There is ongoing controversy regarding the association between vesicoureteric reflux (VUR), recurrent urinary tract infections (UTI), and renal damage. Despite this, routine work up for VUR is still recommended after febrile UTI in most children. The present article reviews the indications and imaging modalities available for VUR diagnosis. Alternative newer techniques like MR cystography and voiding urosonography are discussed. The increasing evidence of the role of DMSA scans in managing children with VUR is highlighted. The goals of an imaging procedurein general are to confirm the diagnosis suspected with a high degree ofsensitivity and specificity, to aid treatment and allow prognostication. On theother hand, it is obligatory for the treating physician to analyze the risksand benefits of the diagnostic procedure and understand the natural history ofthe disease in question to establish whether or not the diagnosis and treatmentof a condition would alter long term outcome or impact management decisions.The diagnosis of vesicouretericreflux (VUR) is a relatively straightforward and well-established procedure.However, the underlying rationale for identifying VUR to prevent recurrentpyelonephritis (PN) and long-term renal damage has been vigorously questionedin the recent literature –4. CouplThe current article reviews theavailable modalities for evaluating VUR, suggests protocols for investigatingchildren with suspected VUR, and presents the recent evidence justifying theserecommendations.An ideal test for VUR detectionwould be one involving no radiation, no bladder catheterization, no sedation,low cost, high sensitivity, and one which provides complete anatomical details.The traditional method for diagnosing VUR is the fluoroscopic voiding cystourethrogram(VCUG). Currently, radionuclide cystography (RNC) remains the primaryalternative to a VCUG in evaluating VUR. The objection to performing a VCUG isrelated to the high radiation exposure of a traditional VCUG which is believedto be about 100 times that of a RNC. However, with the judicious use of digitaland pulsed fluoroscopy with meticulous image coning, the radiation exposurefrom a VCUG has been significantly reduced . DespiteP < .001).Several studies have compared thesensitivity of VCUG and RNC and concluded that RNC is at least as sensitive asor more than a VCUG for detecting VUR –10. SükaThe indirect RNC offers thepossibility of detecting VUR without bladder catheterization and presumably ina more physiological setting with natural bladder filling. The additionaladvantage is the ability to assess upper tract differential function anddrainage with the injected radioisotope. Although a few reports have shown acomparable degree of sensitivity between the direct and indirect RNC, theconsensus is that due to an inability to study the filling phase with anindirect RNC there is a considerable false negative rate with indirect RNC–15. TherThe sonographic evaluation of VURfollowing intravesical instillation of UScontrast agent has graduallypopularized VUS over the last decade. In a comprehensive review of VUS whencompared with VCUG, Darge showed that in 1338 patients with 2893 refluxingunits, VUS showed a diagnostic accuracy of 78%–96% . The oveMR cystography involves intravesical administration ofgadolinium with imaging using MR during filling and voiding. The relativebenefits of the procedure are that it can evaluate VUR without ionizingradiation and additionally give important information about renal-acquiredcortical defects and differentiate acquired cortical defects from congenitaldysplasia. It must be borne in mind that dysplasia and scarring are differententities diagnosed on histopathological examination. In this paper we refer tothem as congenital and acquired cortical defects. The potential drawbacksinclude a lower sensitivity as compared to VCUG, higher costs, and the need forsedation or anesthesia to perform the study. Takazakura et al. showed 90%sensitivity and a 96% specificity of MRVCUG and all children with grade 3 ormore VUR were identified using this modality . Lee et Rubenstein et al. in 2003introduced a novel but controversial technique to identify VUR in childrenpresenting with febrile UTIs and negative VCUG . The inhThe primary indications forevaluating children for VUR are discussed in this section. There has beenconsiderable evolution of our knowledge about VUR management over the lastseveral years. The role of antibiotic prophylaxis in preventing recurrentinfections has been challenged along with an increasing awareness of thedevelopment of antibiotic resistance –3. This The American Academy of Pediatrics: Committee on Quality Improvement recommends a VCUG for all childrenaged between 2 months to 2 years old following the first febrile UTI . The ratPrimary VUR is the commonestheritable disorder of the genitourinary tract and is inherited as a Mendeliandominant with partial expression . SeveralA tailored approach for siblingstherefore could be an RNC or a VUS in siblings younger than the toilet-trainedage and US as the initial screening modality for all older siblings. In thepresence of any US evidence of cortical damage a VCUG is recommended in children under 5 years ofage as they form the subset most at risk of renal damage. Symptomatic siblingsat any age are evaluated with a VCUG.VUR is suspected antenatally in thepresence of ureteric dilatation and/or hydronephrosis (HN) or following the diagnosisof ectopic kidneys, multicystic dysplastic kidney, and unilateral renalagenesis wherein there is an increased incidence of contra lateral VUR. VanEerde et al. performed a meta-analysis to review the value of antenatal HN inpredicting postnatal VUR . HN was A routine VCUG is recommended inthe work up of children with multicystic dysplastic kidneys (MCDK) based on thereported 15%–25% prevalence of VUR in children with MCDK –40. MillThe incidence of VUR in childrenwith unilateral renal agenesis (URA) is slightly higher than MCDK and variesbetween 24%–28% , 41–43. Primary VUR occurs in less than 1%of the general population but up to 50% of children who present with a UTIwill have VUR . TherefoThere is a considerable debateregarding the initial investigation following a febrile UTI with severalstudies highlighting the emerging role of DMSA scan vis a vis the VCUG. Therationale for this argument stems from the recent evidence which has downgradedthe importance of VUR as a sole factor in causing long-term renal damage. Infact, our aggressive management of VUR over the last several decades has notimpacted long-term renal outcome. Craig et al. reviewed the Australiaand New Zealand Dialysisand Transplant Registry between 1971 and 1998 and noted that over the decades,despite a more aggressive identification and treatment of VUR, refluxnephropathy continued to remain a cause of ESRD in about 14% of childrenregistered . This seRecent studies have demonstratedthat acquired renal scarring correlates best with recurrent UTI and not withVUR and primary VUR is neither sufficient nor essential for renal damage. Theexception to this rule is secondary reflux associated with bladder outletobstruction or high-pressure neurogenic bladders. Gordon et al. performed ameta-analysis to determine the value of VUR diagnosis to predict renal damagein children hospitalized with UTI . The anaMingin et al. retrospectivelyreviewed records of children who underwent DMSA scans following a febrile UTIor antenatal HN . 88% of In 303 children less than 2 years of ageevaluated with VCUG and DMSA scans after an episode of UTI, Hansson et al.found that 51% had an abnormal DMSA scan and 46% with a positive DMSA scanhad no evidence of VUR on VCUG . There wP = .04). Garin et al. performed arandomized prospective trial in 218 children with or without VUR who presentedwith PN, comparing prophylaxis with no prophylaxis [The role of VUR, especially lowergrades, as a predisposing factor for recurrent UTI is also controversial. Nuutinen and Uhari noted a higher rate ofrecurrent UTIs in children with grade III–V VUR in comparison with childrenwith grade I-II VUR . It is nphylaxis .The facThe problem in interpreting studiesattempting to clarify this aspect is the lack of a standardized definition of afebrile UTI and the variability in the methodology of obtaining urine samples.The ongoing randomized intervention for children with vesicoureteric reflux (RIVUR)study is a multicenter, double blinded, randomized, placebo controlled trialwhich aims to answer the ongoing controversy regarding the role of antibioticprophylaxis in preventing recurrent febrile/symptomatic UTI in children withVUR diagnosed after a UTI.in children presented with a UTI, up to 50% of children may have evidence of upper tract damage without evidence of VUR on a VCUG;the rate of spontaneous resolution of VUR is higher in children with low-grade VUR and a normal DMSA scan;a positive DMSA scan at diagnosis predicts a higher rate of recurrent UTI or breakthrough infections in children with VUR;VUR identification has not altered the ESRD rate related to reflux nephropathy.The idea behindthese studies is to encourage a more selective approach in investigatingchildren who present with a first UTI, contrary to the AAP practice guidelines.A DMSA would be the initial investigation and all children with an abnormalDMSA will then proceed to a VCUG. This would identify the majority of childrenwith dilating/significant VUR who would then benefit from antibioticprophylaxis, thus reducing both the number of VCUGs and number of children onantibiotic prophylaxis. Such a selective approach is justifiable with oneobjection being that boys with a potential posterior urethral valve presentedoutside the neonatal period may be missed with this approach. However, this maybe unlikely if a US study is simultaneously performed as part of the routine work up.In summary:MRU is increasingly being advocatedas a single imaging modality, which can be used to provide information obtainedon a VCUG and DMSA scan. The primary advantage of the MRU is its ability todistinguish between renal dysplasia  and acquiredscarring  . In addiThe ALARA (as low as reasonably achievable)concept has stressed the importance of minimizing radiation exposure inchildren being followed conservatively after diagnosis of VUR . ThompsoPersistence of VUR is more likelyin high-grade VUR, in children with bilateral disease  and when reflux is diagnosed in the older child. The value of the VCUGand RNC in predicting VUR resolution has been studied. It has been demonstratedthat when VUR occurs at less than 60% of expected bladder capacity and thereflux volume is more than 2% of bladder capacity, the resolution is poor, 59. KnuVUR is a heterogenous disorder, andits diagnosis and management continues to remain one of the most controversialproblems in pediatric urology. There is a realization that rather than adisease entity, VUR is a marker of overall urinary tract dysfunction, which maypredispose to UTI. The primary goal for the treating physician should continueto remain preservation of renal function and preventing the relatively smallpercentage of acquired renal defects associated with VUR. There has been aparadigm shift in the earnestness with which the diagnosis of VUR is soughtafter based on an increasing body of evidence which suggests that acquiredrenal defects are often not related to VUR and that our current modalities fordiagnosing VUR are associated with unacceptable radiation exposure and bladdercatheterization. The newer modalities do hold promise but further work iswarranted before they can replace the existing well-established techniques.


