Eating your mitochondria-when too much of a good thing turns bad.

How mitophagy is turned on to remove damaged or excess mitochondria from cells has been well-studied, but less is known about how the pathway is turned off to avoid "over-eating" of mitochondria under basal conditions. Three new studies now reveal the disease-associated FBXL4 protein as an important negative regulator of constitutive mitophagy, controlling the stability of mitophagy receptors BNIP3 and NIX.

BNIP3L/NIX regulates both mitophagy and pexophagy.

Mitochondria and peroxisomes are closely related metabolic organelles, both in terms of origin and in terms of function. Mitochondria and peroxisomes can also be turned over by autophagy, in processes termed mitophagy and pexophagy, respectively. However, despite their close relationship, it is not known if both organelles are turned over under similar conditions, and if so, how this might be coordinated molecularly. Here, we find that multiple selective autophagy pathways are activated upon iron chelation and show that mitophagy and pexophagy occur in a BNIP3L/NIX-dependent manner. We reveal that the outer mitochondrial membrane-anchored NIX protein, previously described as a mitophagy receptor, also independently localises to peroxisomes and drives pexophagy. We show this process happens in vivo, with mouse tissue that lacks NIX having a higher peroxisomal content. We further show that pexophagy is stimulated under the same physiological conditions that activate mitophagy, including cardiomyocyte and erythrocyte differentiation. Taken together, our work uncovers a dual role for NIX, not only in mitophagy but also in pexophagy, thus illustrating the interconnection between selective autophagy pathways.

Comparing PVP and Polymeric Micellar Formulations of a PEGylated Photosensitizing Phthalocyanine by NMR and Optical Techniques.

Phthalocyanines are ideal candidates as photosensitizers for photodynamic therapy (PDT) of cancer due to their favorable chemical and photophysical properties. However, their tendency to form aggregates in water reduces PDT efficacy and poses challenges in obtaining efficient forms of phthalocyanines for therapeutic applications. In the current work, polyvinylpyrrolidone (PVP) and micellar formulations were compared for encapsulating and monomerizing a water-soluble zinc phthalocyanine bearing four non-peripheral triethylene glycol chains (Pc1). 1H NMR spectroscopy combined with UV-vis absorption and fluorescence spectroscopy revealed that Pc1 exists as a mixture of regioisomers in monomeric form in dimethyl sulfoxide but forms dimers in an aqueous buffer. PVP, polyethylene glycol castor oil (Kolliphor RH40), and three different triblock copolymers with varying proportions of polyethylene and polypropylene glycol units (termed P188, P84, and F127) were tested as micellar carriers for Pc1. 1H NMR chemical shift analysis, diffusion-ordered spectroscopy, and 2D nuclear Overhauser enhancement spectroscopy was applied to monitor the encapsulation and localization of Pc1 at the polymer interface. Kolliphor RH40 and F127 micelles exhibited the highest affinity for encapsulating Pc1 in the micellar core and resulted in intense Pc1 fluorescence emission as well as efficient singlet oxygen formation along with PVP. Among the triblock copolymers, efficiency in binding and dimer dissolution decreased in the order F127 > P84 > P188. PVP was a strong binder for Pc1. However, Pc1 molecules are rather surface-attached and exist as monomer and dimer mixtures. The results demonstrate that NMR combined with optical spectroscopy offer powerful tools to assess parameters like drug binding, localization sites, and dynamic properties that play key roles in achieving high host-guest compatibility. With the corresponding adjustments, polymeric micelles can offer simple and easily accessible drug delivery systems optimizing phthalocyanines' properties as efficient photosensitizers.

Exploring different stakeholders' perspectives on ward rounds in paediatric oncology: a qualitative study.

