Tinospora cordifolia (Willd.) Miers. The adaptogenic effect of three samples of Guduchi ghrita, prepared using plain ghee (clarified butter) obtained from three different sources was studied in albino rats and compared with expressed juice of stem of Guduchi. The test preparations were evaluated against forced–swimming induced hypothermia, gastric ulceration and changes in the hematological parameters. The test drug given in the form of 'ghrita' produced better effect in comparison to the expressed juice. Among the three 'ghrita' preparations evaluated, only the 'Solapur Guduchi ghrita' (SGG) was found to produce significant inhibition of stress hypothermia and gastric ulceration. The other two preparations 'Nanded Guduchi ghrita' (NGG), and 'Wardha Guduchi ghrita' (WGG) could produce only a marginal effect. In hematological parameters 'Guduchi' juice produced better reversal of the stress-induced changes in comparison to the test 'ghrita' preparations. The present study provides evidence highlighting the importance of formulation factors for the expression of biological activity.This study was undertaken to investigate the impact of formulation factors and adjuvants on the expression of biological activity of  Ghrita',also known as clarified butter, is a traditional adjuvant/ vehicle described in Ayurveda.[ghrita' with plant material is renowned for enhancing the therapeutic efficacy of the plant ingredients. In this reference, a specific word 'Sahasraveerya,' depicting infinite potency or power of 'ghrita,' is used by Charaka. The 'ghrita' is believed to be made more potent through its processing with substances possessing different properties. More interestingly, 'Ghrita' also possesses the property of 'yogavaahitwa' by way of which it enhances the properties of substances with which it is processed.[Tinospora cordifolia (Willd.) Miers., (Menispermaceae) is described in Ayurveda as a 'rasaayana'[Guduchi ghrita', is significantly superior to the antipyretic effect observed in 'Guduchi' juice-administered group.[Guduchi' juice-administered group was comparatively faster in appearance but was short lived. It appeared late but sustained for a longer period up to 12 hours, in all the three 'Guduchi ghrita' groups. The present study was conducted to evaluate the adaptogenic activity and hematologic effects of different formulations of Tinospora cordifolia in ghrita (ghee) obtained from various sources and the expressed juice of the stem of Guduchi.'Ayurveda. Processirocessed. Tinosporasaayana' and receasaayana'4 The plaasaayana' Our earled group. The antiTinospora cordifolia (Willd.) Miers were collected during winter (November 2005), as this is the suitable season for collection of plants,[Mature stems of f plants, Collectighrita', clarified butter or ghee prepared in the traditional way from cow's milk, were obtained from three different places namely Wardha, Nanded and Solapur and were used as the base materials for preparing 'Guduchi ghrita'.Three samples of 'Guduchi ghrita' each from 1 kg of plain ghee were prepared by one person at one place for this study in accordance with the formulation prescribed by 'Vaagbhata and explained by Indu'.[kalka' i.e. paste of 'Guduchi', 'ghrita' and 'swarasa' of 'Guduchi' was 1:4:16 respectively as prescribed by Indu.[Guduchi' is hard in consistency and does not yield 'swarasa' in adequate quantity by the method of crushing and squeezing the material. Hence the method described by Sharngadhara*[Guduchi swarasa'. The stem of 'Guduchi' was crushed and overnight soaked in double the quantity of water. The contents were then filtered through muslin cloth. The filtrate so obtained was used as 'Guduchi swarasa' (Expressed juice). All the ingredients were placed in a container, and were heated on a gas stove to evaporate aqueous part. The contents were stirred regularly to avoid burning. Heating was stopped when the 'ghrita' passed all the tests described in the text, such as disappearance of froth, formation of a wick, burning of 'ghrita' without crackling noise etc.[Guduchi ghrita' was collected in a clean autoclaved glass bottle and stored in a cool place. The test 'Guduchi ghritas' were named after the places from which the plain 'ghrita' samples were obtained, as Nanded Guduchi Ghrita (NGG), Wardha Guduchi Ghrita (WGG) and Solapur Guduchi Ghrita (SGG). The three samples of plain 'ghrita' used and the three samples of test formulations were subjected to preliminary physical and chemical investigations. Noted observations are shown in Tables Guduchi' sample was tested for bitterness value. Macro and microscopic studies of the sample were also carried out. High performance thin layer chromatograph profile of the plant material used, and the three final formulation products, was recorded.[ghritas', sourced from different places, the same plant material was used for all the preparations.Three samples of 'by Indu'. The prop by Indu. The stemngadhara* was usedoise etc. The contrecorded. Since thCharles Foster strain albino rats of either sex weighing between 140 and 200g, procured from the institute's animal house were used. They were maintained on 'Amrut' brand rat/mice pellets and exposed to ambient temperature, humidity and natural day and night cycles. All the experiments were carried out between 8 am and 12 noon. The studies were conducted in conformity with the guidelines of the Institutional Animal Ethics Committee after obtaining its permission.Guduchi ghrita' as practiced traditionally is 10 g per person. (The dose of 'ghrita' preparations as prescribed by Ayurvedic Formulary of India (AFI) is 12 g. However 10 g dose was taken for this study as an average dose.)[The human dose of 'ge dose.)ad libitum ii) 'Guduchi swarasa' , and iii) WGG 900 mg/kg body weight orally for 7 days, iv) NGG 900 mg/kg body weight orally for 7 days, v) SGG 900 mg/kg body weight orally for 7 days, vi) Plain 'ghrita' . The samples were used for estimation of hematological parameters. Hematological parameters were estimated with the help of an auto cell counter , which along with RBC counts, additionally gives size based quantification of RBC (micro and macro). After careful laparotomy, the stomach was excised out by opening along the greater curvature. It was washed gently with tap water. The inner surface of the stomach was carefully observed with a magnifying lens for the number of ulcers and severity of ulceration in glandular area as well as in lumen.  The seve0‐No visible ulcer1‐Approximate ulcerated area up to 1 mm. in diameter2‐Approximate ulcerated area up to 2 mm. in diameter3‐Approximate ulcerated area up to 3 mm. in diameter4‐Approximate ulcerated area more than 3 mm. in diameter5‐Perforation of the gastric wallSeverity of the ulceration and the number of ulcers in each rat stomach were recorded for calculating ulcer index.P< 0.05 was considered to be significant. In case of Ulcer index Mann Whitney U test was employed. The analysis was carried out using Sigma Stat version 3.1 and Graph pad 3.The data are presented as Mean ± Standard Error of Mean (SEM) for 6 animals per group. Comparison between control and test drug administered groups was made by one way ANOVA followed by Dunnet's multiple 't' test or by unpaired 't' test. The data pertaining to the effect of different types of treatments on forced-swimming-stress-induced hypothermia are shown in ghrita'-, test ghritas, and 'Guduchi swarasa'-administered groups an apparent decrease in hypothermia was observed in comparison to water control. However, the decrease observed in pooled plain ghrita and test ghritas was found to be statistically significant. When test ghritas were compared to pooled plain 'ghrita' control group, the decrease observed in SGG was also found to be significant. However, the observed difference in other groups was non-significant.Marked hypothermia was observed in water control rats subjected to forced-swimming stress. In pooled plain 'ghrita'-administered group, the severity of ulceration was 27.8% less in comparison to normal control. Severity of ulceration increased slightly in WGG- and moderately in NGG administered group. However, in SGG-administered group, significant decrease was observed in comparison to water control. When the data of SGG treated group was compared with pooled plain ghrita control group though 40% decrease in severity was noted, the difference was found to be statistically non-significant. Surprisingly in 'Guduchi swarasa' administered group, the severity