Mosquitoes that breed in temporary pools in remote areas that dry up seasonally are especially difficult to control through chemical or biological means. The annual killifish has been suggested as a means of eradicating the aquatic stages of mosquitoes in transient pools because they can maintain permanent populations in such habitats by undergoing suspended animation or diapause during the embryonic stages to survive periodic drought. However, very little is known about the predatory activity of annual killifish and their usefulness in mosquito control.Nothobranchius guentheri, native to Tanzania, was used in this investigation. Food preference was tested under laboratory conditions by feeding juvenile killifish with 2nd instar mosquito larvae of Culex quinquefasciatus in the presence of alternative food sources, such as rotifers and chironomid larvae. Semi-field tests were conducted by introduction of hibernating killifish embryos and juvenile fish to artificial ponds in an outdoor open environment that allowed natural oviposition of Cx. quinquefasciatus. Food preference studies show that N. guentheri preferred to prey on mosquito larvae than either chironomid or rotifers. When hibernating killifish embryos were added to ponds simultaneously with the addition of freshwater, the embryos hatched and fed on mosquito larval population resulting in complete elimination of the immature stages. The introduction of juvenile fish to ponds with high density of mosquito larvae resulted in total eradication of the mosquito population due to predation by fish. Complete biocontrol of the mosquito larval population was achieved in the presence of 3 fish per m2 of pond surface area.The annual killifish, The annual killifish provides yet another tool that may be employed in the eradication diseases carried by mosquitoes through vector control, particularly in temporary bodies of freshwater. The fish can be conveniently transported in the absence of water in the form of hibernating embryos. Once introduced either as embryos or juveniles in ponds, the annual killifish can effectively reduce the larval population because of its aggressive predatory activity. Gambusia affinis), native to northeastern United States, was eventually selected for worldwide mosquito control because of its high larvivorous capacity, high fecundity and adaptability to new environments. However, the success of Gambusia in mosquito control was overshadowed by its intrinsic aggressive nature that drove many other native species of aquatic organisms to the brink of extinction [Gambusia in providing the same mosquito larval control in permanents streams and pools [Vector control using pesticides remains an important component of all mosquito control program worldwide. However, the persistent use of pesticides caused the development of chemically resistant substrains of mosquitoes. This chemical resistance in mosquitoes is increasing with new reports of resistance emerging in various areas -3. For ttinction . More rend pools -7.Mosquito larval control in breeding sites comprising pools that dry up seasonally limits the use of conventional fish. Transporting fish to remote areas is both logistically challenging and expensive. For these reasons, attention had been focused on a unique group of freshwater fish, collectively known as annual killifish, native to Africa and South America that can maintain populations in seasonal pools. These killifish can survive prolonged drought, sometimes lasting as much as five years, by undergoing suspended animation or diapause at three stages of their embryonic development ,9. At thNothobranchius taeniopygus to combat malaria in seasonal swamps in Tanganyika and Kenya [The first record of annual killifish being used for this purpose was during World War II when Vanderplank initiated the introduction of nd Kenya . In 1952nd Kenya . Years lnd Kenya ,19. Somend Kenya ,20. HoweNothobranchius guentheri, an annual killifish indigenous to Tanzania. A hypothesis regarding its survival strategy in nature has already been proposed [N. guentheri and ways these fish might be employed in mosquito control.Much more is now known about the life cycle of proposed . What isNothobranchius guentheri, was established at the Poseidon Sciences field laboratory in the Philippines where all the experiments described in this paper was conducted. This population was derived from the original population maintained in the United States since 1975 and interbred recently with N. guentheri breeding pairs supplied by Mr. Fred Behrman of Athens Aquatics  and Mr. Chris Butcher . The killifish were maintained in 10 gallon aquaria according to previously described methods [Artemia for the first 2 weeks. Thereafter, the fry were fed live fruit fly larvae (Drosophila melagonaster) until they reach the juvenile size of 1.5 cm in length.The breeding population of the annual killifish,  methods . The spaCulex quinquefasciatus. These mosquitoes were allowed to naturally oviposit in the experimental ponds. Other aquatic species present in the ponds in significant numbers were chironomids or midge larvae (Chironomus plumosus) and rotifers (Brachionus sp.).Laboratory and semi-field studies were conducted in the island of Panay in the Philippines  during the rainy season from May 2009 and ending in October, 2009. The water temperature ranged from 27° to 30°C, with pH in the range from 7.0 to 8.0. The dissolved oxygen level was 4 ppm. The artificial ponds were constructed near banana trees, with the overhanging leaves providing partial shade. The nearby trees also provided shade and leaves were permitted to fall naturally onto the experimental ponds. The only species of mosquitoes occurring naturally at this location was N. guentheri, the prey organisms and the killifish were kept in glass tanks (11 cm depth × 29 cm length × 23 cm width) containing 7 liters of pond water. Freshwater rotifers were collected from an existing pond by passing the water through a fine mesh net. The concentrated freshwater rotifers were placed in the glass tanks filled with pond water. The rotifer count was determined by taking a 1 ml sample of the water and counting the number of rotifers under the microscope. 2nd instar larvae of Cx. quinquefasciatus were collected from ponds and also introduced in the glass tank either separately or in combination with rotifers in the presence of 1 male fish, which was starved for 24 hours prior to the experiment. The rotifer and mosquito larval consumption was obtained at 30 min and 120 min after killifish introduction to the tank.To study the food preference of nd instar larvae of Cx. quinquefasciatus were manually collected from the artificial pond and introduced into the test aquaria with killifish as described above. The number of larvae remaining in the tank after a given time period was determined.Chironomid larvae and 2All laboratory tests were conducted during the light phase of the artificial photoperiod (14 h light phase: 10 hour dark phase) at water temperature of 27 ± 1.5°C. All freshwater used in this study was obtained from an artificial well.Cx. quinquefasciatus, the only mosquito species found in the pond, was characterized according to the stages (instars and pupae). The larvae and pupae were placed in shallow pans for counting and returned back to the pond within 3 hours. For simplicity, the total mosquito count represents all larvae and pupae combined. The chironomids were counted by determining the total number of intact cocoons found underneath leaves in the bottom substrate. The rate of growth of the annual killifish in the ponds was determined by measuring the standard length as the distance from the tip of the snout to the end of the caudal peduncle.All of the mosquito larvae and pupae were harvested from each test pond by using fine mesh dip nets. Given the small size of the test ponds, it was possible to harvest all mosquito larvae to obtain more reliable data on larval abundance. N. guentheri can hatch, survive and feed on mosquito larvae, 1 m × 1 m × 0.3 m plastic lined ponds were constructed using 0.75 mm polyethylene plastic sheeting. These artificial ponds were filled with freshwater initially to a height of 5 cm. The killifish embryos in peat moss were introduced to the pond on the same day. Each group is composed of 5 ponds. No other food was provided to the fries and the pond water was allowed to be augmented by the daily rains to a final height of 0.3 m. The bottom substrate was composed of sandy-muddy soil at a thickness of 1 cm. Cx. quinquefasciatus was allowed to naturally oviposit eggs simultaneously with the introduction of the fish embryos.To determine if newly introduced embryos of Cx. quinquefasciatus was allowed to naturally oviposit in the ponds. After one week, the ponds showed substantial presence of mosquito larvae at which time a total of 5 juvenile killifish at 25 days of age  measuring 1.5 cm in length were placed in each pond. The total number of mosquito larvae and pupae was determined at weekly intervals. The controls represented ponds that did not receive any killifish. Each test group consisted of 3 ponds. After 28 days, the killifish in the experimental ponds were removed to determine the time for mosquito population to recover in the absence of fish. Also at day 28, five killifish  were added to the control ponds to determine the effect of the killifish in ponds with an already high mosquito density. No mortalities were observed during the transfer of the killifish and during length measurements.Artificial ponds (1 m × 2 m) were constructed and filled with water to height of 0.3 m. 2 ponds at densities from 0 to 5 fish per pond. Each test group was composed of 5 ponds. Cx. quinquefasciatus was allowed to naturally oviposit eggs for 1 week to provide a natural population of mosquito larvae. The total number of larvae present in the ponds was determined 4 days after fish introduction.To determine the fish density required to eradicate mosquito larval population in the ponds, juvenile male annual killifish were added to 1 mThe statistical significance between control and experimental groups was evaluated using Student's t test.N. guentheri, when presented with mosquito larvae and rotifers. There was no difference between males and females as far as food preference was concerned when corrected for body size (data not shown). The data for both sexes were therefore combined. When mosquito larvae and rotifers were given simultaneously as food source, the killifish preferentially consumed the mosquito larvae. After 120 min, the average consumption of mosquito larvae was 61.3% while only 24.1% of the rotifers were eaten. However, when presented with rotifers alone, the killifish doubled their consumption of rotifers. The difference in the food consumption between the rotifers alone and rotifers provided simultaneously with mosquito larvae were statistically significant (P < 0.0001). When presented with mosquito larvae alone, the killifish consumed the same amount of larvae as those killifish given both rotifers and larvae in combination. A similar pattern was observed when killifish was presented with mosquito larvae alone or in combination with chironomid larvae , lesser water boatmen (Coryxa sp.) and various small crustaceans [With the current emphasis on integrated vector management for malaria control the use of local species of annual killifish may represent an additional tool in the control of malaria vector species since the distribution of malaria  overlapst Africa . In natustaceans . Mosquitstaceans .nd instar mosquito larvae at 0.6 mm was not markedly different from the mean length for chironomid at larvae at 0.7 mm. Rotifers were much smaller prey measuring approximately 0.2 mm. The annual killifish would eat rotifers and chironomids as alternate food source if no other food was available, but overwhelmingly preferred mosquito larvae. Like most annual killifish, N, guentheri is a surface feeder and specially adapted to feed on aquatic invertebrates at or near the surface of the pond. The annual killifish will actively chase after the mosquito larvae upon sensing movements. Hence, the preference for mosquito larva that spends most of its time at the surface. In contrast, chironomids are bottom dwelling and typically found underneath decaying leaves. Rotifers are free swimming throughout the water column and likely preferred by the young killifish fry during the first week after hatching because of their small size.Food preference for mosquito larvae was evident when either chironomids or rotifers were simultaneously presented as prey. The