Indirect Immunodetection of Fungal Fragments by Field Emission Scanning Electron Microscopy

Submicronic fungal fragments have been observed in in vitro aerosolization experiments. The occurrence of these particles has therefore been suggested to contribute to respiratory health problems observed in mold-contaminated indoor environments. However, the role of submicronic fragments in exacerbating adverse health effects has remained unclear due to limitations associated with detection methods. In the present study, we report the development of an indirect immunodetection assay that utilizes chicken polyclonal antibodies developed against spores from Aspergillus versicolor and high-resolution field emission scanning electron microscopy (FESEM). Immunolabeling was performed with A. versicolor fragments immobilized and fixed onto poly-l-lysine-coated polycarbonate filters. Ninety percent of submicronic fragments and 1- to 2-μm fragments, compared to 100% of >2-μm fragments generated from pure freeze-dried mycelial fragments of A. versicolor, were positively labeled. In proof-of-concept experiments, air samples collected from moldy indoor environments were evaluated using the immunolabeling technique. Our results indicated that 13% of the total collected particles were derived from fungi. This fraction comprises 79% of the fragments that were detected by immunolabeling and 21% of the spore particles that were morphologically identified. The methods reported in this study enable the enumeration of fungal particles, including submicronic fragments, in a complex heterogeneous environmental sample.     

Crystal Structure of Human Protein N-Terminal Glutamine Amidohydrolase,an Initial Component of the N-End Rule Pathway

The N-end rule states that half-life of protein is determined by their N-terminal amino acid residue. N-terminal glutamine amidohydrolase (Ntaq) converts N-terminal glutamine to glutamate by eliminating the amine group and plays an essential role in the N-end rule pathway for protein degradation. Here, we report the crystal structure of human Ntaq1 bound with the N-terminus of a symmetry-related Ntaq1 molecule at 1.5 Å resolution. The structure reveals a monomeric globular protein with alpha-beta-alpha three-layer sandwich architecture. The catalytic triad located in the active site, Cys-His-Asp, is highly conserved among Ntaq family and transglutaminases from diverse organisms. The N-terminus of a symmetry-related Ntaq1 molecule bound in the substrate binding cleft and the active site suggest possible substrate binding mode of hNtaq1. Based on our crystal structure of hNtaq1 and docking study with all the tripeptides with N-terminal glutamine, we propose how the peptide backbone recognition patch of hNtaq1 forms nonspecific interactions with N-terminal peptides of substrate proteins. Upon binding of a substrate with N-terminal glutamine, active site catalytic triad mediates the deamination of the N-terminal residue to glutamate by a mechanism analogous to that of cysteine proteases.     

Classification of Chinese herbs based on the cluster analysis of delayed luminescence

Traditional Chinese material medica are an important component of the Chinese pharmacopeia. According to the traditional Chinese medicinal concept, Chinese herbal medicines are classified into different categories based on their therapeutic effects, however, the bioactive principles cannot be solely explained by chemical analysis. The aim of this study is to classify different Chinese herbs based on their therapeutic effects by using delayed luminescence (DL). The DL of 56 Chinese herbs was measured using an ultra‐sensitive luminescence detection system. The different DL parameters were used to classify Chinese herbs according to a hierarchical cluster analysis. The samples were divided into two groups based on their DL kinetic parameters. Interestingly, the DL classification results were quite consistent with classification according to the Chinese medicinal concepts of ‘cold’ and ‘heat’ properties. In this paper, we show for the first time that by using DL technology, it is possible to classify Chinese herbs according to the Chinese medicinal concept and it may even be possible to predict their therapeutic properties. Copyright © 2015 John Wiley & Sons, Ltd.     

Arboreal ants as key predators in tropical lowland rainforest trees

Ants numerically dominate the canopy fauna of tropical lowland rain forests. They are considered to be key predators but their effects in this regard have only rarely been studied on non-myrmecophytes. A conspicuously low abundance of less mobile, mainly holometabolous arthropods like Lepidoptera larvae corresponds with ant dominance, while hemimetabolous highly mobile nymphs occur regularly and in large numbers in the trees. This is in contrast to the temperate regions where ants are mostly lacking on trees and holometabolous larvae are frequent. In this study we experimentally measured ant predation in the trees by offering caterpillars as baits. Fifty-four ant species were tested, of which 46 killed caterpillars and carried them away to their nests while only eight species ignored the offered larvae. Insecticidal knockdown fogging of ten trees after finishing the prey experiments showed that on average 85% of ant individuals per tree were predacious. With the analysis of another 69 foggings and meticulous observations in many other trees this suggests that arboreal ants are responsible for the low abundance of less mobile arthropods in tropical lowland rain forest canopies. Ant predation was significantly lower in a disturbed forest indicating that human disturbance induces a change in the functional interactions in these ecosystems.     