was more than doubled in comparison to pooled plain 'ghrita' group. Because of the variation of the data and large SEM, the observed difference was found to be non-significant.Moderate–to-severe ulceration in the stomach was observed in control rats subjected to forced-swimming stress. In pooled plain 'The data related to the effect of test preparations on stress induced changes in WBC related parameters can be seen in ghrita' administered group, the decrease in count was marginally less in comparison to stress control group. In test preparation-administered groups, the observed decrease was further less in comparison to the stress control group and was statistically significant employing unpaired 't' test. This difference was not reflected when ANOVA test was applied, however this test demonstrated significant difference between pooled plain 'ghrita' administered group and SGG treated group.Rats subjected to forced-swimming stress showed marked decrease (52.94%) in total WBC count in comparison to non-stressed control rats. In pooled plain 'ghrita' and SGG treated group. The stress-induced decrease observed in lymphocyte count was not reversed by treatment with either pooled plain 'ghrita' or test 'Guduchi ghrita' preparations. Weak reversal of the stress-induced decrease in granulocyte count was observed in 'Guduchi' juice and NGG-administered groups. However, the reversal did not reach statistically significant level. Other preparations had no effect on this parameter. Moderate reversal of stress-induced decrease in monocyte count was observed in 'Guduchi' juice administered group but it was statistically non-significant. No reversal could be observed in other test preparations-administered groups.Forced-swimming stress was found to decrease lymphocyte, granulocyte and monocyte counts in comparison to normal control rats, which was statistically significant in pooled plain 'guduchi juice treated group showed significant decrease in this elevation against stress water control when compared using unpaired 't' test.Though absolute count of lymphocyte was found to be decreased  in stressed animals, in comparison to normal control when the data were presented as percentages, significant increase in lymphocyte percentage and significant decrease in granulocyte and monocyte percentages were observed in stress control rats in comparison to normal control rats. The elevated lymphocyte percentage due to stress was not significantly reversed by any of the treatment mode employed (ANOVA). However, the Guduchi' juice, which did not reach statistically significant level (ANOVA). However, when compared using unpaired 't' test, it showed significant increase against stress water control. Similar non-significant reversal was observed in NGG and WGG administered groups.The stress-induced decrease in granulocyte percentage was antagonized by the administration of 'Guduchi juice reversed the stress induced decrease, which was not significant statistically.In case of monocyte percentage, Mean Corpuscular Volume (MCV) and Hematoctrit (HCT)% were found to be apparently increased in stress water control in comparison to normal water control groups. Although, unpaired 't' test showed that the observed increase is significant, ANOVA test did not show this difference.ghrita group, 'Guduchi' juice and NGG administered groups. Elevation of HCT (%) was found to be significantly reversed in the 'Guduchi' juice and SGG administered groups. The reversal observed in pooled ghrita, NGG and WGG administered group was found not significant using ANOVA although when unpaired 't' test was applied the observed difference in means was significant in the pooled plain ghrita and NGG groups as well.The apparent elevation observed in MCV was found to be significantly reversed in the pooled plain There was increase in MCHC level in rats subjected to forced-swimming stress as compared to normal rats. There was no significant effect on this parameter in any of the treated groups.ghrita', WGG and SGG administered groups. The moderate reversal observed in NGG-administered and 'Guduchi' juice-administered groups was statistically non-significant.Micro RBC formation was not observed in any of the groups studied. An increase in macro RBC count was observed in stress water control in comparison to normal water control. The increase observed in macro RBC count was found to be unaffected in pooled plain 'ghrita is summarized in Tables Guduchi ghrita'.The data related to physico-chemical investigation of Bhaishajya kalpana' in Ayurveda, has an important place in Ayurvedic therapeutics. Clear-cut guidelines, such as parts of plants to be used, processes to be followed for formulations, along with indications regarding disease- and patient-specific formulations, have been prescribed in ancient Ayurvedic texts. [The science of drug formulation, termed as 'c texts.  It is beghrita' is 99.5% milk fat. It is a complex lipid of glycerides, free fatty acids, phospholipids, sterols, sterol esters, fat-soluble vitamins, tocopherol, carbonyls, hydrocarbons, carotenoids, small amounts of charred casein, traces of minerals like calcium, phosphorus, iron, copper etc. Ayurveda describes that 'Ghrita', is a useful adjuvant/ vehicle. Administering the plant material incorporated in a ghrita is known to enhance the therapeutic efficacy of the plant ingredient. Characteristics of 'ghrita' suitable for processing have also been described in ancient Ayurvedic classics.[ghrita has been described to exert therapeutic effects like 'rasaayana' (adaptogenic activity). Moreover, 'ghrita' has been described to absorb the properties of the material with which it is processed ('sanskaraanuvartana').[Chemically, 'classics. Besides artana').Guduchi' in the form of ' ghrita' exerts better adaptogenic activity compared to 'Guduchi swarasa' (expressed juice) in the forced-swimming stress- induced hypothermia and gastric ulceration model. In fact, increased ulceration was observed in the group that received 'Guduchi'-expressed juice alone. Interestingly adaptogenic activity observed was different in the three formulations. SGG was effective in providing protection against hypothermia as well as production of gastric ulcers, while WGG and NGG failed to produce a significant effect. The reason behind the observed difference among the three test formulations is not clear from our results. It is to be noted that the same type of expressed juice from the same batch was used for the preparation of 'Guduchi ghrita'; only the plain 'ghrita' used was different. This clearly indicates that the quality of plain 'ghrita' used can have important influence on the activity expressed. The chemical- or formulation-related basis for the observed difference needs to be probed. It is very important to obtain such information considering that this difference will have serious implications in clinical settings.Our study indicates that administration of 'Guduchi' juice, whereas NGG was less effective. It is to be noted that the activity profile of the test formulations is different in different sets of parameters. This indicates possible involvement of different factors and existence of difference in the test formulations' ability to modulate them. However, one feature that stands out is that SGG was able to produce anti-stress activity against majority of the parameters recorded. This indicates that the SGG has special attributes that potentiate the adaptogenic activity of 'Guduchi' juice. The exact nature of the potentiation observed in this study requires further elucidation. The data generated in this study can be taken as an example of the importance of different formulation factors in the expression of biological activity.In the present study, significant elevation in RBC count, macro RBC percentage, MCV, HCT% and significant decrease in MCHC and Hb were observed in stress control group. This contradictory data may be due to increase in the number of immature RBC leading to increased RBC count without concomitant increase in Hb%. It has been observed that kidney plays an important role in the regulation of formation of RBC through formation of erythropoietin. Hypoxia in the kidney is the major stimulant for the increased formation of RBC through secretion of erythropoietin. It can b