size of the prey is not likely a major factor since the average size of the 2N. guentheri on culicine larvae strongly suggests that it is a suitable candidate for mosquito control. The results in this study show that a minimum of 3 killifish is adequate to eradicate the larval population from 1 m2 of pond surface area. Whether anopheline mosquitoes will be equally preyed upon like the culicine mosquitoes in this study or whether other annual killifish will have similar aggressive predatory activity remains to be established. Recently, Louca, et al [gambiae do not share the same oviposition behavior and will continue to lay eggs in the presence of fish in the pond [An. gambiae is endemic.The high predatory activity of a, et al  demonstrthe pond . The preNothobranchius habitat is composed of black or gray mud, commonly referred to as "black cotton soil" or vertisols which consist of swelling clays, such as montmorillonite, with high water retaining capacity [Nothobranchius is never observed to maintain populations in habitats with red clays or oxisols. This habitat requirement may limit the type of environments that may sustain Nothobranchius species.The onset of diapause sets the annual killifish apart from conventional fish and provides a novel means of providing biological control in transitory bodies of freshwater. The convenience of transporting and disseminating embryos for mosquito control purposes remain a major advantage particularly in remote areas where insecticide resistance is rampant and places where chemical use is not wanted by the local population. Even if re-introduction of new embryos becomes necessary during the next rainy season, this task can be easily accomplished by local mosquito control officers or even by the local population. The likelihood that introduced eggs will maintain sustainable populations in any new habitats is still open for exploration. The substrate in a typical capacity ,28. NothN. guentheri to other areas, even within Tanzania, may pose some risk to contamination of other transient pools inhabited by other species of Nothobranchius. However, field studies have shown that in many instances Nothobranchius species can be found in natural settings already intermixed with other species of annual killifish [N. guentheri is used for this study merely as a demonstration of the potential of using annual killifish for mosquito control and should be limited for use in its natural geographic range, there are other species, such as N. melanospilus, that have a wider distribution in Tanzania that may be better adapted to the different conditions found in the country .There are, however, ecological issues of concern before annual killifish can be employed for this purpose. Introduction of illifish ,29. WhilN. guentheri is only evident when males compete for the attention of females for reproduction and to establish dominance hierarchies [Another issue of concern is whether introduction of this species in new habitats may pose risks to other indigenous aquatic organisms. Annual killifish survive only in temporary pools and require a period of drying to complete its life cycle. For this reason, annual killifish are never found in permanent bodies of water. Since they are typically the dominant inhabitant in a transient pool, annual killifish may not have evolved behavioral adaptations to compete against other fishes. In multi-species aquaria for example, annual killifish do not survive well. The aggressive nature of rarchies . While iThere are many practical advantages of the annual killifish in mosquito control. First, there is a wide range of indigenous species of annual fish found in South America and sub-Saharan Africa. It is therefore feasible to use indigenous annual killifish species for any given malarial region rather than introducing an exotic species to new habitats. Second, the use of diapausing embryos may allow in suitable areas to have recurring populations in transient bodies of freshwater so that re-introductions will not be necessary, thereby reducing cost of maintaining mosquito control. Vectors of malaria and dengue propagate in both permanent and in transient pools, such as tire tracks, water collecting stations, animal footprints and depressions created by mining activities. Transporting conventional live fish for biocontrol measures in such situations will be difficult and costly. On the other hand, transporting thousands of annual killifish embryos in peat moss or in a more convenient delivery system would make it easier and more economical. Third, the annual killifish are small and less likely to be used as food source. The small size also allows easy access to shallow areas where mosquito larvae tend to congregate. Fourth, the annual killifish is not aggressive against other fish, cannot survive in permanent bodies of freshwater and likely pose minimal ecological issues. And finally fifth, as shown in this report, the annual killifish is sufficiently larvivorous to exert substantial control of the immature stages of the mosquito population. All of these characteristics satisfy World Health Organization's recommendations on the selection of larvivorous fish for mosquito control .Nothobranchius guentheri, aggressively preys on culicine larvae preferentially compared to other aquatic food sources under both laboratory and semi-field conditions. Total control of the immature stages of mosquitoes in freshwater can be achieved with the introduction of killifish. More important, introduction of hibernating or diapause embryos serves as a convenient means of disseminating killifish in transient pools. This study suggests that annual killifish may be useful in the future as part of the integrated program in vector control.In summary, this study demonstrates that the annual killifish, The authors declare that they have no competing interests.JRM participated in the design of the study, writing the manuscript and performing the statistical analysis. AQA participated in its design, coordination and helped to draft the manuscript. All authors read and approved the final manuscript.


In this overview the influence of functional bladder disturbances and of its treatment on the resolution of vesicoureteral reflux (VUR) in children is discussed. Historically both bladder dysfunction entities, the overactive bladder (OAB) and the dysfunctional voiding (DV), have been described in conjunction with VUR. Treatment of the dysfunction was also considered to influence spontaneous resolution in a positive way. During the last decades, however, papers have been published which could not support these results. Regarding the OAB, a prospective study with treatment of the bladder overactivity with anticholinergics, did not influence spontaneous resolution ratein children with a dysfunction including also the voiding phase, DV and DES , most studies indicate a negative influence on the resolution rate of VUR in children, both before and after the age for bladder control, both with and without treatment. However, a couple of uncontrolled studies indicate that there is a high short-term resolution rate after treatment with flow biofeedback.It should be emphasized that the voiding phase dysfunctions (DV and DES) are more severe than the genuine filling phase dysfunction (OAB), with an increased frequency of UTI and renal damage in the former groups.To be able to answer the question if treatment of bladder dysfunction influence the resolution rate of VUR in children, randomized controlled studies must be performed. There is a close relationship between bladder dysfunction andvesicoureteral reflux (VUR). This is most evident in children with neurogenicbladder dysfunction, in which the high intravesical pressure due to outflowobstruction induced by detrusor/sphincter dyssynergia is directly responsiblefor the development of the reflux. Another example of VUR as a secondaryphenomenon is in boys with posterior urethral valves, where the reflux issecondary to an anatomical obstruction in the urethra. In most cases ofsecondary reflux, normalisation of bladder function means spontaneousresolution of the VUR. When it comes to primary VUR, a close connection to bladder functionpathology of nonneurogenic origin has also been established. Studies have beenpublished describing children with functional bladder disturbance and VUR aftertoilet-training age. Studies from the 1980s most often dealt with girls whohave overactive bladders (OAB) in combination with reflux, and treatment of thedysfunction positively influenced resolution of the reflux. During the lastdecades, however, bladder dysfunction including the voiding phase, such asdysfunctional voiding and dysfunctional elimination syndrome, has been reportedto have a negative influence on VUR resolution in some studies. In otherstudies, treatment of the dysfunction has improved the resolution rate. Children with high-grade congenital reflux have also been shown to haveabnormal bladder function in about half of the cases. This dysfunction wascharacterised by an overdistended bladder and incomplete emptying. Thedysfunction per se had a negative influence on spontaneous resolution of VUR, which didnot improve despite treatment.In this overview of bladder dysfunction and VUR, only primary reflux isdiscussed. There are some contradictory results in the literature available, asindicated above, which make an overview interesting. One of the major problems is the fact that the level of evidence in almost all papers is low, most have level three, a few have level two, and no paper was identified as level one.When studying the literature about bladder dysfunctionin refluxing children, one finds that there is still confusion emanating fromdifferences in terminology , diagnostic procedures , degree of dysfunction, andso on. In this review, two bladder dysfunction entities are addressed inchildren after the age for bladder control: overactive bladder (OAB) anddysfunctional voiding (DV). The ICCS definitions of these entities are used.This means that dysfunctional voiding is only used in the sense of adysfunction during the voiding phase, characterised by increased activity inthe pelvic floor during voiding. The term can only be applied if repeat uroflowmeasurements show a staccato or interrupted pattern . DysfuncThe overall term recommended in the standardisation document for bladderdysfunction is lower urinary tract (LUT) dysfunction. This term is used as asynonym for bladder dysfunction in the present overview.In the early 1990s, bladder dysfunction was reported in small infantswith high-grade reflux. The dysfunction was characterised by low bladdercapacity, high voiding pressure, and overactivity during filling –4. LaterDyscoordination at voiding was often seen in these young children withVUR, a finding that can be suggested as the reason for the high capacitybladder with incomplete emptying.However, investigations of nonrefluxing children haveshown that dyscoordination at voiding was seen in healthy infants, both incystometric  and freeInterestingly, a bladder dysfunction pattern that wassimilar to the above-mentioned high capacity bladder with incomplete emptyingdynamics was described as early as 1987 by Griffiths and Scholtmeijer , 10. TheHigh-capacity bladder has also been reported in otherstudies in children with VUR after the infant year, especially in boys  andtogeThe reported prevalence of bladder dysfunction in aVUR population varies. When diagnosed using invasive urodynamic investigations,higher figures were generally found (38%–75%) in contrast to what was seenwhen nonurodynamic investigations were used (18%–52%) . ThisvaThe earliest studies of bladder dysfunction mostlydealt either with dysfunctional voiding or the overactive bladder. Recentstudies often give the prevalence of both dysfunctions together in childrenwith VUR. The advantages of separating the dysfunctions are that differenttreatment modalities can be evaluated, as well as differences in results whenit comes to effects on VUR resolution. The disadvantage is that thedysfunctions often are combined and sometimes difficult to separate.The first reports about bladder dysfunction and VURcame in the 1970s. Hinman and Baumann and AlleA few years later, Koff and Murtagh and TaylIn studies of larger cohorts of children with VUR, theprevalence of all bladder dysfunction together was reported to be between 18%and 50%, using questionnaires and flow measurements for the diagnose , 14, 16.The concept dysfunctional elimination syndrome (DES)was introduced by Koff et al. in 1998 , includiThese latter results were in line with what was seenin a followup