Identification and Localization of a Rickettsia sp. in Bemisia tabaci (Homoptera: Aleyrodidae)

Whiteflies (Homoptera: Aleyrodidae) are sap-sucking insects that harbor “Candidatus Portiera aleyrodidarum,” an obligatory symbiotic bacterium which is housed in a special organ called the bacteriome. These insects are also home for a diverse facultative microbial community which may include Hamiltonella, Arsenophonus, Fritchea, Wolbachia, and Cardinium spp. In this study, the bacteria associated with a B biotype of the sweet potato whitefly Bemisia tabaci were characterized using molecular fingerprinting techniques, and a Rickettsia sp. was detected for the first time in this insect family. Rickettsia sp. distribution, transmission and localization were studied using PCR and fluorescence in situ hybridizations (FISH). Rickettsia was found in all 20 Israeli B. tabaci populations screened but not in all individuals within each population. A FISH analysis of B. tabaci eggs, nymphs, and adults revealed a unique concentration of Rickettsia around the gut and follicle cells, as well as a random distribution in the hemolymph. We postulate that the Rickettsia enters the oocyte together with the bacteriocytes, leaves these symbiont-housing cells when the egg is laid, multiplies and spreads throughout the egg during embryogenesis and, subsequently, disperses throughout the body of the hatching nymph, excluding the bacteriomes. Although the role Rickettsia plays in the biology of the whitefly is currently unknown, the vertical transmission on the one hand and the partial within-population infection on the other suggest a phenotype that is advantageous under certain conditions but may be deleterious enough to prevent fixation under others.     

Use of arboreal and terrestrial space by a small mammal community in a tropical rain forest in Borneo, Malaysia

Aim Small mammals were live‐trapped in a primary rain forest to evaluate the relative distribution of species to each other and to microhabitat properties on the ground and in the canopy. Location Kinabalu National Park in Borneo, Sabah, Malaysia. Methods Seven trapping sessions were conducted along two grids with 31 trap points at distances of 20 m on the ground and in the lower canopy at an average height of 13.5 m. Results Species diversity and abundance of small mammals proved to be high: 20 species of the families Muridae, Sciuridae, Tupaiidae, Hystricidae, Viverridae and Lorisidae were trapped, with murids being dominant in both habitat layers. The terrestrial community was significantly more diverse with 16 captured species (Shannon–Wiener's diversity index = 2.47), while 11 species were trapped in the canopy ( = 1.59). The Whitehead's rat, Maxomys whiteheadi, and the red spiny rat, Maxomys surifer, dominated the terrestrial community whereas the large pencil‐tailed tree mouse, Chiropodomys major, was by far the most abundant species in the canopy. Other abundant species of the canopy community, the dark‐tailed tree rat, Niviventer cremoriventer, and the lesser treeshrew, Tupaia minor, were also abundant on the ground, and there was no clear boundary between arboreal and terrestrial species occurrences. Main conclusions As most species were not confined to specific microhabitats or habitat layers, species seemed to rely on resources not necessarily restricted to certain microhabitats or habitat layers, and separation of species probably resulted mainly from a species’ concentrated activity in a preferred microhabitat rather than from principal adaptations to certain habitats. Ecological segregation was stronger in the more diverse terrestrial community, though microhabitat selection was generally not sufficient to explain the co‐occurrences of species and the variability between local species assemblages. Constraints on small mammal foraging efficiency in the three‐dimensional more complex canopy may be responsible for the similarity of microhabitat use of all common arboreal species. Community composition was characterized by mobile species with low persistence rates, resulting in a high degree of variability in local species assemblages with similar turnover rates in both habitats.     

Challenges in Providing Counselling to MSM in Highly Stigmatized Contexts: Results of a Qualitative Study from Kenya

The role of men who have sex with men (MSM) in the African HIV epidemic is gaining recognition yet capacity to address the HIV prevention needs of this group is limited. HIV testing and counselling is not only a critical entry point for biomedical HIV prevention interventions, such as pre-exposure prophylaxis, rectal microbicides and early treatment initiation, but is also an opportunity for focused risk reduction counselling that can support individuals living in difficult circumstances. For prevention efforts to succeed, however, MSM need to access services and they will only do so if these are non-judgmental, informative, focused on their needs, and of clear benefit. This study aimed to understand Kenyan providers'' attitudes towards and experiences with counselling MSM in a research clinic targeting this group for HIV prevention. We used in-depth interviews to explore values, attitudes and cognitive and social constructs of 13 counsellors and 3 clinicians providing services to MSM at this clinic. Service providers felt that despite their growing experience, more targeted training would have been helpful to improve their effectiveness in MSM-specific risk reduction counselling. They wanted greater familiarity with MSM in Kenya to better understand the root causes of MSM risk-taking (e.g., poverty, sex work, substance abuse, misconceptions about transmission, stigma, and sexual desire) and felt frustrated at the perceived intractability of some of their clients'' issues. In addition, they identified training needs on how to question men about specific risk behaviours, improved strategies for negotiating risk reduction with counselling clients, and improved support supervision from senior counsellors. This paper describes the themes arising from these interviews and makes practical recommendations on training and support supervision systems for nascent MSM HIV prevention programmes in Africa.     