Endoscopic treatment for vesicoureteral reflux (VUR) has become an established alternative to long-term antibiotic prophylaxis and ureteral reimplantation. We present the outcome of endoscopic treatment with dextranomer/hyaluronic acid copolymer (Deflux) for VUR in children by a single surgeon at our institute from October 2003 to October 2009. We reviewed the cases of 150 patients , 56 girls (37%) and 94 boys (63%), with a mean age of 2.2 years and a median followup of 2.5 years (range 3–68 months). Among the 239 ureters treated, 67.4% (161/239) were cured with a single injection, and a second and third injection raised the cure rate to 86.6% (207/239) and 88.3% (211/239), respectively. None had postoperative ureteral obstruction. Vesicoureteric reflux (VUR) is a common problem encountered by pediatric urologists. Traditionally, if medical management with low-dose antibiotic prophylaxis failed, the only alternative was ureteral reimplant surgery . Since MWe retrospectively reviewed all cases of subtrigonal injection performed with Deflux from October 2003 to October 2009 at Taichung Veteran General Hospital. All patients who entered into the study had vesicoureteric reflux, as determined by voiding cystourethrogram (VCUG). The radiological grading of VUR is according to the international system introduced by the International Reflux Study Committee in 1985 . A totalOne hundred and fifty patients, 56 girls (37%) and 94 boys (63%), with a mean age of 2.2 years (range 1 mo–10 yr) underwent subtrigonal injection with Deflux from October 2003 to October 2009. Median followup duration was 2.5 years and ranged from 3 to 68 months. Eighty patients (53%) had bilateral VUR, 61 (41%) had unilateral VUR, and 9 (6%) had new contralateral VUR for a total of 239 ureters treated with a median followup of 2.5 years (range 2–62 mo). There are 9 (3.8%) ureters graded as I, 44 (18.4%) as II, 113 (47.3%) as III, 57 (23.8%) as IV, and 16 (6.7%) as V. The VCUG or radionuclide cystogram followup showed that the overall cure rate was 88.3% (211/239). The detail of the patients' data is shown in There were 73 ureters with high grade VUR, including 57 with grade IV VUR and 16 with grade V VUR. Overall cure rate was 80.8% (59/73), 5 (6.9%) ureters were downgraded to grade I, and 9 (12.3%) converted to open ureteral reimplantation. Complete resolution of vesicoureteral reflux after a single injection occurred in 33 ureters (45%), and 26 (36%) required more than 1 injection to correct VUR. No patients had postoperative vesicoureteral obstruction, gross hematuria, and febrile UTI.Our series demonstrates an overall patient cure rate of 88.3% 211/239) of the ureters among those who were cured needed more than a single injection. In addition, approximate 5% (12/239) was downgraded to grade I, and no further treatment was needed. These results are acceptable when we compare the cure rate obtained by other series shown in 1/239 of The complication rate in this series was low. Contralateral low-grade de novo VUR was present in 6% (9/239) of the patients treated for unilateral VUR—an incidence rate comparable to a previous report . ConversEndoscopic correction is a safe, effective, and minimally invasive outpatient procedure for VUR in children. It demonstrated a cure rate of approximately 92% (138/150) of patients and 88% (211/239) of the ureters by using the bulking agent, Deflux by an experienced surgeon. Even high grade VUR, complex VUR, and failed open ureteroneocystostomy do not seem to adversely affect results.


Natural landscapes are critical for species persistence and PAs can play a major role in conservation and in climate policy. Such contributions may be harder than expected to implement if new PAs are constrained to the same kinds of locations that PAs currently occupy.About an eighth of the earth's land surface is in protected areas (hereafter “PAs”), most created during the 20Quantitatively extending the perception that PAs occupy “rock and ice”, we show that across 147 nations PA networks are biased towards places that are unlikely to face land conversion pressures even in the absence of protection. We test each country's PA network for bias in elevation, slope, distances to roads and cities, and suitability for agriculture. Further, within each country's set of PAs, we also ask if the level of protection is biased in these ways. We find that the significant majority of national PA networks are biased to higher elevations, steeper slopes and greater distances to roads and cities. Also, within a country, PAs with higher protection status are more biased than are the PAs with lower protection statuses.In sum, PAs are biased towards where they can least prevent land conversion (even if they offer perfect protection). These globally comprehensive results extend findings from nation-level analyses. They imply that siting rules such as the Convention on Biological Diversity's 2010 Target [to protect 10% of all ecoregions] might raise PA impacts if applied at the country level. In light of the potential for global carbon-based payments for avoided deforestation or REDD, these results suggest that attention to threat could improve outcomes from the creation and management of PAs. Initiatives to establish new protected areas (PAs) to conserve natural landscapes for species habitat and climate-change mitigation are underway worldwide Many goals and constraints influenced past PA locations. Given that, have we maximized conservation priorities? To maximize anything, PAs must have impact, i.e. change land-use outcomes. That is, land use would have to differ from what would have occurred in a PA-free world. To change the rate of loss of natural land cover, PAs have to be located where they can prevent forest clearing et al.'s Before explaining such choices or considering changing them, though, we must ask whether the global “rock and ice” perception is correct. Previous national-level studies suggest the adage has a basis in reality All of these previous global studies have ignored political boundaries. That is a vital omission. Ecological processes cross borders but most PAs do not. The Convention on Biological Diversity's (CBD) 2010 Target aims to protect 10% of global terrestrial ecoregions We provide the first comprehensive global assessment of the distributions across space of all of the national PA networks of any significant magnitude (>100 km2). We ask whether it is true on the whole, across many countries, that national protection networks have evolved over time to be found disproportionately on higher, steeper, more remote, agriculturally unsuitable lands. We ask too whether the variation in the level of protection across the PAs within a national network is correlated with these indicators of low threat.2 or more of terrestrial surface (147 countries) and quantify the network's elevation, slope, agricultural suitability, distance to roads, and distance to urban areas, as well as its species richness  Materials and For summarizing many countries, additional description is required. Here, we empirically model the probability that a one kmFor all of our variables, we present summaries of such significance across all countries. This lets us summarize global trends in national decisions. For each variable, are biased towards higher elevation, steeper slope, and greater distance from roads and cities (away from suitable land). Yet it is in line with other variables as a majority of countries have networks on less suitable lands. A slight majority of national networks is biased away from the species-rich ecosystems.The majority of national networks d cities . The treBias in PA locations could be driven by a particular subset of the network. Six PA-management categories are defined by the International Union for the Conservation of Nature (IUCN). Greater human intervention is permitted as status moves from I to VI. Put another way, lower-numbered categories are designated as more protected. Category I-II PAs tend to be large We find  that prohow different protected lands are, in a way that brings together all the differences along the dimensions the table lists (e.g. slope and distance). Further it cannot show whether the difference between protected and unprotected lands is more or less than the difference between PAs with higher and lower protection status.To make those comparisons, we require a single index of similarity across all locations. The impact-evaluation literature regularly employs a probability of receiving intervention We find that locations currently within the protected area networks had on average a 24% chance of being protected (keeping in mind that on average only 10% of the