study at the age of 7 years of 20 children who presented duringinfancy with grade 4-5 VUR and bladder dysfunction, diagnosed at that time. Thedysfunction was characterised by high bladder capacity and incomplete emptying.At the followup, these children had infrequent voiding, and often did not voidat school or in the morning if not prompted by parent or other guardian.Constipation had been or was still a problem in the majority of these children. The repHowever, in other studies it has not been possible todiagnose dysfunctional elimination syndrome more often in children with VURthan in control groups. Shaikh et al.  investigIn studies where the inclusion criterion was idiopathic bladderdysfunction, the prevalence of VUR was between 14% and 47% , 29, 30The registration of severity of voiding LUTdysfunction and its response to treatment with regard to symptoms is oftenhighly subjective, since the definition of how often the symptoms areexperienced is seldom given. Furthermore, in most cases a number of symptomsare included. Using a symptom score has been suggested in order to overcomethis obstacle. Upadhyay and coworkers  reportedThe results of treatment of overactivity in relationto VUR resolution are conflicting. Most studies do not have a control group,include only a small number of patients, are retrospective, and have nonuniformways of diagnosing overactivity, which might explain the different results.Many studies suggest increased spontaneous resolution after such treatment , 32, 33 Conversely, Willemsen and Nijman  showed, Whether spontaneous resolution of VUR in a group withuntreated OAB is different from a group without OAB cannot be established from thestudies available.There are very few studies reporting on VUR andtreatment of isolated DV, while there are more on DV and detrusor overactivityseen together.Kibar et al.  reportedHomsy et al.  reportedSnodgrass  noted a However, including studies in which bladderdysfunction was characterised by a dysfunctional voiding pattern, data supportthe assumption that there is a decreased spontaneous resolution of VUR inchildren with this dysfunction, especially when seen in combination withhigh-grade VUR.Yeung et al.  showed, DES in children with VUR was also correlated to alower resolution rate , despiteIt has previously been suggested that reimplantationof the ureter into the bladder in a child with major voiding dysfunctioncarries a high risk of failure. The dysfunction that carries the high risk is asevere form of dysfunctional voiding, induced by functional obstruction duringvoiding , 38. ReRecurrent UTIs have been shown in many studies to behigher in VUR patients with bladder dysfunction than in VUR children withoutsuch dysfunction , 15, 20.Snodgrass  showed aMost of the studies reporting on children with VUR andthe OAB dysfunction have not found any difference in numbers of children withrenal damage in the groups with and without the dysfunction , 18. In The bladder function pattern with high capacitybladders and incomplete emptying seen at follow up in children presentingduring the infant year with high-grade VUR , 10 is The extra volume load induced by the refluxing urinevolumes, which circulate between the bladder and the upper urinary tract, mightalso be a factor of importance for the high capacity bladder. In such cases,the bladder problems should more or less disappear after surgical treatment ofthe reflux. Investigation of bladder function in a group of children ages 7-8years who had been surgically treated for high-grade reflux at the age ofmedian 4 years did not support this theory. These children were diagnosed earlyas having a bladder dysfunction characterised by high-capacity bladders withincomplete emptying. At the follow-up investigation, they still had highcapacity bladders with few voiding per day but their emptying ability hadimproved, with quite low volumes of residual urine . The reThe connection between the overactive bladder dysfunction pattern and reflux is less clear. It is difficult to consider bladder overactivity the cause of reflux, since it causes only intermittentincreases in bladder pressure, which is not thought to induce reflux if thejunction is competent. Only a concomitant obstruction inducing a continuouspressure problem in the bladder is considered to be able to induce VUR inparallel to what is seen in children with the NBD or anatomical urethralobstruction, for example, in boys with PUV. The other possibility is that thereis only marginal competence in the valve mechanism, and in these cases thedetrusor contractions against a contracted sphincter may induce VUR. If thislatter causality exists, it might explain why renal damage seldom is seen inchildren with an OAB : the preVUR is associated with both OAB and dysfunctionalvoiding, with different entities as described above. However, we can onlyspeculate about the precise causative mechanisms between the respectivedysfunction and VUR. There are divergent opinions concerning whether thetreatment of the overactive bladder influences the rate of spontaneousresolution. There are as yet no randomised studies investigating the effect onthe reflux of treatment of the OAB versus no treatment. To my knowledge, thereare no studies comparing a group of children with VUR and untreated OAB with agroup of children with VUR and a stable bladder.In children with dysfunctional voiding and VUR, it iseasier to see a causative connection, especially in the more severe forms ofVUR, since this can be considered parallel to neurogenic bladder dysfunction.It is not known whether this is an acquired dysfunction as most authors suggestor if it is a congenital anomaly and part of a complex that also includes VUR.Treatment of the dysfunctional voiding increases thespontaneous resolution rate as has been suggested in some studies, but not inothers. Since there are no randomised studies available comparing resolutionrates in treated children with untreated children, this cannot be established.However, what is known is that dysfunctional voiding, dysfunction elimination syndrome,and the similar dysfunction seen in children with high-grade congenital reflux,all have negative influences on the spontaneous resolution rate of VUR whenuntreated, and lead to an increased risk for recurrent UTI.Since there seems to be a lower resolution rate inchildren with dysfunctional voiding than in those with OAB, it is important todistinguish between the diagnoses when comparing VUR resolution rates ofchildren with and without dysfunction. OAB in its genuine form seems to be amuch more benign dysfunction than dysfunctions including incomplete emptying ofthe bladder. However, it should be remembered that bladder overactivity anddysfunctional voiding are often seen together.In summary, the question if treatment of bladderdysfunction improves prognosis for spontaneous resolution of reflux cannot beanswered from the studies available. This is true for the overactive bladder,the dysfunctional voiding, as well as the dysfunctional elimination syndrome.Randomised studies have to be performed to give an answer. In these studies alsothe definitions from the ICCS standardisation document have to be used, toavoid confusion about terminology. Maybe the use of a scoring system of bladderdysfunction symptoms would be useful as well. However, treatment of bladder dysfunction should ofcourse be recommended, especially in cases with dysfunctional voiding and DES.One reason is that the success of surgical treatment of the reflux, bothendoscopic and open, probably depends on the bladder function status. The mostobvious reason for treating the bladder dysfunction in these refluxing childrenis, of course, as in nonrefluxing children, the symptoms of urgency, urinaryincontinence, constipation, UTI, and so on.


Climate change has the potential to have many significant impacts on aeroallergens such as pollen and mould spores, and therefore related diseases such as asthma and allergic rhinitis. This paper critically reviews this topic, with a focus on the potential adaptation measures that have been identified to date. These are aeroallergen monitoring; aeroallergen forecasting; allergenic plant management; planting practices and policies; urban/settlement planning; building design and heating, ventilating, and air-conditioning (HVAC); access to health care and medications; education; and research. Although this impacts research has been reviewed by several authors recently, an overview and critical review of the adaptation responses has not yet been conducted. This is the aim of this paper. In the following sections, climate change (both observed in the recent past and projected into the future) is described, followed by a summary of the potential impacts of these changes on aeroallergens and allergic respiratory diseases, with the latter placed in context through a synthesis of the research on the public health importance of these diseases. The major focus of the paper is a review of adaptation responses to these potential impacts, including: aeroallergen monitoring; aeroallergen forecasting; allergenic plant management; planting practices and policies; urban/settlement planning; building design and heating, ventilating, and air-conditioning (HVAC); access to health care and medications; education; and research.2.2]) has increased from a pre-industrial value of about 280 parts per million (ppm) to 386.30 ppm in 2009 . The impacts of climate change on aeroallergens, and in particular pollen, include impacts on pollen production and atmospheric pollen concentration, pollen season, plant and pollen spatial distribution, pollen allergenicity, and similar impacts on mould spores. Details of, and evidence for, these impacts is provided in the following sections.Climate change impacts on many physical, biological and human systems. Although human health is clearly part of the latter system(s), many diseases are intimately related to the environment, and therefore impacts of climate change on physical and biological systems may also result, indirectly, in impacts on human health. Climate change has had, and will continue to have, many significant adverse impacts on human health. The impacts of climate change on allergic respiratory diseases such as asthma and allergic rhinitis, via impacts on aeroallergens such as pollen and mould spores, are now emerging as one of the major indirect impacts of climate change on public health. The impacts of climate change on aeroallergens were reviewed by Beggs  and moreet al. , and theet al.  and Shea [et al. . Further [et al.  recently3.1.2] and temperature can result in increases in pollen production and atmospheric pollen concentrations. Experimental research where ragweed (Ambrosia artemisiifolia) has been grown at pre-industrial, current, and potential future [CO2]s has found that pollen production in this species is significantly increased at current compared to pre-industrial [CO2] and also at potential future compared to current [CO2] . Similamosphere .Betula)    produced pollen that was significantly more allergenic than pollen produced by plants grown at both current and pre-industrial [CO2]. There is also some evidence to suggest that higher air temperature can increase birch pollen allergenicity s, produced nearly three times more spores and more than twice the total antigenic protein per plant than at lower [CO2]s. Earlier observational research by Corden and Millington  and temperature on pollen and allergen production of more plant species is required. Similarly, modelling and surveillance of future changes in allergenic plant ranges is required.Continued research is required. This includes further climate model studies of future changes in thunderstorms, which can lead to short-term outbreaks of asthma symptoms . ContinuFinally, more research into adaptation in this area is required . This is6.Climate change has the potential to impact on aeroallergens such as pollen and mould spores, including increases in production and atmospheric concentrations, earlier pollen and mould spore season starts, increased allergenicity, and changes in at least plant and pollen spatial distribution. These biological impacts of climate change will likely have significant adverse impacts on allergic respiratory diseases such as asthma and allergic rhinitis unless appropriate adaptation measures are implemented. A range of such adaptation measures have now been identified. The outcomes of some of these measures, such as modified planting practices and policies, would not eventuate for many years, and they should therefore be implemented as soon as possible.