RATIONAL/AIMS AND OBJECTIVES: Ward rounds are a core routine for interprofessional communication and clinical care planning: Health care professionals and patients meet regularly and it encourages patients to actively participate. In paediatric oncology, the long treatment process, the serious diagnosis, and involvement of both patients and their parents in shared-decision-making require specific ward round skills. Despite its high value for patient-centred care, a universal definition of ward round is lacking. Little is known about attitudes and expectations of different participants towards a 'good' ward round. This study aims to capture experiences and expectations of different stakeholders to better understand ward round needs in paediatric oncology and serve as a basis to improve future ward rounds. METHOD: Semi-structured interviews were conducted with patients, parents, nurses and medical doctors of a paediatric oncology ward until theoretical saturation (13 interviews). A standardised qualitative analysis using the phenomenological framework defined by Colaizzi was used to identify important aspects in the interviews. RESULTS: Three major themes were identified in the interviews: [1] Structure and Organisation; [2] Communication; [3] Education. Further analysis revealed 23 categories and elucidated several opportunities and unmet needs recognized by stakeholders: Ward round functions in comforting families in stressful situations, and relationship building. Interviewees expressed their concerns about missing structures. Families pleaded for smaller ward round teams and layperson language. Health care professionals underscored the lack of ward round training. Paediatric patients stated that ward round scared them without proper explanation. All interviewees emphasized the need for professionalization of the ward round in the setting of paediatric oncology. CONCLUSION: This study gives important insights into ward round functions and organisational requirements. It addresses special challenges for ward round participants in paediatric oncology, such as consideration of the emotional aspect of cancer treatment or the limits of shared decision making. Furthermore, this study underscores the great significance of ward rounds in paediatric oncology, with an emphasis on communication and relationship-building. Although performed universally, ward rounds are poorly explored or evaluated. This structured analysis synthesizes important expectations of different WR stakeholders, revealing opportunities of improvement and stressing the need for guidelines, training, and preparation.

STARD3 mediates endoplasmic reticulum-to-endosome cholesterol transport at membrane contact sites.

StAR-related lipid transfer domain-3 (STARD3) is a sterol-binding protein that creates endoplasmic reticulum (ER)-endosome contact sites. How this protein, at the crossroad between sterol uptake and synthesis pathways, impacts the intracellular distribution of this lipid was ill-defined. Here, by using in situ cholesterol labeling and quantification, we demonstrated that STARD3 induces cholesterol accumulation in endosomes at the expense of the plasma membrane. STARD3-mediated cholesterol routing depends both on its lipid transfer activity and its ability to create ER-endosome contacts. Corroborating this, in vitro reconstitution assays indicated that STARD3 and its ER-anchored partner, Vesicle-associated membrane protein-associated protein (VAP), assemble into a machine that allows a highly efficient transport of cholesterol within membrane contacts. Thus, STARD3 is a cholesterol transporter scaffolding ER-endosome contacts and modulating cellular cholesterol repartition by delivering cholesterol to endosomes.

Identification of MOSPD2, a novel scaffold for endoplasmic reticulum membrane contact sites.

Membrane contact sites are cellular structures that mediate interorganelle exchange and communication. The two major tether proteins of the endoplasmic reticulum (ER), VAP-A and VAP-B, interact with proteins from other organelles that possess a small VAP-interacting motif, named FFAT [two phenylalanines (FF) in an acidic track (AT)]. In this study, using an unbiased proteomic approach, we identify a novel ER tether named motile sperm domain-containing protein 2 (MOSPD2). We show that MOSPD2 possesses a Major Sperm Protein (MSP) domain which binds FFAT motifs and consequently allows membrane tethering in vitro MOSPD2 is an ER-anchored protein, and it interacts with several FFAT-containing tether proteins from endosomes, mitochondria, or Golgi. Consequently, MOSPD2 and these organelle-bound proteins mediate the formation of contact sites between the ER and endosomes, mitochondria, or Golgi. Thus, we characterized here MOSPD2, a novel tethering component related to VAP proteins, bridging the ER with a variety of distinct organelles.

Characterization of the use of shock wave therapy among equine veterinarians.

Extracorporeal shock wave therapy (ESWT) research has prioritized mechanism of action and efficacy. Data regarding frequency of use and clinical opinion are not available. A web-based survey was offered to members of the American Association of Equine Practitioners; 144 responses were obtained. Frequency of ESWT use by respondents was as follows: daily by 8.3% (12/144), at least once weekly by 36.8% (53/144), at least once per month by 22.9% (33/144), less than once per month by 19.4% (28/144), and never by 12.5% (18/144) of respondents. The most common reason for use was to treat ligamentous injuries. Opinion of efficacy was variable.

