A Genetic Screen to Isolate Toxoplasma gondii Host-cell Egress Mutants

The widespread, obligate intracellular, protozoan parasite Toxoplasma gondii causes opportunistic disease in immuno-compromised patients and causes birth defects upon congenital infection. The lytic replication cycle is characterized by three stages: 1. active invasion of a nucleated host cell; 2. replication inside the host cell; 3. active egress from the host cell. The mechanism of egress is increasingly being appreciated as a unique, highly regulated process, which is still poorly understood at the molecular level. The signaling pathways underlying egress have been characterized through the use of pharmacological agents acting on different aspects of the pathways^1-5. As such, several independent triggers of egress have been identified which all converge on the release of intracellular Ca²⁺, a signal that is also critical for host cell invasion^6-8. This insight informed a candidate gene approach which led to the identification of plant like calcium dependent protein kinase (CDPK) involved in egress⁹. In addition, several recent breakthroughs in understanding egress have been made using (chemical) genetic approaches^10-12. To combine the wealth of pharmacological information with the increasing genetic accessibility of Toxoplasma we recently established a screen permitting the enrichment for parasite mutants with a defect in host cell egress¹³. Although chemical mutagenesis using N-ethyl-N-nitrosourea (ENU) or ethyl methanesulfonate (EMS) has been used for decades in the study of Toxoplasma biology^11,14,15, only recently has genetic mapping of mutations underlying the phenotypes become routine^16-18. Furthermore, by generating temperature-sensitive mutants, essential processes can be dissected and the underlying genes directly identified. These mutants behave as wild-type under the permissive temperature (35 °C), but fail to proliferate at the restrictive temperature (40 °C) as a result of the mutation in question. Here we illustrate a new phenotypic screening method to isolate mutants with a temperature-sensitive egress phenotype¹³. The challenge for egress screens is to separate egressed from non-egressed parasites, which is complicated by fast re-invasion and general stickiness of the parasites to host cells. A previously established egress screen was based on a cumbersome series of biotinylation steps to separate intracellular from extracellular parasites¹¹. This method also did not generate conditional mutants resulting in weak phenotypes. The method described here overcomes the strong attachment of egressing parasites by including a glycan competitor, dextran sulfate (DS), that prevents parasites from sticking to the host cell¹⁹. Moreover, extracellular parasites are specifically killed off by pyrrolidine dithiocarbamate (PDTC), which leaves intracellular parasites unharmed²⁰. Therefore, with a new phenotypic screen to specifically isolate parasite mutants with defects in induced egress, the power of genetics can now be fully deployed to unravel the molecular mechanisms underlying host cell egress.     

Population Changes of Heterodera glycines and Soybean Yields Resulting from Soil Treatment with Alachlor,Fenamiphos, and Ethoprop

The population dynamics of Heterodera glycines as influenced by alachlor, fenamiphos, and ethoprop alone and in herbicide-nematicide combinations were studied in the field. Numbers of H. glycines juveniles and eggs were higher at midseason and harvest where nematicides were applied. Fenamiphos alone or in combination with alachlor provided better control of H. glycines and greater seed yields than treatments with ethoprop. Numbers of H. glycines eggs at harvest in 1980 were positively correlated with numbers of juveniles at planting in 1981 and negatively related to seed yield in 1981.     

Diet of the Indochinese silvered langur (Trachypithecus germaini) in Kien Luong Karst area,Kien Giang Province

The Indochinese silvered langur (Trachypithecus germaini) is distributed to the west of Mekong River in Cambodia, Lao PDR, Thailand and Vietnam. During a two‐year study, from May 2014 to May 2016, we collected 320.44 hr of behavior, with 17,040 feeding bouts recorded (142 hr) for T. germaini on Chua Hang Karst Mountain, Kien Luong District, Kien Giang Province, Vietnam. Feeding accounted for 45% of the Indochinese silvered langurs’ activity budget. The plant diet of the Indochinese silvered langurs was principally composed of young leaves (58%), followed by mature leaves (9.5%), fruits (22.7%), flowers (4.7%), buds (3.3%), petioles (1.2%), and other (0.5%). A total of 58 plant species were fed on by the silvered langurs, and leaves of eight species (Phyllathus reticulatus, Ficus rumphii, Ficus tinctoria, Ficus microcarpa, Cayratia trifolia, Streblus ilicifolia, Combretum latifolium, and Streblus asper) were fed on throughout the year. P. reticulatus was most frequently eaten (13.9% feeding time, n = 1,733). Food selection differed significantly between months and seasons. The Indochinese silvered langurs ate 27 plant species in the wet season compared with 23 plant species in the dry season. Leaf chemical composition of two food categories, 16 eaten species (with 10 most frequently consumed species and six least consumed species), and four noneaten species, were analyzed. Feeding samples from eaten species in the Indochinese silvered langurs's diet contained lower amounts of condensed tannin, lignin, protein, ash, and lipids, but a higher amount of total sugar compared with samples from noneaten species. Furthermore, the most frequently consumed species contained lower amounts of lignin compared with the less frequently consumed species. Using a generalized linear model with five variables, including neutral detergent fiber (NDF), total sugar, lignin, lipid, and calcium (Ca) indicated that NDF positively correlated and lignin content negatively correlated with feeding records in the diet of these langur.     