country is protected). In contrast, the much larger group of unprotected locations had an 8% chance. Those percentages, however, are unweighted averages across many countries that are quite different in total size (the average country is 8% of the largest country) as well as in network size. When weighting the likelihood-of-protection numbers for each country by its national network size, the protected locations had a 32% chance of being protected while the unprotected had only a 14% chance. Either way, PAs are on lands more than twice as likely to be protected than unprotected lands. That is a considerable difference.Since weighting clearly matters, we also consider the medians. For protected locations, the median chance of being protected is 21% while for unprotected locations is it 6%, similar results to those above in confirming a significant difference. Looked at another way, looking across all the countries the median ratio of these two probabilities of being protected (for protected versus unprotected locations) is 3.0. By this measure of bias in location, protected lands were three times as likely to be protected as unprotected lands.Such an index is most useful for comparison purposes. It can be used, for example, to examine whether the differences just discussed are similar to those between higher and lower protection status . Using the same probabilities of being protected, we find that higher status locations had on average a 25% chance of being protected while lower status had a 20% chance (and the network-weighted averages are 27% for high status and 25% for lower). The median probability of protection for high-protection PAs is 18% while the median for lower categorized PAs is 15%. The median of the ratios of these probabilities is 1.3. This is much less bias than when we simply compared all protected versus unprotected lands.Across 147 countries' national networks, protected areas are indeed non-randomly located on the landscape. The same types of biases also hold, although less so, for the more highly protected area compared with less highly protected areas. All of these results corroborate prior descriptions of specific PA network biases towards, for instance, “rock and ice” One area of research where our results are directly relevant is in the quantification of the “conservation success” or effectiveness of protected areas. Interest in this area has been growing rapidly This same concern applies to the differing impacts across PA management categories. Location bias means that much of the observed habitat retention in PA Categories I and II Such results also naturally raise the question of why some PA networks are representative while others are not. An exploratory examination finds no impact of total population or density of population or GDP on the sign and magnitude of coefficients reported above. What this likely means is that the issue of where protection is located is too variable, even within a country or region, to be easily summarized at a global scale. For example, PAs within the United States are (on average) preferentially located on marginal lands Fortunately, many recent PAs have been (and others will be) created under consistent and locally contextualized frameworks. Systematic conservation planning et al. Our results support the idea that targeting and blocking threat may deserve higher priority in the future creation and management of PAs. The issue of locating protection in areas of greatest threat has received the most attention in the discussion of biodiversity hotspots 2 resolution. We used ArcGIS 9.1 to harmonize projections, cell size, and extent and used Python 2.4 in order to remove all marine areas and to create individual text files for each variable for each country. All further analyses were done in R 2.7.1 All datasets are global in scale, in raster (grid) format, and projected into Albers Equal Area projection at a one kmThe analytical framework we used was a general linear model (package “glm” in R 2.7.1) with a probit link because the regressions we run here involve binary outcomes. In the first set of regressions for , we are 2 or more, were included. When two PAs overlapped, we assigned that area the highest IUCN classification of the two. Due to high potential error rates Information on PA location came from the 2007 World Database on Protected Areas (WDPA) 2 or more of categories I – II and 100 km2 or more of categories III – VI. To analyze protection over time . The SRTM gathered elevation data on a near-global scale, generating a very complete high-resolution elevation database. We calculated slope values from the SRTM elevation dataset. All slope values are degrees from horizontal. Distance to roads was calculated from a vector road network extracted from the VMAP Level0 dataset We obtained elevation data from the Shuttle Radar Topography Mission (SRTM) Figure S12 of protected area. See Global maps of predictors of all categories of protection for elevation, slope, distance to roads, distance to urban areas, agricultural suitability, and species richness. Red indicates that the variable was a significant and positive factor in a regression model explaining protection. Yellow shows a significant and negative association, black indicates the variable was not a significant predictor for that country, while grey shows those countries with less than 100 km(3.01 MB TIF)Click here for additional data file.Figure S22 of protected area. See Global maps of predictors of IUCN Category I or II protection within the entire protected area network for a country elevation, slope, distance to roads, distance to urban areas, agricultural suitability, and species richness. Red indicates that the variable was a significant and positive factor in a regression model explaining protection. Yellow shows a significant and negative association, black indicates the variable was not a significant predictor for that country, while grey shows those countries with less than 100 km(2.93 MB TIF)Click here for additional data file.


Vesicoureteral reflux is uncommonly diagnosed and treated after puberty. The natural history of uncorrected VUR after puberty is not documented. Postpubertal patients with recurrent pyelonephritis and VUR should be considered for treatment. Ureteral reimplantation, endoscopic injections, and laparoscopic or robotic ureteral reimplantation may be utilized. Endoscopic injection is an appealing option for these patients. The role of laparoscopic or robotic ureteral reimplantation in these patients is evolving. Vesicoureteral reflux (VUR) is a common finding in children with urinarytract infections (UTIs). The incidenceof VUR associated with UTIs drops significantly in older children, particularlyafter the age of 5 . It has There hasbeen a long-standing observation of the association of febrile UTIs and thedevelopment of renal scarring . The risThere has also been a change in VUR management by some pediatric urologists that is thepolar opposite of stopping prophylaxis and observing patients. Instead offollowing patients for resolution of their VUR, primary therapy with endoscopicinjections after the diagnosis of VUR is being offered . Touted The primary motivation for treatmentof VUR before puberty has been over concerns of increased risk of UTIs whenfemale patients become sexually active or pregnant. To my knowledge, however, the natural historyof women with persistent VUR during pregnancy has not been documented. Pyelonephritis is a known risk to pregnantwomen and strongly associated with bacteruria. Large series have reported rates of 2% of pregnant women . InpregThe incidence of VUR after pubertyis significantly lower than in young infants with febrile UTIs . It has Most postpubertal patients with VUR will present with UTIs. Since the majority of patients will not haveVUR and assuming that VUR is an important risk factor for pyelonephritis, whatare the factors that should prompt an evaluation for VUR in a postpubertalpatient? First and foremost should be a history of recurrent febrile urinarytract infections, as these patients if found to have reflux, have a good chanceof having their symptoms alleviated if VUR is found and treated. Suspicionshould be raised if there is a history of prior VUR, febrile infections as ayoung child, or a family history of VUR. Due to the high association of renal scarring and VUR, patients who havea history of recurrent febrile UTIs and evidence of renal scarring on imagingstudies should be