La recherche sur la thérapie extra-corporelle par ondes de choc (ESWT) a priorisé le mécanisme d'action et l'efficacité. Les données concernant la fréquence d'utilisation et l'opinion clinique ne sont pas disponibles. Un sondage sur le web fut offert aux membres de l'American Association of Equine Practitionners; 144 réponses furent obtenues. La fréquence d'utilisation d'ESWT par les répondants était la suivante : quotidiennement par 8,3 % (12/144), au moins une fois semaine par 36,8 % (53/144), au moins une fois par mois 22,9 % (33/144), moins d'une fois par mois par 19,4 % (28/144) et jamais par 12,5 % (18/144) des répondants. La raison la plus fréquente pour son utilisation était pour traiter des blessures aux ligaments. Les opinions sur son efficacité étaient variables.(Traduit par Dr Serge Messier).

Sacrococcygeal luxation and complete tail amputation following a tail pull injury in a horse.

A 17-year-old Quarter horse mare was presented because of traumatic luxation of the fifth sacral and first coccygeal vertebrae resulting in loss of sensation, motor function, and perfusion of the tail. The case was complicated by an associated tail head hematoma. Due to the severity of the injury, tail amputation was performed at the level of the luxation. Tail amputations in horses at the sacrococcygeal junction following a suspected tail pull injury are infrequently reported in the literature.

Luxation sacrococcygienne et amputation complète de la queue à la suite d'une blessure par traction de la queue chez un cheval. Une jument Quarter horse âgée de 17 ans fut présentée pour cause de luxation traumatique de la cinquième vertèbre sacrée et de la première vertèbre coccygienne résultant en une perte de sensation, de fonction moteur, et de perfusion de la queue. Le cas était compliqué par l'association d'un hématome de la tête de la queue. Compte tenu de la sévérité de la blessure, l'amputation de la queue fut effectuée au site de la luxation. Les amputations de la queue chez les chevaux à la jonction sacrococcygienne à la suite d'une blessure suspectée causée par traction de la queue ne sont rapportées que peu fréquemment dans la littérature.(Traduit par Dr Serge Messier).

Comparison Between Doppler-Echocardiography and Uncalibrated Pulse Contour Method for Cardiac Output Measurement: A Multicenter Observational Study.

OBJECTIVES: Echocardiography and pulse contour methods allow, respectively, noninvasive and less invasive cardiac output estimation. The aim of the present study was to compare Doppler echocardiography with the pulse contour method MostCare for cardiac output estimation in a large and nonselected critically ill population. DESIGN: A prospective multicenter observational comparison study. SETTING: The study was conducted in 15 European medicosurgical ICUs. PATIENTS: We assessed cardiac output in 400 patients in whom an echocardiographic evaluation was performed as a routine need or for cardiocirculatory assessment. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: One echocardiographic cardiac output measurement was compared with the corresponding MostCare cardiac output value per patient, considering different ICU admission categories and clinical conditions. For statistical analysis, we used Bland-Altman and linear regression analyses. To assess heterogeneity in results of individual centers, Cochran Q, and the I statistics were applied. A total of 400 paired echocardiographic cardiac output and MostCare cardiac output measures were compared. MostCare cardiac output values ranged from 1.95 to 9.90 L/min, and echocardiographic cardiac output ranged from 1.82 to 9.75 L/min. A significant correlation was found between echocardiographic cardiac output and MostCare cardiac output (r = 0.85; p < 0.0001). Among the different ICUs, the mean bias between echocardiographic cardiac output and MostCare cardiac output ranged from -0.40 to 0.45 L/min, and the percentage error ranged from 13.2% to 47.2%. Overall, the mean bias was -0.03 L/min, with 95% limits of agreement of -1.54 to 1.47 L/min and a relative percentage error of 30.1%. The percentage error was 24% in the sepsis category, 26% in the trauma category, 30% in the surgical category, and 33% in the medical admission category. The final overall percentage error was 27.3% with a 95% CI of 22.2-32.4%. CONCLUSIONS: Our results suggest that MostCare could be an alternative to echocardiography to assess cardiac output in ICU patients with a large spectrum of clinical conditions.