Normal serum biochemical and hematological parameters in Macaca fascicularis.

The effects of age, sex, pregnancy, were analyzed and data from fasted and fed animals were compared in a population of cynomolgus macaques. No significant sex effects were observed for biochemical values and no changes were found in male hematological parameters in relation to age. Most values of females during pregnancy were within normal ranges. Comparison between fed and fasted animals showed that several biochemical parameters (e.g., ALT, glucose, CPK, LDH) and several hematological parameters (e.g., monocytes, eosinophils, basophils, hemoglobin, MCV, MCHC, and MCH) were affected by food intake.     

The relative importance of lethal and non-lethal effects of fish on insect colonisation of ponds

1. We hypothesised that adult insects actively monitor potential habitats for the presence of fish by means of chemical cues and avoid sites that pose significant risks. This was examined by quantifying colonisation of insects in outdoor pools with no fish (controls), fish (direct predation effect) or caged fish (chemical predator cues).
2. A significant direct effect of predation was found, but no indirect effect (avoidance of chemical cue pools), on the total biomass of colonising insects. However, predatory insects avoided fish-cue pools, thus releasing non-predatory insects from predation. This resulted in significantly greater biomass of non-predatory insects in fish-cue pools than control pools.
3. Fish reduced the number of species of colonising insects in pools through predation. This negative influence of fish implies that caution is necessary when stocking wetlands and ponds with fish if the goal is to maximise biodiversity.
4. Our data suggest that although predatory aquatic insects may use chemical signals to assess the quality of potential habitats with respect to predation risk, direct predation is the main method by which fish affect insect assemblages in ponds. Because fish and invertebrate predators may both have strong effects on prey mortality, behavioural adjustment by insects to the actual predator regime within a habitat should be more important than avoiding colonisation of habitats with fish.     

NAD hydrolysis: Chemical and enzymatic mechanisms

The pyridine nucleotide have important non-redox activities as cellular effectors and metabolic regulators [1–3]. The enzyme-catalyzed cleavage of the nicotinamide-ribosyl bond of NAD⁺ and the attendant delivery of the ADPRibosyl moiety to acceptors is central to these many diverse biological activities. Included are the medically important NAD-dependent toxins associated with cholera, diphtheria, pertussis, and related disease [4]; the reversible ADPRibosylation-mediated biological regulatory systems [5,6]; the synthesis of poly (ADPRibose) in response to DNA damage or cellular, division [7]; and the synthesis of cyclic ADPRibose as part of an independent, calcium-mediated regulatory system[8]. As will be presented in this chapter, all evidence points to both the chemical and enzyme-catalyzed cleavage of the nicotinamide-ribosyl bond being dissociative in character via an oxocarbenium intermediate.     

Hard-sphere/pseudo-particle modelling (HS-PPM) for efficient and scalable molecular simulation of dilute gaseous flow and transport

Continuum methods are not accurate enough for flows at high Knudsen numbers, whereas rigorous molecular dynamics (MD) methods are too costly for simulations at practical dimensions. Hard-sphere (HS) model is a simplified MD method efficient for dilute gaseous flow but is of poor parallelism due to its event-driven nature, which sets a strong limitation to its large-scale applications. In this work, pseudo-particle modelling, a time-driven modelling approach is coupled with HS model to construct a scalable parallel method capable of simulating flows and transport processes at high Knudsen numbers without losing necessary molecular details in describing their macro-scale behaviours. The method is validated in several classical simulation cases and its performance is evaluated to be favourable. To demonstrate the potential applications of this method, we also simulate the diffusion of small molecules in multi-scale porous media which is related to catalysis, material preparation and micro chemical engineering in the long term.