Expanding the DNA alphabet in the fruit fly

DNA integrity is under the control of multiple pathways of nucleotide metabolism and DNA damage recognition and repair. Unusual sets of protein factors involved in these control mechanisms may result in tolerance and accumulation of non-canonical bases within the DNA. We investigate the presence of uracil in genomic DNA of Drosophila melanogaster. Results indicate a developmental pattern and strong correlations between uracil-DNA levels, dUTPase expression and developmental fate of different tissues. The intriguing lack of the catalytically most efficient uracil-DNA glycosylase in Drosophila melanogaster may be a general attribute of Holometabola and is suggested to be involved in the specific characteristics of uracil-DNA metabolism in these insects.     

Mechanisms Involved in the Nociception Triggered by the Venom of the Armed Spider Phoneutria nigriventer

Background

The frequency of accidental spider bites in Brazil is growing, and poisoning due to bites from the spider genus Phoneutria nigriventer is the second most frequent source of such accidents. Intense local pain is the major symptom reported after bites of P. nigriventer, although the mechanisms involved are still poorly understood. Therefore, the aim of this study was to identify the mechanisms involved in nociception triggered by the venom of Phoneutria nigriventer (PNV).

Methodology/Principal Findings

Twenty microliters of PNV or PBS was injected into the mouse paw (intraplantar, i.pl.). The time spent licking the injected paw was considered indicative of the level of nociception. I.pl. injection of PNV produced spontaneous nociception, which was reduced by arachnid antivenin (ArAv), local anaesthetics, opioids, acetaminophen and dipyrone, but not indomethacin. Boiling or dialysing the venom reduced the nociception induced by the venom. PNV-induced nociception is not dependent on glutamate or histamine receptors or on mast cell degranulation, but it is mediated by the stimulation of sensory fibres that contain serotonin 4 (5-HT₄) and vanilloid receptors (TRPV1). We detected a kallikrein-like kinin-generating enzyme activity in tissue treated with PNV, which also contributes to nociception. Inhibition of enzymatic activity or administration of a receptor antagonist for kinin B₂ was able to inhibit the nociception induced by PNV. PNV nociception was also reduced by the blockade of tetrodotoxin-sensitive Na⁺ channels, acid-sensitive ion channels (ASIC) and TRPV1 receptors.

Conclusion/Significance

Results suggest that both low- and high-molecular-weight toxins of PNV produce spontaneous nociception through direct or indirect action of kinin B₂, TRPV1, 5-HT₄ or ASIC receptors and voltage-dependent sodium channels present in sensory neurons but not in mast cells. Understanding the mechanisms involved in nociception caused by PNV are of interest not only for better treating poisoning by P. nigriventer but also appreciating the diversity of targets triggered by PNV toxins.     

Symptomatic Versus Inapparent Outcome in Repeat Dengue Virus Infections Is Influenced by the Time Interval between Infections and Study Year

Four dengue virus serotypes (DENV1-4) circulate globally, causing more human illness than any other arthropod-borne virus. Dengue can present as a range of clinical manifestations from undifferentiated fever to Dengue Fever to severe, life-threatening syndromes. However, most DENV infections are inapparent. Yet, little is known about determinants of inapparent versus symptomatic DENV infection outcome. Here, we analyzed over 2,000 DENV infections from 2004 to 2011 in a prospective pediatric cohort study in Managua, Nicaragua. Symptomatic cases were captured at the study health center, and paired healthy annual samples were examined on a yearly basis using serological methods to identify inapparent DENV infections. Overall, inapparent and symptomatic DENV infections were equally distributed by sex. The mean age of infection was 1.2 years higher for symptomatic DENV infections as compared to inapparent infections. Although inapparent versus symptomatic outcome did not differ by infection number (first, second or third/post-second DENV infections), substantial variation in the proportion of symptomatic DENV infections among all DENV infections was observed across study years. In participants with repeat DENV infections, the time interval between a first inapparent DENV infection and a second inapparent infection was significantly shorter than the interval between a first inapparent and a second symptomatic infection. This difference was not observed in subsequent infections. This result was confirmed using two different serological techniques that measure total anti-DENV antibodies and serotype-specific neutralizing antibodies, respectively. Taken together, these findings show that, in this study, age, study year and time interval between consecutive DENV infections influence inapparent versus symptomatic infection outcome, while sex and infection number had no significant effect. Moreover, these results suggest that the window of cross-protection induced by a first infection with DENV against a second symptomatic infection is approximately 2 years. These findings are important for modeling dengue epidemics and development of vaccines.     

A high-throughput media design approach for high performance mammalian fed-batch cultures

An innovative high-throughput medium development method based on media blending was successfully used to improve the performance of a Chinese hamster ovary fed-batch medium in shaking 96-deepwell plates. Starting from a proprietary chemically-defined medium, 16 formulations testing 43 of 47 components at 3 different levels were designed. Media blending was performed following a custom-made mixture design of experiments considering binary blends, resulting in 376 different blends that were tested during both cell expansion and fed-batch production phases in one single experiment. Three approaches were chosen to provide the best output of the large amount of data obtained. A simple ranking of conditions was first used as a quick approach to select new formulations with promising features. Then, prediction of the best mixes was done to maximize both growth and titer using the Design Expert software. Finally, a multivariate analysis enabled identification of individual potential critical components for further optimization. Applying this high-throughput method on a fed-batch, rather than on a simple batch, process opens new perspectives for medium and feed development that enables identification of an optimized process in a short time frame.     