Touché! STARD3 and STARD3NL tether the ER to endosomes.

Membrane contact sites (MCSs) are subcellular regions where the membranes of distinct organelles come into close apposition. These specialized areas of the cell, which are involved in inter-organelle metabolite exchange, are scaffolded by specific complexes. STARD3 [StAR (steroidogenic acute regulatory protein)-related lipid transfer domain-3] and its close paralogue STARD3NL (STARD3 N-terminal like) are involved in the formation of contacts between late-endosomes and the endoplasmic reticulum (ER). The lipid transfer protein (LTP) STARD3 and STARD3NL, which are both anchored on the limiting membrane of late endosomes (LEs), interact with ER-anchored VAP [VAMP (vesicle-associated membrane protein)-associated protein] (VAP-A and VAP-B) proteins. This direct interaction allows ER-endosome contact formation. STARD3 or STARD3NL-mediated ER-endosome contacts, which affect endosome dynamics, are believed to be involved in cholesterol transport.

Fluorescence lifetime imaging and phasor analysis of intracellular porphyrinic photosensitizers applied with different polymeric formulations.

The fluorescence lifetime of a porphyrinic photosensitizer (PS) is an important parameter to assess the aggregation state of the PS even in complex biological environments. Aggregation-induced quenching of the PS can significantly reduce the yield of singlet oxygen generation and thus its efficiency as a medical drug in photodynamic therapy (PDT) of diseased tissues. Hydrophobicity and the tendency to form aggregates pose challenges on the development of efficient PSs and often require carrier systems. A systematic study was performed to probe the impact of PS structure and encapsulation into polymeric carriers on the fluorescence lifetime in solution and in the intracellular environment. Five different porphyrinic PSs including chlorin e6 (Ce6) derivatives and tetrakis(m-hydroxyphenyl)-porphyrin and -chlorin were studied in free form and combined with polyvinylpyrrolidone (PVP) or micelles composed of triblock-copolymers or Cremophor. Following incubation of HeLa cells with these systems, fluorescence lifetime imaging combined with phasor analysis and image segmentation was applied to study the lifetime distribution in the intracellular surrounding. The data suggest that for free PSs, the structure-dependent cell uptake pathways determine their state and emission lifetimes. PS localization in the plasma membrane yielded mostly monomers with long fluorescence lifetimes whereas the endocytic pathway with subsequent lysosomal deposition adds a short-lived component for hydrophilic anionic PSs. Prolonged incubation times led to increasing contributions from short-lived components that derive from aggregates mainly localized in the cytoplasm. Encapsulation of PSs into polymeric carriers led to monomerization and mostly fluorescence emission decays with long fluorescence lifetimes in solution. However, the efficiency depended on the binding strength that was most pronounced for PVP. In the cellular environment, PVP was able to maintain monomeric long-lived species over prolonged incubation times. This was most pronounced for Ce6 derivatives with a logP value around 4.5. Micellar encapsulation led to faster release of the PSs resulting in multiple components with long and short fluorescence lifetimes. The hydrophilic hardly aggregating PS exhibited a mostly stable invariant lifetime distribution over time with both carriers. The presented data are expected to contribute to optimized PDT treatment protocols and improved PS-carrier design for preventing intracellular fluorescence quenching. In conclusion, amphiphilic and concurrent hydrophobic PSs with high membrane affinity as well as strong binding to the carrier have best prospects to maintain their photophysical properties in vivo and serve thus as efficient photodynamic diagnosis and PDT drugs.

Mitochondria and peroxisomes: partners in autophagy.