evaluated for VUR. To evaluate for VUR a standard VUCG may beperformed, or if there are symptoms worrisome for voiding dysfunction avideourodynamics study. In patients with evidence of renal scarring or a smallkidney on ultrasound, a DMSA renal scan should be obtained to evaluate forrenal scarring and the differential function. Typically, the grade of VUR is an important factor for predictingresolution and risk of renal scarring; however, in postpubertal patients it hasnot been studied as extensively. It is expected that the rates of spontaneousresolution will be lower after puberty.One of the more humbling experiences for a pediatric urologist is their first ureteralreimplantation done on a female after puberty. These difficulties start asfemales approach puberty. During puberty, the pelvis widens and deepens in thefemale. The trigone assumes a deeper retropubic location which makes access tothe ureteral orifices and the mobilization of the ureters more difficult. Additionally, the plexus of vein running across the surface of the bladder enlarge and are more proneto troublesome bleeding during ureteral dissection. Though not well documented,most pediatric urologists would agree that, if a child truly needed ureteralreimplantation for correction of their VUR, then the operation is bestperformed if they are operated on during childhood rather than after puberty. Experience would tell us that they recover quicker and that technically thesurgery should be more successful, however, there are not series documentingpoorer results after ureteral reimplantation for patients treated afterpuberty. Options for treatment of VUR inpatients after puberty include intra- or extra-vesical ureteral reimplantation,endoscopic injection, and laparoscopic or robotic reimplantation. In patients with unilateral VUR to a poorlyfunctioning kidney nephrectomy may be an alternative choice to ureteralreimplantation. This can be performed laparoscopically with rapid recovery andshort hospitalization.As mentioned previously, the postpubertal changes in women make the surgical access to thetrigone more challenging and limit exposure; however, ureteral reimplantationcan be performed in women successfully. In males, the changes are less dramatic. Published results of ureteral reimplantation in postpubertal patientsare sparse due to the limited number of patients in whom this surgery isindicated. From a technical standpoint,it is prudent to position the patient over the break in the OR table. This allowsthe table to be flexed if necessary to open the pelvis and improve retropubicexposure. This is similar to thepositioning of a male undergoing radical retropubic prostatectomy. The size and body mass index will play a rolewith obese patients creating more difficulties with exposure. Proper surgicalplanning and the use of larger and more flexible fixed retractors such as aBookwalter rather than the Denis-Browne retractor will facilitate theprocedure. Both ureteral advancements and ureteroneocystotomy procedures  can be performed but limiteddata is available on their use in the treatment of VUR postpuberty –20. TherAlthough controversy remains about the role of endoscopic injection for VUR as analternative to ureteral reimplantation in young children, there are few“reimplanters” who would completely dismiss this option in patients afterpuberty. This technique has been practiced for over 20 years and a variety of bulkingagents have been injected including polytetrafluoroethylene paste,gluteraldehyde cross-linked bovine collagen, polydimethysiloxane, and detranomer/hyaluronic acid copolymer (D/HA) . In the Minimally invasive ureteral reimplantation for VUR has been performed for over a decade;however its widespread uses have been slow to spread. Proponents oflaparoscopic surgery would cite the limited laparoscopic experience of mostpediatric urologists as a primary factor for this slow adaptation. However,many would argue against relearning to do a procedure which has rapid recovery,high success rate, low complication rate, and is done through an inconspicuousincision. A low, transverse abdominal incision leaves a scar that often blendsinto the natural creases of the abdominal skin leaving a barely visible mark. Ifplaced low enough it is covered by undergarments and is not disfiguring. Giventhe questionable benefits and technical difficulties the laparoscopic techniquehas been slow to gain acceptance. Onepopulation where this approach actually may be a real asset is in treating VURafter puberty.Both intra- and extra-vesical laparoscopic techniques for reimplantation have been described in children –28. In tRobotic assisted laparoscopic surgery has become a widely practiced urologic techniquefor performing radical prostatectomy . The surThe natural history of untreated VURin postpubertal patients is unknown. Treatment of VUR should be considered in those patients with VUR andrecurrent febrile UTIs. The optimal method to treat VUR is not clear. Endoscopic injection is a minimally invasive approach which has a good successrate for treating VUR and offers some benefits over ureteral reimplantation. The role of laparoscopic and robotic ureteral reimplantation is evolving andmay offer some advantages over open ureteral reimplantation in postpubertalpatients.


Micturating cystourethrogram (MCUG) is advocated in all patients with antenatally detected hydronephrosis to diagnose vesicoureteral reflux (VUR). In this study, the authors have tried to identify if MCUG is really needed for all these patients. They followed 55 children with antenatal hydronephrosis (ANH) by adopting a selective approach to performing MCUG. Ultrasound examination was done at one week for all unilateral hydronephrosis. It was done at two to three days postnatally in patients with bilateral hydronephrosis. All these patients were periodically followed up every two to three months by ultrasound. If the Antero-posterior (AP) diameter was more than 15 mm or more than 10 mm with calyceal dilatation on two months' ultrasound, Mercapto-acetyl Triglycine-3 renogram was done to rule out pelviureteric junction (PUJ) obstruction. None of the patients with AP diameter of less than 10 mm had antibiotic prophylaxis. In this study, 29 patients had MCUGs done to rule out VUR, based on the presence of any of the following ultrasound findings: bilateral hydronephrosis, ureteric dilatation, renal scarring, bladder wall thickness greater than 5 mm or presence of duplex system or ureterocele. Out of these only eight patients had VUR, of which resolution occurred in five cases. The remaining three patients did not require reimplantation of ureter. However, surgical correction was needed in one patient who underwent bilateral pyeloplasty and two who needed upper pole heminephrectomies. In 26 patients of ANH, followed without MCUGs, 18 had spontaneous resolution, five required pyeloplasty for increasing hydronephrosis and three had Multicystic Dysplasia of Kidneys (MCDK) on follow-up scans. Minimum follow-up in this study was three years. The authors believe that VUR in most antenatally diagnosed hydronephrotic kidneys is more physiological than pathological and hence resolves without long-term renal damage. More conservative approach to postnatal investigations of ANH reduces the number of unnecessary tests like MCUGs and does not result in missed renal scars.et al. have shown that neonatal VUR showed complete resolution or improvement in 95% of the cases by 20 months.[et al., studied 160 cases of unilateral HN and noted that in seven patients who were subjected to MCUGs for UTI, only one had documented VUR.[Vesicoureteral reflux is one of the common causes of ANH. There is no significant correlation between the degree of ANH and VUR. This necessitates MCUG being recommended in all cases of ANH including mild varieties to diagnose underlying VUR. This is to detect VUR which may lead to increased urinary tract infections (UTIs). The resultant long-term sequelae of renal scar and decreased renal function can thus be reduced. Farhat 0 months. Thomas ented VUR. in varioIn this study, the authors did not use prophylactic antibiotics in patients with renal pelvis diameter of less than 10 mm, who still may be having VUR, the possibility of which is already mentioned in their reference. The VUR per se is not the cause of infection but only facilitates the ascending infections, and hence the tendency to split neonatal VUR and reflux in older children, is an assertive suggestion.