Spatial Assessment of Amphibian Chytrid Fungus (Batrachochytrium dendrobatidis) in South Africa Confirms Endemic and Widespread Infection

Chytridiomycosis has been identified as a major cause of global amphibian declines. Despite widespread evidence of Batrachochytrium dendrobatidis infection in South African frogs, sampling for this disease has not focused on threatened species, or whether this pathogen poses a disease risk to these species. This study assessed the occurrence of Bd-infection in South African Red List species. In addition, all known records of infection from South Africa were used to model the ecological niche of Bd to provide a better understanding of spatial patterns and associated disease risk. Presence and prevalence of Bd was determined through quantitative real-time PCR of 360 skin swab samples from 17 threatened species from 38 sites across the country. Average prevalence was 14.8% for threatened species, with pathogen load varying considerably between species. MaxEnt was used to model the predicted distribution of Bd based on 683 positive records for South Africa. The resultant probability threshold map indicated that Bd is largely restricted to the wet eastern and coastal regions of South Africa. A lack of observed adverse impacts on wild threatened populations supports the endemic pathogen hypothesis for southern Africa. However, all threatened species occur within the limits of the predicted distribution for Bd, exposing them to potential Bd-associated risk factors. Predicting pathogen distribution patterns and potential impact is increasingly important for prioritising research and guiding management decisions.     

Virological Efficacy in Cerebrospinal Fluid and Neurocognitive Status in Patients with Long-Term Monotherapy Based on Lopinavir/Ritonavir: An Exploratory Study

Background

Data on suppression of HIV replication in the CNS and on the subsequent risk of neurocognitive impairment using monotherapy with boosted protease inhibitors are limited.

Methods

Ours was an exploratory cross-sectional study in patients on lopinavir/ritonavir-based monotherapy (LPV/r-MT) or standard triple therapy (LPV/r-ART) for at least 96 weeks who maintained a plasma viral load <50 copies/mL. HIV-1 RNA in CSF was determined by HIV-1 SuperLow assay (lower limit of detection, 1 copy/mL). Neurocognitive functioning was assessed using a recommended battery of neuropsychological tests covering 7 areas. Neurocognitive impairment (NCI) was determined and also a global deficit score (GDS) for study comparisons.

Results

Seventeen patients on LPV/r-MT and 17 on LPV/r-ART were included. Fourteen (82.4%) patients on LPV/r-MT and 16 (94.1%) on LPV/r-ART had HIV-1 RNA <1 copy/mL in CSF (p = 0.601). NCI was observed in 7 patients on LPV/r-MT and in 10 on LPV/r-ART (41% vs 59%; p = 0.494). Mean (SD) GDS was 0.22 (0.20) in patients on LPV/r-MT and 0.47 (0.34) in those on LPV/r-ART (p = 0.012).

Conclusions

Suppression of HIV in CSF is similar in individuals with durable plasma HIV-1 RNA suppression who are receiving LPV/r-MT or LPV/r-ART for at least 96 weeks. Findings for HIV-1 replication in CSF and neurocognitive status indicate that this strategy seems to be safe for CNS functioning.     

A Simple and Fast Non-Radioactive Bridging Immunoassay for Insulin Autoantibodies

Type 1 diabetes (T1D) is an autoimmune disease which results from the destruction of pancreatic beta cells. Autoantibodies directed against islet antigens are valuable diagnostic tools. Insulin autoantibodies (IAAs) are usually the first to appear and also the most difficult to detect amongst the four major islet autoantibodies. A non-radioactive IAA bridging ELISA was developed to this end. In this assay, one site of the IAAs from serum samples is bound to a hapten-labeled insulin (GC300-insulin), which is subsequently captured on anti-GC300 antibody-coated 96-well plates. The other site of the IAAs is bound to biotinylated insulin, allowing the complex to be detected by an enzyme-streptavidin conjugate. In the present study, 50 serum samples from patients with newly diagnosed T1D and 100 control sera from non-diabetic individuals were analyzed with our new assay and the results were correlated with an IAA radioimmunoassay (RIA). Using IAA bridging ELISA, IAAs were detected in 32 out of 50 T1D children, whereas with IAA RIA, 41 out of 50 children with newly diagnosed T1D were scored as positive. In conclusion, the IAA bridging ELISA could serve as an attractive approach for rapid and automated detection of IAAs in T1D patients for diagnostic purposes.     

Pistil Starch Reserves at Anthesis Correlate with Final Flower Fate in Avocado (Persea americana)

A common observation in different plant species is a massive abscission of flowers and fruitlets even after adequate pollination, but little is known as to the reason for this drop. Previous research has shown the importance of nutritive reserves accumulated in the flower on fertilization success and initial fruit development but direct evidence has been elusive. Avocado (Persea americana) is an extreme case of a species with a very low fruit to flower ratio. In this work, the implications of starch content in the avocado flower on the subsequent fruit set are explored. Firstly, starch content in individual ovaries was analysed from two populations of flowers with a different fruit set capacity showing that the flowers from the population that resulted in a higher percentage of fruit set contained significantly more starch. Secondly, in a different set of flowers, the style of each flower was excised one day after pollination, once the pollen tubes had reached the base of the style, and individually fixed for starch content analysis under the microscope once the fate of its corresponding ovary (that remained in the tree) was known. A high variability in starch content in the style was found among flowers, with some flowers having starch content up to 1,000 times higher than others, and the flowers that successfully developed into fruits presented significantly higher starch content in the style at anthesis than those that abscised. The relationship between starch content in the ovary and the capacity of set of the flower together with the correlation found between the starch content in the style and the fate of the ovary support the hypothesis that the carbohydrate reserves accumulated in the flower at anthesis are related to subsequent abscission or retention of the developing fruit.