Mitochondria, often called "the powerhouse" of the cell due to their role as the main energy supplier, regulate numerous complex processes including intracellular calcium homeostasis, reactive oxygen species (ROS) production, regulation of immune responses, and apoptosis. So, mitochondria are a fundamental metabolic hub that also control cell survival and cell death. However, they are not unique in all these functions. Indeed, peroxisomes are small cytoplasmic organelles that also ensure metabolic functions such as fatty acid oxidation and ROS production. This common relationship also extends beyond function as peroxisomes themselves can form from mitochondrial-derived precursors. Given this interconnection between mitochondria and peroxisomes involving biogenesis and function, in our recent work we determined if their turnover was also linked.

PINK1-Dependent Mitophagy Inhibits Elevated Ubiquitin Phosphorylation Caused by Mitochondrial Damage.

Ubiquitin phosphorylation by the mitochondrial protein kinase PTEN-induced kinase 1 (PINK1), upon mitochondrial depolarization, is an important intermediate step in the recycling of damaged mitochondria via mitophagy. As mutations in PINK1 can cause early-onset Parkinson's disease (PD), there has been a growing interest in small-molecule activators of PINK1-mediated mitophagy as potential PD treatments. Herein, we show that N6-substituted adenosines, such as N6-(2-furanylmethyl)adenosine (known as kinetin riboside) and N6-benzyladenosine, activate PINK1 in HeLa cells and induce PINK1-dependent mitophagy in primary mouse fibroblasts. Interestingly, pre-treatment of HeLa cells and astrocytes with these compounds inhibited elevated ubiquitin phosphorylation that is induced by established mitochondrial depolarizing agents, carbonyl cyanide m-chlorophenyl-hydrazine and niclosamide. Together, this highlights N6-substituted adenosines as progenitor PINK1 activators that could potentially be developed, in the future, as treatments for aged and sporadic PD patients who have elevated phosphorylated ubiquitin levels in the brain.

In vivo genotoxicity of furan in F344 rats at cancer bioassay doses.

Furan, a potent rodent liver carcinogen, is found in many cooked food items and thus represents a human cancer risk. Mechanisms for furan carcinogenicity were investigated in male F344 rats using the in vivo Comet and micronucleus assays, combined with analysis of histopathological and gene expression changes. In addition, formamidopyrimidine DNA glycosylase (Fpg) and endonuclease III (EndoIII)-sensitive DNA damage was monitored as a measure of oxidative DNA damage. Rats were treated by gavage on four consecutive days with 2, 4, and 8mg/kg bw furan, doses that were tumorigenic in 2-year cancer bioassays, and with two higher doses, 12 and 16mg/kg. Rats were killed 3h after the last dose, a time established as producing maximum levels of DNA damage in livers of furan-treated rats. Liver Comet assays indicated that both DNA strand breaks and oxidized purines and pyrimidines increased in a near-linear dose-responsive fashion, with statistically significant increases detected at cancer bioassay doses. No DNA damage was detected in bone marrow, a non-target tissue for cancer, and peripheral blood micronucleus assays were negative. Histopathological evaluation of liver from furan-exposed animals produced evidence of inflammation, single-cell necrosis, apoptosis, and cell proliferation. In addition, genes related to apoptosis, cell-cycle checkpoints, and DNA-repair were expressed at a slightly lower level in the furan-treated livers. Although a mixed mode of action involving direct DNA binding cannot be ruled out, the data suggest that furan induces cancer in rat livers mainly through a secondary genotoxic mechanism involving oxidative stress, accompanied by inflammation, cell proliferation, and toxicity.

Characterizing the surface quality and droplet interface shape for microarray plates.

The variation in the surface quality of microarray plates was examined by measuring the contact angles of 480 droplets on five microarray plates. It was found that the measured contact angle did not accurately predict the droplet shape for moderate Bond numbers (~0.5 ≤ N(B) ≤ 5). By defining an apparent contact angle using the ratio of the contact radius to the height, the variance in the predicted interface shape decreased by greater than a factor of 3 for both local and globally averaged characteristics. The error in the predicted droplet height was also reduced by 3 orders of magnitude.

Flavor release of the tomato flavor enhancer, 2-isobutylthiazole, from whey protein stabilized model dressings.

