The highly diverged trypanosomal MICOS complex is organized in a nonessential integral membrane and an essential peripheral module

The mitochondrial contact site and cristae organization system (MICOS) mediates the formation of cristae, invaginations in the mitochondrial inner membrane. The highly diverged MICOS complex of the parasitic protist Trypanosoma brucei consists of nine subunits. Except for two Mic10‐like and a Mic60‐like protein, all subunits are specific for kinetoplastids. Here, we determined on a proteome‐wide scale how ablation of individual MICOS subunits affects the levels of the other subunits. The results reveal co‐regulation of TbMic10‐1, TbMic10‐2, TbMic16 and TbMic60, suggesting that these nonessential, integral inner membrane proteins form an interdependent network. Moreover, the ablation of TbMic34 and TbMic32 reveals another network consisting of the essential, intermembrane space‐localized TbMic20, TbMic32, TbMic34 and TbMic40, all of which are peripherally associated with the inner membrane. The downregulation of TbMic20, TbMic32 and TbMic34 also interferes with mitochondrial protein import and reduces the size of the TbMic10‐containing complexes. Thus, the diverged MICOS of trypanosomes contains two subcomplexes: a nonessential membrane‐integrated one, organized around the conserved Mic10 and Mic60, that mediates cristae formation, and an essential membrane‐peripheral one consisting of four kinetoplastid‐specific subunits, that is required for import of intermembrane space proteins.     

Characterization of the NADH:ubiquinone oxidoreductase (complex I) in the trypanosomatid Phytomonas serpens (Kinetoplastida)

NADH dehydrogenase activity was characterized in the mitochondrial lysates of Phytomonas serpens, a trypanosomatid flagellate parasitizing plants. Two different high molecular weight NADH dehydrogenases were characterized by native PAGE and detected by direct in-gel activity staining. The association of NADH dehydrogenase activities with two distinct multisubunit complexes was revealed in the second dimension performed under denaturing conditions. One subunit present in both complexes cross-reacted with the antibody against the 39 kDa subunit of bovine complex I. Out of several subunits analyzed by MS, one contained a domain characteristic for the LYR family subunit of the NADH:ubiquinone oxidoreductases. Spectrophotometric measurement of the NADH:ubiquinone 10 and NADH:ferricyanide dehydrogenase activities revealed their different sensitivities to rotenone, piericidin, and diphenyl iodonium.     

Knowledge,attitudes, and practices regarding schistosomiasis infection and prevention: A mixed-methods study among endemic communities of western Uganda

IntroductionIn Uganda, schistosomiasis (re)infections have continued to remain high despite the implementation of mass drug administration and sensitization campaigns aimed at controlling the disease. This could imply that there are some barriers to the implemented preventive measures. We conducted a mixed-methods study in Kagadi and Ntoroko districts around Lake Albert to assess knowledge, attitudes, and practices regarding schistosomiasis and to explore and understand perspectives regarding the disease.Materials and methodsSemi-structured survey questionnaires were administered to 337 household adults selected through systematic random sampling. We also interviewed 12 participants and held 28 focus-group discussion sessions with 251 individuals respectively. Quantitative data was analysed using frequencies, percentages, and chi-square tests for associations, while themes and sub-themes were used to analyse qualitative data respectively.FindingsA total of 98.5%, 81.3%, and 78.5% had heard about schistosomiasis, and knew the main transmission modes and symptoms, respectively. The majority (75.8%) said avoiding contact with water was a preventative way, while 67.5% said observing signs and symptoms was a form of diagnosis. Furthermore, 98.4% and 73.4% said it was important to defecate in latrines and to avoid contact with contaminated water respectively. However, it is difficult to avoid contact with lake water because it is the only source of livelihood, especially for fisher communities. Open defecation is commonly practiced along the lake due to insufficient space and difficulties in the construction of latrines. Myths and misconceptions reported include; lake water is safe, gassing in water causes transmission, fetching water early in the morning and from deep water is safe, and feces in the lake water act as a bait for catching fish.Conclusions and recommendationsDespite adequate knowledge of schistosomiasis and a positive attitude towards its prevention, existing myths and misconceptions, coupled with persistent risky water, sanitation, and hygiene practices still pose a challenge. A more robust community-based awareness intervention using bottom-up participatory approaches, accompanied by the provision of clean and safe water sources and increasing latrine coverage, could provide lasting solutions to these barriers.     

The super-cooling agent icilin reveals a mechanism of coincidence detection by a temperature-sensitive TRP channel

TRPM8, a member of the transient receptor potential family of ion channels, depolarizes somatosensory neurons in response to cold. TRPM8 is also activated by the cooling agents menthol and icilin. When exposed to menthol or cold, TRPM8 behaves like many ligand-gated channels, exhibiting rapid activation followed by moderate Ca(2+)-dependent adaptation. In contrast, icilin activates TRPM8 with extremely variable latency followed by extensive desensitization, provided that calcium is present. Here, we show that, to achieve full efficacy, icilin requires simultaneous elevation of cytosolic Ca2+, either via permeation through TRPM8 channels or by release from intracellular stores. Thus, two stimuli must be paired to elicit full channel activation, illustrating the potential for coincidence detection by TRP channels. Determinants of icilin sensitivity map to a region of TRPM8 that corresponds to the capsaicin binding site on the noxious heat receptor TRPV1, suggesting a conserved molecular logic for gating of these thermosensitive channels by chemical agonists.     

A reverse genetic approach for generating gene replacement mutants in<Emphasis Type="Italic"> Ustilago maydis</Emphasis>

We describe a versatile strategy for generating gene replacement mutants in the phytopathogenic fungus Ustilago maydis. The system includes the choice of 32 different insertion cassettes for genetic engineering purposes, such as gene disruption and more sophisticated insertions of reporter genes, heterologous promoters or combinations of the two. PCR-amplified flanking sequences needed for homologous recombination are ligated to the respective insertion cassettes via Sfi I sites. As proof of principle we generated two replacement mutants in which the endogenous promoter of the pheromone gene mfa1 drives expression of the Green Fluorescent Protein gene (gfp). Simultaneously, expression of the mfa1 ORF is controlled either by the carbon source-regulated crg1 promoter or the nitrogen source-regulated nar1 promoter. In both cases gfp expression was pheromone-inducible and pheromone expression was only detected when the heterologous promoters were active.Communicated by G. JürgensThe first two authors contributed equally to this work     

Colocalization but differential regulation of neuronal NO synthase and nicotinic acetylcholine receptor in C2C12 myotubes

In mammalian skeletal muscle,neuronal-type nitric oxide synthase (nNOS) is found to be enriched atneuromuscular endplates. Here we demonstrate the colocalization of thenicotinic acetylcholine receptor (nAChR, stained with -bungarotoxin)and nNOS (stained with a specific antibody) in murineC₂C₁₂ myotubes. However, coimmunoprecipitation experiments demonstrated no evidence for a direct protein-protein association between the nAChR and nNOS in C₂C₁₂myotubes. An antibody to the ₁-subunit of the nAChR didnot coprecipitate nNOS, and an nNOS-specific antibody did notprecipitate the ₁-subunit of the nAChR. Treatment ofmice with bacterial LPS downregulated the expression of nNOS inskeletal muscle, and treatment of C₂C₁₂ cellswith bacterial LPS and interferon- markedly decreased nNOS mRNA andprotein expression. In contrast, mRNA and protein of the nAChR (-,-, and -subunits) remained unchanged at the mRNA and proteinlevels. These data demonstrate that nNOS and the nAChR are colocalizedin murine skeletal muscle and C₂C₁₂ cells but differ in their expressional regulation.