Influence of the hypogaeic army ant<Emphasis Type="Italic"> Dorylus (Dichthadia) laevigatus</Emphasis> on tropical arthropod communities

The majority of army ant species forage hypogaeically. Due to the difficulties in observing these ants, their potential influence on hypogaeic and epigaeic arthropod communities has not yet been investigated. As the first hypogaeically foraging army ant studied in detail, we attracted Dorylus laevigatus to areas monitored for their arthropod diversity. Here, for the first time, the same sites were sampled before and after an army ant raid. Furthermore, interactions between D. laevigatus and the five most common ground-nesting ant species were noted and their life-history traits compared, allowing first inferences on possible mechanisms of their coexistence. The occurrence of D. laevigatus within a study plot had no evident effect on the number of arthropod taxa or individuals collected with epigaeic and hypogaeic pitfall traps. Likewise, juvenile arthropods, which are less mobile and thus are potentially easier prey for D. laevigatus, showed no differences in their collected numbers before and after the army ant had visited a plot. However, significantly fewer ant species were collected with hypogaeic traps after D. laevigatus had been within the study plots, indicating a possible predation of D. laevigatus especially on two Pseudolasius and one Pheidole species. The five most common ground-foraging ant species demonstrated their ability to avoid, kill, and even prey on the army ant. The reaction of Lophomyrmex bedoti towards D. laevigatus indicated the former to be a potential prey species, while Pachycondyla sp. 2 showed signs of "enemy specification." Odontoponera diversus and O. transversa actively preyed on D. laevigatus, while Pheidologeton affinis fought with D. laevigatus over resources. All ant species could co-occur with D. laevigatus at palm oil baits. Adding to the differences detected in previous studies between D. laevigatus and epigaeically foraging army ant species, the occurrence of this hypogaeic army ant seems to have less devastating effects on arthropod community compositions than those of epigaeically mass raiding species.     

Influence of Habitat Fragmentation on the Genetic Variability in Leaf Litter Ant Populations in Tropical Rainforests of Sabah, Borneo

Two ant species, Odontomachus rixosus and Pheidole annexus, were studied in the tropical rainforests of Sabah, Malaysia, North Borneo, to analyze the impact of habitat fragmentation on the genetic diversity and population structure of ant populations using RAPD-fingerprinting. Ants were sampled in a contiguous (43,800 ha) and three patches of primary rainforests of varying size (4294, 146 and 20 ha) that were fragmented about 40 years ago. We found a decrease in genetic variability for both species in the fragmented populations compared to the contiguous. Genetic distances between populations resembled the geographical arrangement of populations and can be explained by an effect of isolation by distance. A high degree in population subdivision suggests a lack of meta-population dynamics due to a shortage of gene flow between populations, possibly the result of the high degree of habitat isolation by oil palm plantations. Although the results of this study are limited due to low replication this is the first data on genetic patterns of insect populations in fragmented rainforests and should be seen as starting point for future research. The value of small to medium sized protection areas for conservation needs to be carefully evaluated in the context of this study, as even relatively large areas (4294 ha) may not prevent the critical loss of genetic variability and guarantee long-term survival of organisms.     

High-throughput fluorescence assay for small-molecule inhibitors of autophagins/Atg4

Autophagy is an evolutionarily conserved process for catabolizing damaged proteins and organelles in a lysosome-dependent manner. Dysregulation of autophagy may cause various diseases, such as cancer and neurodegeneration. However, the relevance of autophagy to diseases remains controversial because of the limited availability of chemical modulators. Herein, the authors developed a fluorescence-based assay for measuring activity of the autophagy protease, autophagin-1(Atg4B). The assay employs a novel reporter substrate of Atg4B composed of a natural substrate (LC3B) fused to an assayable enzyme (PLA(2)) that becomes active upon cleavage by this cysteine protease. A high-throughput screening (HTS) assay was validated with excellent Z' factor (>0.7), remaining robust for more than 5 h and suitable for screening of large chemical libraries. The HTS assay was validated by performing pilot screens with 2 small collections of compounds enriched in bioactive molecules (n = 1280 for Lopac? and 2000 for Spectrum? library), yielding confirmed hit rates of 0.23% and 0.70%, respectively. As counterscreens, PLA(2) and caspase-3 assays were employed to eliminate nonspecific inhibitors. In conclusion, the LC3B-PLA(2) reporter assay provides a platform for compound library screening for identification and characterization of Atg4B-specific inhibitors that may be useful as tools for interrogating the role of autophagy in disease models.