The success of such screens and the interpretation of the data depend on the stringent design of RNAi libraries. We describe and validate NEXT-RNAi, a software for the automated design and evaluation of RNAi sequences on a genome-wide scale. NEXT-RNAi is implemented as open-source software and is accessible at  RNA interference (RNAi) screens have become an important tool for the identification and characterization of gene function on a large-scale and complement classic mutagenesis screens by providing a means to target almost every transcript in a sequenced and annotated genome. RNAi is a post-transcriptional gene silencing mechanism conserved from plants to humans and relies on the delivery of exogenous short double-stranded RNAs (dsRNAs) that trigger the degradation of homologous mRNAs in cells ,2. As anAnopheles gambiae and species used to study evolutionary aspects of development, such as Tribolium castaneum, Acyrthosiphon pisum and Schmidtea mediterranea. RNAi libraries will facilitate the functional characterization of genes in these species, either through studying smaller subsets of candidates or on a genomic scale.The availability of genome-wide RNAi libraries for cell-based assays and whole organisms has opened new avenues to query genomes for a broad spectrum of loss-of-function phenotypes ,5. The nThe design of RNAi reagents is key to obtaining reliable phenotypic data in large-scale RNAi experiments. Several recent studies demonstrated that the degradation of non-intended transcripts  and knock-down efficiency depend on the sequence of the RNAi reagent and have to be carefully monitored -13. BaseCaenorhabditis elegans and Drosophila, RNAi can be triggered by long dsRNAs that are intracellularly broken down into short interfering RNAs (siRNAs) ). We found 49 phosphatases expressed at five or more RPKM , indicating that the observed depletion efficiency depended on the targeted mRNA rather than differences in the NEXT-RNAi designs.In large-scale RNAi experiments, the design of genome-wide silencing libraries has remained an important problem due to the flux of gene annotation and novel insights into the mechanisms that influence RNAi efficiency and off-target effects. We present an approach for the rapid design of whole-genome RNAi libraries and the re-annotation of already existing reagent collections. The method is flexible, identifies multiple independent reagents per gene model and has been implemented in an organism-independent manner. The design process is fully automated and can use annotations from various sequence- or model-organism databases as input, thereby enabling the design of RNAi reagents for any sequenced (and annotated) organism.Drosophila phosphatases expressed in our tissue culture model and found that approximately 89% of the reagents caused at least 60% mRNA knock-down. The application of a standardized design pipeline for independent designs leads to reproducible knock-downs in our experiments (correlation of 0.85 between the independent designs).We have designed several independent RNAi libraries for a diverse group of organisms. The automated pipeline yielded designs for more than 95% of all predicted genes in the first round of prediction. All library designs are available as a resource for download from our webpage . We valiDrosophila and human cells. Our analysis of eight genome-wide RNAi libraries for Drosophila revealed differences in genome coverage and predicted quality , most likely depending on two factors: the quality of the underlying genome release and the factors known to influence reagent quality at the time of the library design. Further, reagents in these libraries often share target sites, thus preventing an independent confirmation of phenotypes on a genomic scale. The re-annotation of commercially available human libraries revealed that a substantial part of the siRNAs  either do not target the intended gene or are predicted to silence additional loci, demonstrating that quality control at the level of sequence mapping is crucial for the interpretation of large-scale screens.RNAi screens have become a key tool for functional genomic analyses. The interpretation of the increasing number of published data sets obtained through RNAi screens relies heavily on correctly annotated reagents. Phenotypes derived from large-scale screens should be linked to the sequence of the RNAi reagent rather than the gene model because off-target or splice-variant-specific silencing can rarely be excluded. For the correct interpretation of RNAi screens, and also the comparison between different libraries, reagent-to-gene-model linkages must be re-mapped in regular intervals because most genome annotations are still in flux. NEXT-RNAi can be used to rapidly evaluate and re-annotate existing genome-wide libraries. For example, we have applied the algorithm to re-annotate RNAi libraries for Several tools for the design of RNAi reagents exist  were grown in Express Five SFM (Invitrogen) supplemented with 20 mM Glutamax I, 100 U/ml penicillin, 100 μg/ml streptomycin. Total RNA was extracted using Trizol (Invitrogen), followed by Rneasy cleanup , including on-column DNAse digest. mRNA was isolated with the MicroPoly(A)Purist kit  and the RNAseq library was prepared according to Illumina's mRNA Sequencing Sample Preparation Guide. Paired-end reads were aligned to the g Tophat  and RPKMg Tophat  based onin vitro transcription as described in  repeats; DRSC: TH and MB developed the concept. TH wrote the software and performed all calculations presented in the manuscript. TH and TS carried out the experimental validation of RNAi reagents. TH and MB wrote the manuscript.Detailed NEXT-RNAi workflow for the (a) design and (b) evaluation of dsRNAs and siRNAs.Click here for fileet al. NEXT-RNAi predictions of siRNA efficiencies using both the 'rational' and 'weighted' methods for 2,431 siRNAs tested by Huesken  repeats; UTR = untranslated region; SNP = single nucleotide polymorphism.Click here for fileDrosophila and human RNAi libraries re-annotated by NEXT-RNAiSummary statistics for . CAN = CA[ACGT] repeats; UTR = untranslated region; SNP = single nucleotide polymorphism.Click here for fileDrosophila RNAi libraries in Figure Raw data for comparison of including number of genes targeted by each library, number of genes targeted by both the compared libraries and number of genes targeted with independent designs .Click here for fileDrosophila phosphatases for the knock-down validation study presented in Figure Primer sequences and target gene information for the independent long dsRNAs designed against 49 Click here for fileDrosophila phosphatases from RNA-sequencing of D.Mel-2 cells and knock-downs measured after RNAi with two independent designs by quantitative RT-PCR  values for 49 Additional file Click here for fileDrosophila phosphatasesResults for knock-down validation of two independent RNAi reagents against 49 . Target-genes were sorted for the measured mRNA knock-down of design one.Click here for fileDescriptions and default values of design parameters used for NEXT-RNAi version 1.31.Click here for file


Leishmania lack RNAi activity and Argonaute or Dicer genes, we show that Leishmania braziliensis and other species within the Leishmania subgenus Viannia elaborate active RNAi machinery. Strong attenuation of expression from a variety of reporter and endogenous genes was seen. As expected, RNAi knockdowns of the sole Argonaute gene implicated this protein in RNAi. The potential for functional genetics was established by testing RNAi knockdown lines lacking the paraflagellar rod, a key component of the parasite flagellum. This sets the stage for the systematic manipulation of gene expression through RNAi in these predominantly diploid asexual organisms, and may also allow selective RNAi-based chemotherapy. Functional evolutionary surveys of RNAi genes established that RNAi activity was lost after the separation of the Leishmania subgenus Viannia from the remaining Leishmania species, a divergence associated with profound changes in the parasite infectious cycle and virulence. The genus Leishmania therefore offers an accessible system for testing hypothesis about forces that may select for the loss of RNAi during evolution, such as invasion by viruses, changes in genome plasticity mediated by transposable elements and gene amplification (including those mediating drug resistance), and/or alterations in parasite virulence.RNA interference (RNAi) pathways are widespread in metaozoans but the genes required show variable occurrence or activity in eukaryotic microbes, including many pathogens. While some  Leishmania major and Trypanosoma cruzi. The genome of L. braziliensis, a member of the early diverging Leishmania subgenus Viannia, retained key genes required for RNAi such as an Argonaute. We demonstrated that in fact L. braziliensis shows strong RNAi activity with reporter and endogenous genes affecting flagellar function. These data suggest that RNAi may be productively applied for functional genomic studies in L. braziliensis. We mapped the evolutionary point at which RNAi was lost in lineage leading to Leishmania and Crithidia, and establish that RNAi must have been lost at least twice in the trypanosomatids, once on the lineage leading to T. cruzi and independently following the divergence of the Viannia subgenus from other Leishmania species. Lastly, we discuss hypotheses concerning the forces leading to the loss of RNAi in Leishmania evolution, including viral invasion, increased genome plasticity, and altered virulence.RNAi interference pathways play fundamental roles in eukaryotes and provide important methods for the analysis of gene function. Occasionally RNAi has been lost, precluding its use as a tool, as well as raising the question of what forces could lead to loss of such a key pathway. Genomic and functional studies previously showed that within trypanosomatids protozoans RNAi was absent in both  Argonaute and Dicer suggests that this pathway may have been present in the common eukaryote ancestor ArgonauteIn metazoans, RNAi interference and related pathways play many key roles including regulation of mRNA levels and translation, chromatin silencing, programmed DNA rearrangements, genome surveillance, and defense against invading viruses. The phylogenetic distribution of key genes required for RNA interference such as Trypanosoma brucei), South American trypanosomes (T. cruzi) and a lineage encompassing a number of genera associated with insects or plants, ultimately leading to the mammalian parasite LeishmaniaT. brucei contain an active RNAi pathway and genes, including an Argonaute “slicer”  contains orthologs of T. brucei AGO1, DCL1 and DCL2L. braziliensis, and explored its utility as a genetic tool. Furthermore, we made evolutionary comparisons to map when the RNAi pathway was lost, and we discuss potential selective forces impacting on the parasite that may have contributed to the demise of RNAi during Leishmania evolution.The trypanosomatid protozoa comprise three major lineages, broadly grouped as the African trypanosomes , which in trypanosomes are 24–26 nt long i siRNAs .L. braziliensis transfection, when using episomal constructs previously developed in one of our labs that function effectively in many Leishmania species, and in many laboratories SSU) locus, using the appropriately digested DNA from pIR1SAT-based vectors, or derivatives thereof trans-splicing and 3′ polyadenylation