A tomato flavor enhancer, 2-isobutylthiazole (IBT), was added (5 mg/kg) to dressings emulsified with either a whey protein concentrate-80 (WPC-80), a WPC-80 hydrolysate or ß-lactoglobulin at high pressure (70 MPa) at either 20 or 75 °C. The short (2-4 min), high-temperature treatment left the proteins essentially unchanged. IBT addition gave a dominant, green tomato flavor that masked the intrinsic odor of the WPC-80 hydrolysate but enhanced bitter flavor. The sensory IBT odor intensity was determined by oil level (5-30%) and pH; pH 4.0 gave higher IBT odor than pH 6.5. The green (IBT) odor release correlated with the sensory viscosity (p = 0.001) and with instrumentally determined complex modulus (p = 0.001), but not to the dressings' microstructure. The presence of small (<<1.5 µm) oil particles that were difficult to identify from images may explain why no correlation between green odor and microstructure was found. Headspace analysis significantly detected differences in the release of IBT from the different protein types: WPC-80 dressings released the most and ß-lactoglobulin the least amounts of IBT into headspace. As this difference in release of IBT among proteins could not be verified by sensory analysis, it may bear no relevance for perception.

Integrated trajectories of the maternal metabolome, proteome, and immunome predict labor onset.

Estimating the time of delivery is of high clinical importance because pre- and postterm deviations are associated with complications for the mother and her offspring. However, current estimations are inaccurate. As pregnancy progresses toward labor, major transitions occur in fetomaternal immune, metabolic, and endocrine systems that culminate in birth. The comprehensive characterization of maternal biology that precedes labor is key to understanding these physiological transitions and identifying predictive biomarkers of delivery. Here, a longitudinal study was conducted in 63 women who went into labor spontaneously. More than 7000 plasma analytes and peripheral immune cell responses were analyzed using untargeted mass spectrometry, aptamer-based proteomic technology, and single-cell mass cytometry in serial blood samples collected during the last 100 days of pregnancy. The high-dimensional dataset was integrated into a multiomic model that predicted the time to spontaneous labor [R = 0.85, 95% confidence interval (CI) [0.79 to 0.89], P = 1.2 × 10-40, N = 53, training set; R = 0.81, 95% CI [0.61 to 0.91], P = 3.9 × 10-7, N = 10, independent test set]. Coordinated alterations in maternal metabolome, proteome, and immunome marked a molecular shift from pregnancy maintenance to prelabor biology 2 to 4 weeks before delivery. A surge in steroid hormone metabolites and interleukin-1 receptor type 4 that preceded labor coincided with a switch from immune activation to regulation of inflammatory responses. Our study lays the groundwork for developing blood-based methods for predicting the day of labor, anchored in mechanisms shared in preterm and term pregnancies.

Phorbol ester-induced actin assembly in neutrophils: role of protein kinase C.

The shape changes and membrane ruffling that accompany neutrophil activation are dependent on the assembly and reorganization of the actin cytoskeleton, the molecular basis of which remains to be clarified. A role of protein kinase C (PKC) has been postulated because neutrophil activation, with the attendant shape and membrane ruffling changes, can be initiated by phorbol esters, known activators of PKC. It has become apparent, however, that multiple isoforms of PKC with differing substrate specificities exist. To reassess the role of PKC in cytoskeletal reorganization, we compared the effects of diacylglycerol analogs and of PKC antagonists on kinase activity and on actin assembly in human neutrophils. Ruffling of the plasma membrane was assessed by scanning EM, and spatial redistribution of filamentous (F)-actin was assessed by scanning confocal microscopy. Staining with NBD-phallacidin and incorporation of actin into the Triton X-100-insoluble ("cytoskeletal") fraction were used to quantify the formation of (F)-actin. [32P]ATP was used to detect protein phosphorylation in electroporated cells. Exposure of neutrophils to 4 beta-PMA (an activator of PKC) induced protein phosphorylation, membrane ruffling, and assembly and reorganization of the actin cytoskeleton, whereas the 4a-isomer, which is inactive towards PKC, failed to produce any of these changes. Moreover, 1,2-dioctanoylglycerol, mezerein, and 3-(N-acetylamino)-5-(N-decyl-N-methylamino)-benzyl alcohol, which are nonphorbol activators of PKC, also promoted actin assembly. Although these effects were consistent with a role of PKC, the following observations suggested that stimulation of conventional isoforms of the kinase were not directly responsible for actin assembly: (a) Okadaic acid, an inhibitor of phosphatases 1 and 2A, potentiated PMA-induced protein phosphorylation, but not actin assembly; and (b) PMA-induced actin assembly and membrane ruffling were not prevented by the conventional PKC inhibitors 1-(5-isoquinolinesulfonyl)-2-methylpiperazine, staurosporine, calphostin C, or sphingosine at concentrations that precluded PMA-induced protein phosphorylation and superoxide production. On the other hand, PMA-induced actin assembly was inhibited by long-chain fatty acid coenzyme A esters, known inhibitors of nuclear PKC (nPKC). We conclude that PMA-induced actin assembly is unlikely to be mediated by the conventional isoforms of PKC, but may be mediated by novel isoforms of the kinase such as nPKC.