Atherosclerosis is a complex pathological condition caused by a number of mechanisms including the accelerated proliferation of vascular smooth muscle cells (VSMCs). Diabetes is likely to be an important risk factor for atherosclerosis, as hyperglycemia induces vascular smooth muscle cell (VSMC) proliferation and migration and may thus contribute to the formation of atherosclerotic lesions. This study was performed to investigate whether PGC-1α, a PPARγ coactivator and metabolic master regulator, plays a role in regulating VSMC proliferation and migration induced by high glucose.PGC-1α mRNA levels are decreased in blood vessel media of STZ-treated diabetic rats. In cultured rat VSMCs, high glucose dose-dependently inhibits PGC-1α mRNA expression. Overexpression of PGC-1α either by infection with adenovirus, or by stimulation with palmitic acid, significantly reduces high glucose-induced VSMC proliferation and migration. In contrast, suppression of PGC-1α by siRNA mimics the effects of glucose on VSMCs. Finally, mechanistic studies suggest that PGC-1α-mediated inhibition of VSMC proliferation and migration is regulated through preventing ERK1/2 phosphorylation.These results indicate that PGC-1α is a key regulator of high glucose-induced proliferation and migration in VSMCs, and suggest that elevation of PGC-1α in VSMC could be a useful strategy in preventing the development of diabetic atherosclerosis. Atherosclerosis and microvascular diseases are the major vascular complications of diabetes, and constitute the principal causes of morbidity and mortality among diabetics Recent studies demonstrated that TZDs, the PPARγ ligands, decreased cardiovascular risks via exerting direct effects on vascular cells, for example, inhibition of VSMC proliferation and migration PGC-1α is a transcriptional coactivator of PPARγ and regulates expression of many genes coding for mitochondrial proteins High glucose has been reported to increase rat VSMC growth and movement The glucose-induced VSMC proliferation and migration was found to be associated with the inhibition of the expression of PGC-1α. As shown in We also examined PGC-1α mRNA expression in the blood vessel media (VSMC is the only cell type in this layer) in normal and STZ injected rat arterial samples. In STZ-treated rats, PGC-1α mRNA level was decreased by 66±14% compared to the control group .To further study the role of PGC-1α in VSMC proliferation and migration, VSMCs were infected with adenovirus driving the overexpression of PGC-1α. After 24 h PGC-1α mRNA and protein level was markedly elevated. As shown in 6 cell/dish versus 0.98±0.03×106, P<0.001, n = 6, 6 versus 1.05±0.06×106 cells/dish, P>0.05, n = 6, 15 mmol/L glucose was used to stimulate VSMC growth and movement, the number of Ad-GFP infected cells, cultured in 15 mmol/L glucose were nearly 1.5-fold higher than cells cultured at 5.5 mmol/L glucose . Following PGC-1α knockdown, the inhibitory effect of palmitic acid on VSMC proliferation and migration was abolished. As shown in High glucose stimulates VSMC proliferation and migration through the activation of MAPK ERK signaling PGC-1α, an important transcriptional coactivator, plays a key role in energy metabolism A large body of work has established that chronic hyperglycemia promotes VSMC proliferation and migration and contributes to the progress of diabetic atherosclerosis As the most prevalent saturated FFA in circulation, palmitic acid has been shown to down-regulate PGC-1α expression in muscle cells It was previously shown that high glucose induces mitogenesis in VSMCs through increasing extracellular signal-regulated kinase (ERK) activity In summary, our results suggest that high glucose increases VSMC proliferation and migration through PGC-1α. Experimental elevation of PGC-1α  inhibits high glucose-induced VSMC proliferation and migration. Thus, our results reveal a novel function of PGC-1α as a regulator of VSMC proliferation and migration, and provide a potential strategy of treatment for diabetic atherosclerosis.Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health . All animals were treated in accordance with guidelines established by the Nanjing University Institutional Animal Care and Use Committee.The investigation conforms to the Sprague-Dawley rats weighing about 180–200 g were made diabetic by a single injection of streptozotocin  according to a previously described method Primary vascular smooth muscle cells were isolated from the thoracic aortas of 3- to4-week-old male Sprague-Dawley rats and characterized morphologically and immunohistochemically as described previously Recombinant adenoviruses expressing PGC-1α GFP fusion protein and GFP alone were kindly provided by Dr. Daniel P. Kelly . VSMCs were infected with purified adenovirus at an MOI (multiplicities of infection) of 50 to obtain 95–100% efficiency, as determined by GFP expression 24 h after infection.Three siRNA sequences targeting different sites of rat PGC-1α cDNA were designed and synthesized by Genesil . Control sequence which could not target PGC-1α cDNA was also included as a negative control. siRNA was transfected into VSMCs using LipofectAmine reagent  according to the manufacture's manual. Of several sequences tested for PGC-1α knock-down, the sequence with the best interfering effect was selected as described previously 5 cells/dish) and were allowed to grow to subconfluence, then were growth-arrested by serum deprivation for 12 h. Cells were then divided into several groups and were applied to different stimulation. After 48 h, cells were resuspended with 0.05% trypsin and 0.02% EDTA, and the cell number was determined with hemocytometer.Cell counting analysis was performed for evaluation of the effect of glucose on VSMC proliferation. In separate experiments, VSMCs were seeded in 6 cm plastic dishes . A reference point was created on the bottom of the plate in the field of the wound using direct microscopic visualization. The measurement of the width of the gap after 48 h was subtracted from that at 0 h to give the distance the cells migrated. The results of the five readings from each well were averaged. Independent stimulation experiments were repeated three times. Boyden chamber cell migration assay was performed using transwell chambers with fibronection-coated 8-µm-pore-size polycarbonate membranes (BD Biosciences). Preconfluent VSMCs treated in the same way were then suspended in DMEM–0.5% FBS to a concentration of 4×105 cells/mL. Different fatty acids or serum free DMEM (0.6 mL) were added to the lower compartment. A 0.1-mL cell suspension  was added to the upper compartment, and cells were then incubated at 37°C (95% air–5% CO2). 6 h later, non-migrated cells were removed with a cotton swab, and the migrated cells were fixed with paraformal-dehyde for 30 min and stained with crystal violet. Cell migration was quantified by blind counting of the migrated cells on the lower surface of the membrane of 5 fields per chamber under microscope.Migration of VSMCs was investigated by a standard 5′-AGGTCCCCAGGCAGTAGAT-3′ (sense) and 5′-CGTGCTCATTGGCTTCATA (antisense), probe: 5′-fam-ATGAATCAAGCCACT ACAGACACC-tamra-3′; primers for rat β-actin (GenBank access number V01217) were 5′-AGGGAAATCGTGCGTGAC-3′ (sense) and 5′-CGCTCATTGCCGATAGT G-3′ (antisense), probe: 5′-fam-CTGTGCTATGTTGCCCTAGACTTC-tamra-3′; Amplification conditions were: one cycle of 95°C for 5 minutes followed by 40 cycles of 95°C for 30 seconds, 60°C for 1 minute, and final one cycle of 72°C for 2 minutes. Relative abundance of mRNA was determined from the CT values and was normalized to the value of the house-keeping gene.Total RNA was isolated from rat arterial samples or rat VSMCs using RNeasy kit  according to the manufacturer's instructions. cDNA was synthesized and subjected to PCR amplification with the ABI Prism 7000 sequence detection system  as described For Western blot analysis, total proteins were applied to 7.5% SDS-PAGE gel electrophoresis for PGC-1α detection, and rabbit polyclonal PGC-1 antibody  was used as primary antibody. Samples containing 50 µg total proteins were applied to 12.5% SDS-PAGE gel electrophoresis for phosphorylated ERK1/2 detection, and phospho-specific mouse anti-human ERK1/2 antibody (BD Biosciences), mouse anti-human ERK antibody (BD Biosciences) and mouse monoclonal GAPDH antibody (Santa Cruz) were used as primary antibody.All results are expressed as means±SEM. Data were analyzed using a one-way ANOVA and Student-Newman-Keuls tests for multiple comparisons or Student's test for unpaired data. In all cases P≤0.05 was taken as statistically significant.