produces capped mRNAs that can direct protein synthesis We then developed a green fluorescent protein (GFP)-based RNAi reporter assay for siRNA formation, as well as target mRNA and protein levels. Initially we experienced unexpected difficulty in GFP65-StL) was flanked by Leishmania sequences required for efficient 5′ and 3′ end mRNA formation, and was expressed following integration into the parasite small subunit ribosomal RNA locus  construct, containing two copies of an AT-rich GFP reporter (GFP65) in an inverted orientation separated by a short loop . This GFGFP65 probe showed that expression of GFP65-StL gave rise to a variety of products . These data suggested that L. braziliensis expresses a robust Dicer-like activity.Northern blot analysis with a products , lane 2.products . In contGFP65) used in the GFP65-StL construct above, and the second by a GC-rich ORF (GFP+). These genes differ in most 3rd codon positions, but their protein products only differ by a single amino acid. Alignment of these genes showed that the longest tracts of identical nucleotides were less than 14 nt  L. braziliensis or the GFP65-StL transfectant that produces GFP65 siRNAs.We used two GFP reporters, one encoded by the AT-rich ORF  reporter, expressed alone or in combination with a LUC stem-loop construct, revealing strongly-reduced LUC expression , or the genes PFR1 and PFR2, which encode major components of the paraflagellar rod, a component of the trypanosomatid flagellum required for motility StL-transfectants showed a variable decrease in mRNA levels when estimated by qPCR, ranging from no effect (LPG1) to more than 10-fold reduction . This suggests that L. braziliensis requires only low levels of LPG biosynthetic proteins, similar to the relatively small effects of RNAi on trypanosome glycoconjugate biosynthetic genes HGPRT mRNA and protein levels showed 3–4 fold decreases in HGPRT-StL transfectants . Collectively, the strength of the RNAi effect for these phenotypic reporters suggests that RNAi may function sufficiently well to assess the functions of many genes in AGO1 gene in trypanosomes and L. braziliensisL. braziliensis AGO1 in RNAi, we employed the seemingly counterintuitive approach of ‘RNAi of RNAi genes’, where introduction of dsRNAs targeting RNAi pathway genes inhibits RNAi activity, albeit not to the same level seen in null RNAi pathway gene knockouts LUC) and a second encoding a luciferase ORF stem-loop (LUC-StL). This minimized experimental variability and the number of transfections required, allowing the assessment of RNAi efficacy by the introduction of a single construct. When introduced into WT L. braziliensis, the ‘LUC RNAi self reporter’ (LUC-SR) showed low levels of luciferase activity, about 4-fold over background and comparable to that obtained with lines expressing LUC and LUC-StL independently after successive transfections  into the LUC RNAi reporter line (LUC-SR). These transfectants showed an average of 100-fold increased luciferase expression relative to LUCSR transfectants, signifying a considerable reduction in the efficiency of RNAi  but not in Leishmania (Sauroleishmania) tarentolae, L. mexicana, L. major or L. donovani (data not shown). Partial genome sequencing of a close non-parasitic ‘outgroup’, Crithidia fasciculata revealed AGO1, DCL1 and DCL2. To confirm the presence or absence of a functional RNAi pathway, we expressed the GFP65-StL RNA in L. tarentolae, L. mexicana, L. panamensis, L. guyanensis and Crithidia fasciculata, and monitored siRNA formation by Northern blotting. Consistent with the observed distribution of RNAi pathway genes, GFP siRNAs were made only in Crithidia, L. guyanensis and L. panamensis .We explored the prevalence of RNAi pathways in other Trypanosomatid species by comparative genomics. PCR assays detected namensis , S4. TraViannia from the remaining species complexes  or have undergone gene deletion  for known trypanosomatid RNAi genes. Since species retaining only a partial set of intact RNAi genes have not been reported, from these data we cannot identify which essential RNAi pathway gene was lost first at this distant point in Leishmania evolution. Presumably, once a gene critical for RNAi activity was inactivated, the remaining genes of the pathway become superfluous and fall prey to evolutionary drift, as seen in many other metabolic pathways during evolution.Association of these findings with the trypanosomatid evolutionary tree  through omplexes . ImportaL. braziliensis AGO1 gene is not syntenic with that of T. bruceiT. cruzi, and a second time in the lineage leading to Leishmania, subsequent to the divergence of most Leishmania groups from the non-parasitic species Crithidia fasciculata and the Leishmania subgenus Viannia  described earlier, relative to transfectants expressing only LUC. Multiple clonal lines were obtained, and LUC expression was measured in six randomly selected lines , compared to the 300-fold reduction seen in the virus free L. braziliensis M2903 . Significantly, the LRV-free line LgM4147/pX63HYG showed a similar 30-fold efficiency of RNAi in these studies (3.3% LUC SR/LUC). These data suggest that the reduced RNAi efficiency seen in L. guyanensis M4147 does not require the continued presence of the virus.While the RNAi pathway was active in the LRV+ L. braziliensis possesses a functional RNAi pathway, which enables the down-regulation of a variety of reporter and endogenous genes when assayed at the mRNA or protein levels. RNAi of AGO1 was used to confirm a requirement for the sole argonaute gene AGO1 in this process. As seen in many organisms, strong reductions in mRNA expression were seen, often accompanied by phenotypic changes, albeit of variable strength. As anticipated, it was not possible to introduce stem-loop constructs for essential genes such as α- or β-tubulins. Studies of such genes will require the development of inducible expression systems in Leishmania, which while promising have not yet reached the point of utility attained in trypanosomes.Our studies have established that PFR1 and PFR2), closely approximating the phenotypes seen in gene deletion mutants in L. mexicanaLeishmania gene function in the future.Strong phenotypes were produced by the knockdown of two genes implicated in flagellar motility and paraflagellar rod synthesis . Selection for the presence of LRV able to promote parasite survival could thus provide a selective force promoting down-regulation of RNAi activity targeting RNA viruses.We proposed previously that viral pressure could act as a selective force for the loss of RNAi in Leishmania, one prediction is that in extant species or strains now harboring Leishmania LRVs, attenuation of the RNAi response may occur. Here we compared the efficacy of RNAi seen in the virus-free L. braziliensis M2903 used in the majority of our studies with a closely related species L. guyanensis that bears the cytosolic dsRNA virus LRV1-4 L. guyanensis, its activity as assayed with LUC or GFP reporters was attenuated ∼10-fold relative to that seen in virus-free L. braziliensis  Leishmania species. In other eukaryotes the RNAi machinery includes as many as 9 proteins or more L. major to transcribe the antisense chromosomal strand at low levels L. major could be lethal Leishmania species will be a challenging task; nonetheless, efforts to introduce this suite of genes from RNAi proficient L. braziliensis are underway.Our findings provoke the question of whether the RNAi machinery could be transplanted from Leishmania braziliensis. These data provide some optimism for the application of RNAi approaches as a tool for the study of these predominantly asexual organisms, by forward and reverse genetic approaches. While less experimentally developed, L. braziliensis has the potential to emerge as an attractive model, and the advent of RNAi-based tools should provide a further stimulus for this effort. In the long term, delivery of siRNAs targeting essential parasite genes may prove an effective route to chemotherapeutic treatment of RNAi-proficient Leishmania. Lastly, the Leishmania provide an attractive system for testing hypotheses about forces leading to the evolutionary loss of RNAi, including the role of viral pressure, changes in genome plasticity, and virulence. As drug resistance mediated by gene amplification is one manifestation of gene plasticity, these findings have practical implications to parasite chemotherapy.In summary, we have shown that the RNAi pathway is functional in L. braziliensis α-tubulin mRNA plus the first 317 nt of the ORF were PCR-amplified from genomic DNA and inserted between the HindIII and XbaI sites of plasmid vector pPD19.36, which contains two opposing T7 RNA Polymerase promoters L. braziliensis SLACS (LbrM08−V2.0700) was PCR-amplified with primers (LBSLACS1399F: 5′-GCCAGAGAGGTGGTGAGGGTG and LBSLACSORFa-R: 5′-GAGCTCGAGAAAGGTCCACCACCCCGA) from M2903 genomic DNA and TA cloned to generate a sense radiolabeled RNA probe for Northern analysis of small RNAs. For LPG2 (LbrM20_V2.2700) the probe was a PCR fragment (nt 1 to nt 411) amplified with primers SMB3219 and SMB3220 , treated with DNAse and purified using MEGAclear columns (Ambion). Reverse transcription (RT) was performed according to the manufacture instructions using Superscript III First-Strand reverse transcriptase (Invitrogen) in a 20 µl reaction containing 1µg purified RNA. Controls containing the same amount of RNA but lacking reverse transcriptase or template were used to rule out DNA or other contamination. For test RNAs, primers were designed to amplify ∼100 bp amplicons within the target ORF but outside of the stem-fragment, and tested using L. braziliensis gDNA. PCRs were performed using the SYBR Green (Applied Biosystems) and the ABI PRISM 7000 Sequence Detection System instrument (Applied Biosystems). PCR amplifications were performed as follows: 50°C for 2 min and 95°C for 10 sec then followed by 40 cycles of 95°C for 15 sec, 60°C for 1min. The generation of specific PCR products was confirmed by melting curve analysis and agarose gel electrophoresis. Each primer set was individually tested for four StL transfectants . All samples were performed in triplicate. Control samples of H2O were included in each experiment. Amplification of SSU rRNA was used as internal control to normalize the parallel reaction of target amplicons.L. braziliensis M2903 (MHOM/BR/75/M2903), L. guyanensis M4147 (MHOM/BR/75/M4147) and L. panamensis WR120 (MHOM/PA/74/WR120) were obtained from Diane McMahon-Pratt , L. braziliensis strain M2904 from Angela Cruz (U. Sao Paulo Riberao Preto), L. tarentolae strain TarII was obtained from M. Ouellette and B. Papadopoulou , L. mexicana (MNYZ/BZ/62/M379) from David Russell (Cornell University), and Crithidia fasciculata Cf-C1 from Larry Simpson (UCLA). The LRV-bearing strain of L. guyanensis M4147 (MHOM/BR/75/M4147) and a virus free derivative M4147/pX63-HYG Viannia strains used were confirmed by partial and/or complete sequencing of the AGO1 or other genes (not shown).Viannia species were grown in freshly prepared Schneider's Insect Medium (Sigma-Aldrich Cat. No. S9895) supplemented with 10% heat-inactivated fetal bovine serum, 2 mM L-glutamine, 500 units penicillin/ml and 50 µg/ ml− streptomycin (Gibco Cat No. 5070). Other Leishmania and Crithidia were propagated in M199 medium supplemented with 10% heat-inactivated fetal bovine serum, hemin, adenine, biopterin and biotin L. braziliensis were resuspended in 100 µl human