Impact of slow-release Bacillus sphaericus granules on mosquito populations followed in a tropical urban environment.

A floating, slow-release, granular formulation of Bacillus sphaericus (Neide) was used to control mosquito larvae in two suburban areas of two tropical cities: Ouagadougou and Bobo-Dioulasso, Burkina Faso. A circular area of 2 km2, diameter 1,600 m, was treated in each city using a similar, smaller area 1 km away as an untreated control. Mosquito captures were made in houses in four concentric circles, from the periphery to the center; each circle was 50 m in width. Mosquitoes were captured in CDC light traps or from human landings. More than 95% of the mosquitoes were Culex quinquefasciatus (Say) (Diptera: Culicidae). The human landing catches provided twice as many mosquitoes as did the CDC traps/night/house. The treatments resulted in important reductions relative to the control area and to preintervention captures. The reduction was more prominent in the inner circle (up to 90%) than in the outer circle (50-70%), presumably because of the impact of immigrating mosquitoes from nontreated breeding sites around the intervention area. This effect was more pronounced for light trap catches than from human landings. The impact of treatment was also measured as the mean ratio of mosquito density in the two outer circles to that of the two inner circles. This ratio was approximately 1:1 before the intervention and reached 1:0.43 during the intervention. This comparison does not depend on the assumption that, in the absence of intervention, the mosquito population development in the two areas would have been identical, but does depend on the homogeneity of the intervention area. The study showed that it is possible to organize mosquito control in a tropical, urban environment by forming and rapidly training teams of young people to carry out the mosquito control mostly using a biopesticide that can be applied without any tools except an iron bar to lift lids on some cesspits.

Intracellular and Plasma Membrane Cholesterol Labeling and Quantification Using Filipin and GFP-D4.

Cholesterol, a major component of biological membranes, is rapidly trafficked and unevenly distributed between organelles. Anomalies of intracellular cholesterol distribution are the hallmark of a number of lysosomal lipid storage disorders. A major methodological obstacle for studying cholesterol trafficking is tracing this molecule in situ. The use of fluorescent probes that specifically bind cholesterol allows the visualization and imaging of cellular cholesterol. Here, we describe a series of assays optimized for quantifying free cholesterol in cell populations and at the single cell level, both at the plasma membrane and inside cells. These methods use two fluorescent probes: the D4 fragment of perfringolysin O fused to GFP (GFP-D4) and the polyene macrolide filipin. First, we report a robust method for quantifying plasma membrane cholesterol by flow cytometry using the GFP-D4 probe. Second, to optically distinguish and quantify intracellular cholesterol accumulation, we have adapted the classical filipin cholesterol staining protocol. Indeed, we observed that treatment of living cells with methyl-ß-cyclodextrin, a chemical known to extract cholesterol from the plasma membrane, improves the visualization of the intracellular cholesterol pool with filipin. To complement these staining procedures, we developed an image analysis protocol based on image segmentation to quantify, in a robust manner, intracellular cholesterol stained with filipin. Thus, this chapter is a guideline for cellular cholesterol staining and signal quantification.