To investigate the role san-1(ok1580) animal has low penetrating phenotypes including an increased incidence of males, larvae arrest, slow growth, protruding vulva, and defects in vulva morphogenesis. We found that the viability of san-1(ok1580) embryos is significantly reduced when HCP-1 (CENP-F homologue), MDF-1 (MAD-1 homologue), MDF-2 (MAD-2 homologue) or BUB-3 (predicted BUB-3 homologue) are reduced by RNAi. Interestingly, the viability of san-1(ok1580) embryos is not significantly reduced when the paralog of HCP-1, HCP-2, is reduced. The phenotype of san-1(ok1580);hcp-1(RNAi) embryos includes embryonic and larval lethality, abnormal organ development, and an increase in abnormal chromosome segregation . Several of the san-1(ok1580);hcp-1(RNAi) animals displayed abnormal kinetochore (detected by MPM-2) and microtubule structure. The survival of mdf-2(RNAi);hcp-1(RNAi) embryos but not bub-3(RNAi);hcp-1(RNAi) embryos was also compromised. Finally, we found that san-1(ok1580) and bub-3(RNAi), but not hcp-1(RNAi) embryos, were sensitive to anoxia, suggesting that like SAN-1, BUB-3 has a functional role as a spindle checkpoint protein.The C. elegans embryo, HCP-1 interacts with a subset of the spindle checkpoint pathway. Furthermore, the fact that san-1(ok1580);hcp-1(RNAi) animals had a severe viability defect whereas in the san-1(ok1580);hcp-2(RNAi) and san-1(ok1580);hcp-2(ok1757) animals the viability defect was not as severe suggesting that hcp-1 and hcp-2 are not completely redundant.Together, these data suggest that in the  The spindle checkpoint genes regulate chromosome segregation during mitosis and when mutated lead to a variety of human health concerns including cancer ,2. Thus,The spindle-checkpoint signaling pathway is well understood and has been discussed in many reviews ,5-9. BriCaenorhabditis elegans and Drosophila melanogastor. For example, in C. elegans the genes mdf-1 and mdf-2 (MAD1 and MAD2 homologues respectively) have a role in development and germline function [mdf-1(gk2) animals. A genetic screen for suppressors of the weak allele of mat-3, an APC subunit, identified several mdf-2,mdf-1, and san-1/mdf-3 alleles, providing evidence that the metaphase to anaphase transition in meiosis is regulated by the spindle checkpoint [C. elegans embryonic blastomeres [mdf-2 and san-1 (MAD3 homologue) genes are required for response to microtubule-depolymerizing environments such as anoxia-induced suspended animation and nocodazole [Much of the work to elucidate the mechanistic function of the spindle checkpoint proteins has been conducted in yeast and vertebrate cell culture. However, there is increasing evidence that the spindle checkpoint is also required for specific functions in function . Specifieckpoint . The spistomeres . Brieflycodazole ,22. Finacodazole ,21. TogeC. elegans embryos HCP-1, the CENP-F like protein, is localized to the kinetochore during mitosis; yet hcp-1(RNAi) animals do not have any obvious phenotype [In henotype . HCP-1 ahenotype ,24. Howehenotype ,25. The henotype ,25,26; ahenotype . Additiohenotype .san-1 by using RNAi to identify genes that are synthetic lethal with san-1(ok1580). The viability of san-1(ok1580) embryos is significantly reduced when HCP-1, MDF-2 (MAD-2 homologue) or BUB-3  are reduced by RNAi. The viability of mdf-2(RNAi);hcp-1(RNAi) embryos but not bub-3(RNAi);hcp-1(RNAi) embryos was compromised suggesting that HCP-1 interacts with a subset (san-1 and mdf-2) of the spindle checkpoint components. Phenotype analysis of san-1(ok1580);hcp-1(RNAi) embryos supports the idea that HCP-1 functions in the proper alignment of chromosomes and chromosome segregation and that these functions are dependent upon the spindle checkpoint pathway.In this report we further investigate the functional role of the spindle checkpoint gene san-1(RNAi) embryos are viable, yet sensitive to conditions that depolymerize microtubules (anoxia or nocodazole treatment), providing evidence that the san-1 gene product has a role in the spindle checkpoint pathway [san-1 has in C. elegans we obtained a san-1 deletion strain from the C. elegans Knockout Consortium. We verified this deletion by DNA sequence analysis and found that the san-1(ok1580) allele has a 992 kb deletion, which is consistent with what is documented in wormbase. The san-1 gene contains 8 predicted exons, of which the deletion in san-1(ok1580) removes all of exons 3–7 and the first 13 bp of intron 7. Thus, the san-1(ok1580) allele is minimally a catalytic null as the predicted kinase domain and Mad3/Bub1 homology region predicted to be essential for binding to CDC20p is absent (data not shown).Previously, it was shown that  pathway ,22. To isan-1(ok1580) hermaphrodites grown at 24°C have several phenotypes including a higher incidence of males in the population, egg laying defects, and a higher death rate in adult hermaphrodites due to vulva bursting or formation of a bag of worms [san-1(RNAi) was done at 20°C, we characterized the phenotype of san-1(ok1580) animals grown at 20°C. Similar to animals maintained at 24°C, we found that the san-1(ok1580) embryos were viable, yet had some low penetrant phenotypes (Table san-1(ok1580) animals have a larvae arrest and slow growth phenotype (Table san-1(ok1580) adults have a higher incidence of death resulting from defects in vulva morphogenesis (Table san-1(ok1580) animals and determined that the san-1(ok1580) animals contained tissue damage, abnormal vulva anatomy, and an increase in the protruding vulva phenotype  animals is not significantly different (P = .176) (Table san-1(ok1580) animals, indicating that some of the san-1(ok1580) animals were not laying embryos. Consistent with this idea, we observed that 18% (n = 16) of the san-1(ok1580) 1-day old adults had larvae hatching out in the uterus. We analyzed progeny production in san-1(ok1580) hermaphrodites and determined that the average number of progeny produced was significantly decreased in comparison to wild-type hermaphrodites (Table san-1(ok1580) hermaphrodites died as young adults and produced less than 60 progeny, whereas others did not die, yet they still produced significantly less progeny than wild-type hermaphrodites (P = .001). The san-1(ok1580) animals also have a higher incidence of males (Table san-1(ok1580) males have various defects in tail morphology including an increase in tissue damage, and abnormal ray morphology or tissue structure  males for mating indicating that the san-1(ok1580) males are not sterile. In summary, these results are consistent with defects in cell cycle progression leading to abnormal vulva development in the hermaphrodite and somatic cell defects in the adult male [Others have shown that of worms . As prioe Figure , Table 1e Figure . In our ult male .san-1 function in C. elegans we used RNAi to identify additional genes that genetically interact with san-1(ok1580). We were interested in determining if RNAi of specific gene products resulted in a more severe phenotype in the san-1(ok1580) background. This approach is similar in concept to a synthetic lethal screen, an established technique to identify genetic interactions in which the double mutant has a phenotype distinct or more severe than the phenotype(s) of the individual single mutants. The genes that we evaluated included those that are involved with kinetochore assembly or spindle checkpoint function and do not have severe embryonic lethality phenotypes. Thus, we analyzed hcp-1, hcp-2, the putative bub-3 homologue Y54G9A.6, the known spindle checkpoint genes, mdf-1 and mdf-2, and the predicted kinetochore protein kbp-5 [hcp-1/2 genes encode kinetochore-localized proteins required for kinetochore assembly, and animals only have a significant defect in viability when both HCP-1/2 are simultaneously depleted by RNAi [C. elegans Y54G9A.6 predicted gene product suggests there is homology with