T-cell Nucleofector solution (Amaxa Cat No. VPA-1002) mixed with 5 µl of 4 µg/ µl of α-tubulin dsRNA or control dsRNA and subjected to nucleofection with an Amaxa Nucleofector with program U-033 using the kit's cuvette. The transfection mixture was transferred immediately to 10 ml of complete medium and kept in 28°C for 3 hrs. RNA from 9 ml cells was taken for Northern blot analysis with an α-tubulin hybridization probe.For each transfection, 10 ml of log phase SAT marker, parasites were plated on 50–100 µg/ml nourseothricin , and with the PHLEO marker, parasites were plated on 0.2–2 µg/ml phleomycin (Sigma). After colonies emerged  they were recovered and grown to stationary phase in 1 ml media, and passaged thereafter in 10 and 0.1 µg /ml nourseothricin and phleomycin, respectively. The plating efficiency of untransfected L. braziliensis M2903 ranged from 60–95% and the transfection efficiency from 50–220 colonies / 20 µg DNA.Stable transfections were performed using the high voltage (1400V) procedure described previously Crithidia fasciculata strain Cf-C1 by 454 sequencing technology will be described fully elsewhere. Blast searches using L. braziliensis AGO1 were used to identify homologous sequences, which were then assembled manually into several large contigs. PCR primers were designed to amplify missing gaps, and the 5′ end of the mRNA was obtained by RT-PCR using a forward miniexon primer (CFSLB 5′-AAGTATCAGTTTCTGTACTTTATTG) and reverse CfAGO1 specific primer (SMB2895: 5′-AAGCAGTTCGTCTCCACCGTCACCTG). Then a nested PCR was done with CfSLB and CfAGO1 primers (SMB 2894: 5′- GTGATGCCGCCCTCCTCGCGGTCACG). The PCR products were TA cloned and sequenced. The CfAGO1 sequence was deposited in GenBank (EU714010). We noted a polymorphism in the CfAGO1 sequence, introducing a stop codon yielding a truncated protein terminating after amino acid 198. The consequences of this polymorphism (if any) have not been investigated further. The sequence of the L. guyanensis M4147 AGO1 ORF was determined by direct sequencing of the PCR amplicon obtained with primers B2468 (5′-ATGTTGGCGCTAAACGCAGGTTC) and B2469 (5′- CTACAGGTAGTGCATCGTGGGGC), and deposited in GenBank (accession number FJ234150).The generation of whole genome shotgun sequence data from RT-PCR reactions were performed as described above, with two sets of primers to detect LRV viruses described previously XbaI/SmaI, site a, and BglII, site b). High fidelity thermostable polymerases such as recombinant Pfu DNA polymerase (Stratagene) were used for PCR, and constructs were confirmed by restriction mapping and sequencing of all relevant regions. Unless otherwise indicated, all constructs were digested with SwaI and the linear SSU-targeting fragment purified for subsequent transfection by electroporation.The constructs used in this work are derivatives of pIR1SAT (B3541) SAT marker of pIR1SAT with the PHLEO marker . pIR1PHLEO-GFP+(a) (B5793), pIR1PHLEO-GFP65(a) (B5779) and pIR1-GFP65*(a) (B5959) were constructed by generating ORF cassettes of the respective genes and inserting into the XbaI (a) site. The GFP+ ORF was taken from pXG-GFP+ (B2799), GFP65 from pXG-GFP65 (B2355), and GFP65* was obtained by site-specific mutagenesis of pIR1PHLEO-GFP65 , changing nt 193 from T to A, resulting in a S65T mutation. A luciferase (LUC) ORF was amplified using pGL3-basic (Promega) as template, with primers adding flanking BglII sites, and a CCACC initiation sequence preceding the initiation codon. The modified LUC ORF was inserted into pGEM-T (Promega) yielding pGEM-Luciferase (B6033); the LUC ORF was then extracted by BglII digestion and inserted into the BglII site of B5959 to create pIR1PHLEO-GFP65*(a)-LUC(b) (B6034).pIR1PHLEO (B4054) was created by replacing the LPG1 ; LPG2 ; LPG3 ; HGPRT ; α-tubulin ; β-tubulin ; PFR1 ; PFR2 , AGO1  and LUC . These steps yielded constructs pIR1SAT-LPG1-StL (B6128), pIR1SAT-LPG1-StL  (6132), pIR1SAT-LPG2-StL (B6137), pIR1SAT-LPG2-StL (B6138), pIR1SAT-LPG3-StL (B6140), pIR1SAT-HGPRT-StL (B6136), pIR1SAT-HGPRT-StL (B6135), pIR1SAT-PFR1-StL (B6294), pIR1SAT-PFR2-StL (B6282), pIR1SAT-αTub-StL (B6283), pIR1SAT-βTub-StL (B6295) and pIR1SAT-LUC-StL (B6185), or pIR1SAT-LUC-StL (B6190).pIR1SAT-GFP65-StL(b) (B4733) was described previously LUC Stem-Loop into the ‘a’ site of a modified pIR vector (pIR2SAT-LUC-StL(a)-LUC(b) (B6386). This construct is referred to as the ‘LUC RNAi self reporter’ or ‘LUC SR’. For RNAi studies of AGO1, an analogous construct was made with a HYG marker (pIR2HYG-LUC-StL(a)-LUC(b), strain B6447). A pIR1SAT-LbrAGO1-StL(b) construct was used for RNAi tests (B6524).A single construct enabling tests of RNAi activity was generated by inserting the LUC ORF into the ‘b’ site and a L. donovani HGPRT antiserum  as the primary antibody, and detected using goat anti-rabbit IgG as the secondary antibody . Parasites expressing GFPs were analyzed using a Becton-Dickenson FACS Calibur, using fluoroscein excitation/emission parameters. LPG was purified and quantitated from L. braziliensis lines grown in logarithmic phase (4–5×106 cells/ml) as described L. braziliensis LPG Western blots were performed as described elsewhere using anti-GFP  or anti-6 logarithmic phase promastigotes were suspended in 200 µl media containing 30 µg/ ml of luciferin (Biosynth AG) and added to a 96-well plate . After 10 min incubation, the plate was imaged using a Xenogen IVIS photoimager , and luciferase activity quantitated as photons/sec (p/s).10Promastigotes were fixed in 2% paraformaldehyde/2.5% glutaraldehyde  in 100 mM phosphate buffer, pH 7.2 for 1 hr at room temperature. Samples were washed in phosphate buffer and postfixed in 1% osmium tetroxide  for 1 hr. Samples were then rinsed extensively in water prior to en bloc staining with 1% aqueous uranyl acetate  for 1 hr. Following several rinses in water, samples were dehydrated in a graded series of ethanol solutions and embedded in Eponate 12 resin (Ted Pella Inc.). Sections of 95 nm were cut with a Leica Ultracut UCT ultramicrotome , stained with uranyl acetate and lead citrate, and viewed on a JEOL 1200 EX transmission electron microscope .Table S1Primers used for qRT-PCR.(0.05 MB DOC)Click here for additional data file.Figure S1L. braziliensis M2903 promastigotes and Trypanosoma brucei procyclics. This Northern blot was probed with a L. braziliensis SLACS probe and the autoradiogram is shown. Trypanosome SLACs differs greatly in sequence from that of L. braziliensis and as expected no siRNA hybridization is evident. Size standards are in the left track.SLACS-derived siRNAs. siRNA analysis of RNAs from (0.47 MB TIF)Click here for additional data file.Figure S2GFP Reporters ORF nucleotide alignment. An alignment of the AT-rich GFP65 ORF and GC-rich GFP+ nucleotide sequences is shown. The T→A mutation in GFP65* (S65T in the protein) is indicated. Regions of identity are boxed.(2.87 MB TIF)Click here for additional data file.Figure S3L. braziliensis M2903 with lines bearing a Luciferase reporter subsequently transfected with LUC-StL. Luciferase activity (photons/sec or p/s) was measured as described in the L. braziliensis M2903 (Lb WT) and L. braziliensis M2903 expressing luciferase -LUC(b)). Test transfectants of Lb+LUC additionally expressed the LUC-StL in a convergent (SSU:SAT:LUC-StL(b-CONV); Panel A) or divergent orientation (SSU:SAT:LUC-StL(b-DIV); Panel B). GFP expression varied less than 10% amongst experimental samples.Tests of RNAi in (0.33 MB TIF)Click here for additional data file.Figure S4Crithidia. Crithidia fasciculata clone Cf-C1 was electroporated with the targeting fragment from pIR1SAT-HYG(a)-GFP(65)-StL(b), yielding SSU:SAT-HYG-GFP(65)-StL transfectants. These were confirmed by PCR tests for the marker and presence of the inverted GFP65 repeats, and RNA was isolated and subjected to Northern blotting for siRNAs using a GFP65 probe.GFP siRNAs in (0.94 MB TIF)Click here for additional data file.


Mammalian ATAD3 is a mitochondrial protein, which is thought to play an important role in nucleoid organization. However, its exact function is still unresolved.Caenorhabditis elegans (C. elegans) ATAD3 homologue (ATAD-3) and investigate its importance for mitochondrial function and development. We show that ATAD-3 is highly conserved among different species and RNA mediated interference against atad-3 causes severe defects, characterized by early larval arrest, gonadal dysfunction and embryonic lethality. Investigation of mitochondrial physiology revealed a disturbance in organellar structure while biogenesis and function, as indicated by complex I and citrate synthase activities, appeared to be unaltered according to the developmental stage. Nevertheless, we observed very low complex I and citrate synthase activities in L1 larvae populations in comparison to higher larval and adult stages. Our findings indicate that atad-3(RNAi) animals arrest at developmental stages with low mitochondrial activity. In addition, a reduced intestinal fat storage and low lysosomal content after depletion of ATAD-3 suggests a central role of this protein for metabolic activity.Here, we characterize the C. elegans development in vivo. Moreover, our results suggest that the protein is important for the upregulation of mitochondrial activity during the transition to higher larval stages.In summary, our data clearly indicate that ATAD-3 is essential for  Mitochondria are semi-autonomous organelles in eukaryotic organisms, which have a profound role in cell viability through the control of energy production via oxidative phosphorylation (OXPHOS) The mitochondrial DNA (mtDNA) typically forms a circular, double-stranded molecule that spans about 16,569 base pairs. It encodes 37 genes, all of which are essential for normal mitochondrial function  Obviously, gene expression and (timely) replication of mtDNA require a great number of regulatory factors, which are nuclear-encoded and translocate to the mitochondria to execute their tasks. In addition, specific proteins are involved in maintenance and structural organization of mtDNA. A novel candidate with a putative role in theses processes is mammalian ATAD3, an ubiquitously expressed protein, which was initially found in a proteomic screen of rat liver mitochondria  homologue of vertebrate ATAD3. We demonstrate that ATAD-3 is a mitochondrial protein and RNA mediated interference (RNAi) induced depletion leads to severe phenotypes, characterized by early larval arrest, gonadal dysfunction, reduced intestinal fat storage and embryonic lethality. Investigation of mitochondrial physiology revealed a disorganized organellar morphology in young adults. Nevertheless, enzymatic measurements after atad-3(RNAi) demonstrated that arrested L1 larvae had similar OXPHOS activities when compared to wild type L1 larvae. However, we also observed a clear increase in OXPHOS activities at later developmental stages in wild type animals, indicating an activation of mitochondrial biogenesis, which is apparently disturbed in atad-3(RNAi) animals.Here, we characterized ATAD-3, the C. elegans protein, encoded by the predicted open reading frame (pORF) F54B3.3, as a highly conserved homologue of human ATAD3. BLAST analysis Drosophila (CG6815-PA) with overall sequence identity of 58%, 55% and 53%, respectively  motif C. elegans ATAD-3 . However, there was no detectable signal in the cytoplasmic fraction. As further depicted in C. elegans ATAD-3 and human ATAD3, both protein localize to mitochondria.To confirm that ATAD-3 