infected cells produced less IL-12p70, TNF-α, IL-10, IL-6 and high IL-1β in response to lipopolysaccharide and purified protein M. tuberculosis-derived. Reduced T-cell proliferative response and IFN-γ secretion in response to phytohemagglutinin and culture filtrate proteins from M. tuberculosis was also observed in infected cells when compared to non-infected ones. The ability of monocytes differentiated in the presence of M. tuberculosis to control mycobacterial growth in response to IFN-γ stimulation was attenuated, as determined by bacterial plate count; however, they had a similar ability to uptake fluorescent M. tuberculosis and latex beads compared to non-infected cells. Recombinant IL-1β partially altered monocyte differentiation into macrophages; however, treating M. tuberculosis-infected monocytes with IL-1RA did not reverse the effects of infection during differentiation. The results indicated that M. tuberculosis infection altered monocyte differentiation into macrophages and affected their ability to respond to innate stimuli and activate T-cells.