is indeed a mitochondrial protein, we generated anti-ATAD-3 antibodies see . and perions see .. As illC. elegans, we performed RNA mediated interference (RNAi) by “feeding” atad-3(RNAi) animals revealed a clear decrease in the level of protein expression (To further investigate the function of ATAD-3 in pression .C. elegansatad-3(RNAi) animals showed a slightly disorganized mitochondrial network in comparison to control animals fed with HT115 bacteria carrying the “empty” L4440 “feeding”-vector (F) and mitochondrial filamentation (aspect ratio = AR) of mitochondrial regions revealed a slight trend towards a more filamentous network in atad-3(RNAi) animals  animals (see atad-3(RNAi) on mitochondrial mass in adult worms/at this developmental stage.To gain information on potential changes in mitochondrial mass in young adult worms, we performed Western blot analysis of ATAD-3, NUO-2 and GAPDH protein expression levels. Experiments revealed no drastic changes in NUO-2 protein levels  in mals see . MoreoveC. elegans tissue homogenates. Mitochondrial complex I is the largest enzyme of the respiratory chain and constitutes the entry point of electrons into the system. Cirate synthase is a key enzyme of the krebs cycle and previous studies showed that it can be used as an accurate marker of mitochondrial mass in tissue atad-3(RNAi) treatment (atad-3(RNAi) animals did not develop normally but arrested at early larval stages, primarily at the L1 stage. To study the link between CI and CS activities with the stage of development we performed enzymatic measurements on synchronized wild type L1 larvae compared to a mixed stage wild type population. It revealed that wild type L1 worms typically presented low activities of complex I and citrate synthase per milligram of tissue protein, as compared to a mixed stage wild type population. This demonstrates that ATAD-3 deficiency does not alter mitochondrial function in L1 larvae, but suggests that it hinders the transition from L1 stage to later stages where mitochondrial activity is normally enhanced. Accordingly, the drastic increase observed in mitochondrial CI and CS activities at higher larval stages (C. elegans development. These data suggest that atad-3(RNAi) worms cannot perform this transition.To further investigate whether changes in mitochondrial architecture were subject of disturbed mitochondrial function or respiratory chain content, we performed enzymatic measurements of mitochondrial NADH-ubiquinone oxidoreductase (complex I) and citrate synthase (CS) in reatment . Howeverl stages  indicateatad-3 (RNAi) in more detail. Our first observation was that most of atad-3(RNAi) L1 larvae remained small and showed developmental arrest at this stage compared to wild type animals (atad-3(RNAi) plates for 3 days, only 20 became adult, while 11, 16, 31 and 51 arrested in L4, L3, L2 and L1 larval stages, respectively (atad-3(RNAi) animals was comparable to the control (data not shown).Based on the above mentioned observations, we investigated the phenotype caused by  animals . After iectively . Interesatad-3(RNAi) plates, we first noticed a decrease in the reproduction rate. Whereas the wild type control produced about 62 embryos within in the first 12 h, atad-3(RNAi) animals only produced 29 embryos (atad-3(RNAi) induced a very drastic reduction, finally leading to a complete failure of reproduction after 36 h (atad-3(RNAi) treatment of young adults developed apparently normal but showed arrest as L1 larvae. In contrast, atad-3(RNAi) embryos produced by young adults between 24 h–48 h showed an embryonic lethal phenotype with failure of pronuclear migration and abnormal spindle orientation  adults (see above), the overall gonade morphology, as judged by light microscopy, appeared to be rather normal, containing gonadal arms with a distal region, a loop and a proximal region where oocytes are formed (atad-3(RNAi) gonads the anti-ATAD-3 signal was strongly reduced but oogonia retained a rather well organized honeycomb-like pattern  analysis. Both parameters can be measured by the vital dyes Nile Red  animals accumulated significantly less fat than their wild type counterparts (atad-3(RNAi) animals  phenotype arise. Apart from the severe developmental disturbance, the observed alterations in mitochondrial organization are interesting and might indicate a functional defect. Increasing evidence has been provided that mitochondria form a functional reticulum, which on the one hand protects individual mitochondria from stochastic depletion of metabolic substrates or mtDNA and on the other hand mediates the sharing/transport of intramitochondrial constituents like antioxidants C. elegansatad-3(RNAi) animals may indicate additional changes in mitochondrial architecture. However, at the current stage, we cannot rule out that these changes may be due to reduced GFP transport to the mitochondrial matrix.In a combined view of these findings, interesting similarities to our C. elegans L1 larvae displayed very low complex I and citrate synthase activities when compared to a mixed stage wild type population, indicating a low mitochondrial content at this developmental stage. Apparently, atad-3(RNAi) animals mainly arrest at developmental stages with low mitochondrial content. These findings indicate that OXPHOS derived energy requirements are rather low during early larval development, whereas during later development the activation of OXPHOS/biogenesis of mitochondria is initiated to fulfill metabolic demands. These observations are in keeping with other studies showing that mtDNA copy number drastically increases especially after the L3 stage atad-3(RNAi) worms mainly arrest at developmental stages with low mitochondrial tissue content and activity.A remarkable finding of our study was that atad-3(RNAi) was a failure of fertilization. The distal part of the C. elegans gonad is a non-cellularized syncytium where germ line nuclei divide, while in the proximal part cellularization and oocyte growth takes place C. elegans, alterations in mitochondrial function might disrupt normal tissue function . After RNAi against atad-3 no obvious defects became apparent in the distal and proximal region of the gonad, as judged by immunofluorescence and Nomarski optics, respectively (The most obvious tissue-specific dysfunction that we observed after ectively . A furthatad-3(RNAi). These findings might suggest a disturbed balance between fat storage and mobilization, possibly indicating a problem with energy homeostasis and metabolic activity. Importantly, McKay et al. (2003) described that disturbed function of the mitochondrial respiratory chain severely impairs intestinal fat storage in C. elegansC. elegans (e.g. the germ line and the intestine atad-3(RNAi).In line with this idea, we also observed a reduced intestinal fat storage and lysosomal content in young adults after atad-3(RNAi) analysis reveals interesting parallels to phenotypes caused by other mitochondrial dysfunctions in C. elegans . The fragment was cloned in frame in the 6× His-tag expression vector pQE30 (Qiagen), transformed and induced in E. coli strain M15 [pREP4] (Qiagen). Recombinant protein was purified on Ni2+-NTA matrix (Qiagen) and sent to Eurogentec  for immunization. The final bleed of one rabbit was purified using columns and the recombinant protein as a bait. For elution of anti-ATAD-3 antibodies Tris-Cl (pH 8.0) was used.PCR was used to amplify a 900 base pair fragment, encoding the C-terminal 300 amino acids of ATAD-3 (-HPIK… ETAV): forward: atad-3 was obtained from the Ahringer RNAi library (Geneservice Limited). After amplification of a single colony overnight , HT115 (DE3) bacteria  were seeded on NGMamp_tet plates, containing IPTG (1 mM) and further incubated overnight at room temperature (22°C) to allow the expression of double-stranded RNA. HT115 (DE3) bacteria harboring the “empty” KS+based vector, L4440 (containing two T7 promoters flanking a polylinker), were used as a control for RNAi “feeding” experiments.RNAi by “feeding” was performed essentially as described by atad-3(RNAi) bacteria plates. The plates with worms were incubated at room temperature and evaluated after 3 days. The effects on embryonic production were determined by counting the numbers of progeny that managed to hatch on either “WT control” plates or on atad-3(RNAi) bacteria plates.To determine the larval arrest phenotype, wild type L1 stage larvae were either placed on plates seeded with HT115 bacteria containing the “empty” feeding vector L4440 (“WT control”) or on Gravid adults were transferred with a drawn-out pipette to a microscope slide, coated with a thin layer of polylysine in a drop of sterile M9 buffer and cut with a scalpel. Embryos were immediately permeabilized by the freeze-crack method AR) and mitochondrial branching (formfactor = F) were quantified by using a modified computer-assisted approach published by Koopman et al., 2008 . For phenotypic characterization, worms were investigated under a Zeiss Axioplan 2 research microscope fitted with a Hamamatsu Orca-ER camera. To analyze mitochondrial structure, muscle cells of SJ4103 young adults Western blot analysis was performed essentially as described previously For separation of mitochondrial and cytoplasmic fractions, worms were harvested from well grown NGM plates and washed twice with M9 buffer. Washed worms were suspended in MSE buffer . After freeze/thaw three times using liquid nitrogen, worms were homogenized at 4°C, using a glass homogenizer and centrifuged at 4°C, 2000 rpm for 15 minutes. To separate mitochondria from cytoplasmic fraction the supernatant was centrifuged at 4°C, 13000 rpm for 15 minutes .2 spectrophotometer (SAFAS), using standardized and reproducible methods as previously described 2PO4, 5 mM MgCl2, 2 mM KCN, pH 7.2) was supplemented with 2.5 mg/ml BSA, 2 µg/ml antimycin A, 0.1 mM UQ1decylubiquinone and 0.1 mM NADH in a final volume of 1 ml. Enzyme activity was measured by starting the reaction with 20 µg of mitochondrial protein. The decrease in absorption due to NADH oxidation was measured at 340 nm in both the absence and presence of rotenone 10 µg/ml. The difference in both activities indicates the rotenone-sensitive activity of complex I. The extinction coefficient used for NADH was 6.22 mM−1 cm−1. The reduction of 5′,5′-dithiobis(2-nitrobenzoic acid) (DNTB) by citrate synthase (CS) at 412 nm (extinction coefficient 13.6 mM−1 cm−1) was followed in a coupled reaction with coenzyme A and oxaloacetate. A reaction mixture of 20 mM Tris-HCl, pH 8.0, 0.42 mM acetyl-coenzyme A, 0.1 mM DTNB and 20 µg of mitochondrial proteins was incubated at 37°C for 5 min. The reaction was initiated by the addition of 0.5 mM oxaloacetate and the absorbance change monitored for 5 min.Animals were collected in M9 buffer and frozen in liquid nitrogen. Semi-synchronized L1s were obtained by treatment with alkaline hypochloride (2 vol 4 M NaOH : 3 vol 13% NaOCl) . The pelhttp://rsbweb.nih.gov/ij/).Assays were performed essentially as described by others Figure S1Western blot analysis of ATAD-3, NUO-2 and GAPDH protein expression levels. Western blot analysis of ATAD-3, NUO-2 and GAPDH protein expression levels in young adult WT and atad-3(RNAi) animals. Experiments revealed no drastic changes in NUO-2 protein levels in atad-3(RNAi) animals, suggesting no major influence of atad-3(RNAi) on mitochondrial mass in these worms/at this developmental stage.(0.85 MB TIF)Click here for additional data file.