yeast BUB3 , Homo sapiens BUB3 , and D. malanogaster BUB3 . The amino acid identity between the C. elegans Y54G9A.6 predicted gene product and BUB3 proteins is 44% for H. sapiens, 43% for D. melanogastor and 19% for Saccharomyces cerevisiae, as determined using the ClustalW software. Thus, the Y54G9A.6 gene will be referred to as bub-3 in this report. Other gene candidates likely to interact with san-1, such as other spindle checkpoint genes (bub-1), genes that encode products essential for kinetochore assembly and function  and homologues to zw10 and rod , were not analyzed because RNAi of these gene products result in severe embryonic lethality, thus testing a synthetic lethal interaction with san-1(ok1580) is not possible. We chose to focus our analysis instead on genes bub-3, mdf-1, mdf-2, hcp-1, hcp-2 and kbp-5.To further analyze the in kbp-5 . The hcp by RNAi ,25. The san-1, mdf-1, mdf-2, bub-3) or the kinetochore assembly genes  individually did not result in severe embryonic lethality and the majority of animals developed into adults  and mdf-2(RNAi) animals had several phenotypes as adults including death due to vulva bursting or formation of a bag of worms (data not shown), which is consistent with what others have observed [mdf-1(gk2) deletion mutant has a significant embryo lethal phenotype [mdf-1(RNAi) acts as a null. In comparison to san-1(ok1580) animals, the san-1(ok1580);bub-3(RNAi) (P < .04), san-1(ok1580);mdf-1(RNAi) (P < .01) and san-1(ok1580);hcp-1(RNAi) (P < .0003) animals displayed a reduction in the ability for embryos to develop to adulthood ;hcp-1(RNAi) animals died as embryos or young larvae. The san-1(ok1580);hcp-2(RNAi) animals had a decrease in the ability to develop to adulthood, in comparison to san-1(ok1580) animals (P < .05), however the phenotype was not as a severe as the san-1(ok1580);hcp-1(RNAi) animals ;mdf-2(RNAi) had a reduced but not significant difference in viability (P = .073). We did not observe a viability defect in san-1(ok1580);kbp-5(RNAi) animals thus kbp-5 was not further analyzed (data not shown).A reduction in the spindle checkpoint genes  and san-1(ok1580) animals to produce a san-1(ok1580);hcp-2(ok1757) animal. The hcp-2(ok1757) allele is a 1.281 kb deletion, which removes most of exon 5 resulting in a frame-shift; hcp-2 has 8 predicted exons (data not shown). We verified this deletion by DNA sequence analysis. We determined that 26.25% of the F2 offspring (n = 80) from a san-1(ok1580) and hcp-2(ok1757) cross had phenotypes including embryo lethality, larvae lethality, odd morphology and lethargy. The number of F2 progeny with abnormal phenotypes was higher than the expected 1/16 (6.25%) for a homozygous double mutant animal (san-1(ok1580);hcp-2(ok1757)), suggesting that some of the F2 progeny that are a combination of homozygous and heterozygous mutations for either san-1(ok1580) or hcp-2(ok1757) may have an abnormal phenotype. We genotyped 39 F2 animals, using single worm PCR to detect the san-1 and hcp-2 deletions, and determined that the homozygous double mutant (san-1(ok1580);hcp-2(ok1757)) and animals homozygous for the san-1(ok1580) deletion and heterozygous for the hcp-2(ok1757) deletion displayed embryonic and larval lethality phenotypes. The hcp-2(ok1757) animals appeared to be lethargic as adults, as noted by their reduction in motility, yet did not exhibit embryonic lethality and the majority reached adulthood three days after egg laying ;hcp-2(ok1757) double mutant and determined that the majority of the embryos hatched, yet only 7.3 % ± 2.4 of the larvae reached adulthood within three days at 20°C. However, if given four days to develop the majority of san-1(ok1580);hcp-2(ok1757) animals developed to adulthood ;hcp-2(ok1757) animals had morphological defects suggesting that the double mutant had a more severe phenotype then the single mutants (data not shown). Our results indicate that the phenotype observed in san-1(ok1580);hcp-2(ok1757) is not identical to that observed in san-1(ok1580);hcp-1(RNAi) animals.Because RNAi does not always completely remove all of a genes function, we used a genetic deletion of g Figure . We isold Figure . Severalhcp-1(RNAi);hcp-2(RNAi) embryos die during embryogenesis; however, over half of the san-1(ok1580);hcp-1(RNAi) or mdf-2(RNAi);hcp-1(RNAi) animals survive embryogenesis but die as larvae ;hcp-1(RNAi) or mdf-2(RNAi);hcp-1(RNAi) animals is not as severe in comparison to the hcp-1(RNAi);hcp-2(RNAi) animals [mdf-2(RNAi) animals, the mdf-2(RNAi);hcp-1(RNAi) animals had a significant decrease in the ability to develop and survive to adulthood (P < .011). The mdf-2(RNAi);hcp-2(ok1757) animals also had a decrease in the ability to survive to adulthood (P < .025), however the viability defect was not as severe as that observed in mdf-2(RNAi);hcp-1(RNAi) animals  or hcp-1(RNAi) animals, the bub-3(RNAi);hcp-1(RNAi) animals did not have a viability defect ;hcp-2(ok1757) ;hcp-2(RNAi) (data not shown) animals did not have viability defects. Using RT-PCR, in comparison to wild-type, a greater than 5 fold reduction in bub-3 transcript level (P < .029) was observed in bub-3(RNAi) animals, thus demonstrating RNAi efficiency. Together, these results suggest that san-1 genetically interacts with bub-3, mdf-2, mdf-1, hcp-1 and hcp-2 and that C. elegans requires both hcp-1 and either of the checkpoint genes, san-1 or mdf-2 but not bub-3. Furthermore, these results suggest that hcp-1 and hcp-2 have redundant but not identical functions.Nearly all of the e Figure , 2C. Thu animals . In compsan-1(ok1580);mdf-2(RNAi), san-1(ok1580);bub-3(RNAi) and san-1(ok1580);hcp-1(RNAi) animals, we analyzed the animals for developmental defects. The majority of animals, that did not die as embryos, arrested as larvae or had an abnormal gonad as adults ;mdf-2(RNAi), san-1(ok1580);bub-3(RNAi) and san-1(ok1580);hcp-1(RNAi) animals had larvae arrest or slow growth phenotypes ;mdf-2(RNAi) or san-1(ok1580);bub-3(RNAi) animals included abnormal cell morphology in the mitotic region of the gonad, abnormal cell morphology in the meiotic region of the gonad, meiotic cells that had button like cell morphology reminiscent of apoptotic cells, and abnormal oocyte structure ;hcp-1(RNAi) animals died or arrested as larvae. Abnormal gonad morphology was not observed in the bub-3(RNAi) or hcp-1(RNAi) animals. Some of the san-1(ok1580) and mdf-2(RNAi) had abnormal gonad morphology ;hcp-2(RNAi) animals die as embryos whereas many of the san-1(ok1580);hcp-1(RNAi) embryos hatch and later die as young larvae ;hcp-1(RNAi) embryos have developmental defects we used the pharynx as a marker for developmental progression, by analyzing the expression of myo-2::GFP. Briefly, myo-2 encodes a muscle-type specific myosin heavy chain isoform and is expressed during embryogenesis in the primordial pharyngeal muscle cells. Similar to the control embryos   ;hcp-1(RNAi) embryos had either a reduction in myo-2::GFP ;hcp-1(RNAi) embryos have developmental defects.The s Figure , the hcp) Figure  and san-) Figure  animals P Figure  or abnorP Figure . Togethesan-1(ok1580) animals depends upon bub-3 and hcp-1, however it is not clear if, like the san-1,mdf-1 and mdf-2 genes, the bub-3 and hcp-1 gene products function in the spindle checkpoint pathway. It is known that blastomeres of wild-type embryos exposed to oxygen deprivation (anoxia) reversibly arrest cell cycle progression [san-1(RNAi) and mdf-2(RNAi) embryos are sensitive to the microtubule disrupting environments of anoxia or nocodazole treatment. Specifically, san-1(RNAi) and mdf-2(RNAi) embryos exposed to anoxia have a significantly reduced number of metaphase blastomeres and an increase in anaphase bridging and chromosomal abnormalities; supporting the idea that san-1 and mdf-2 function as spindle checkpoint genes [san-1(RNAi) embryos, the san-1(ok1580) embryos exposed to anoxia had a reduced survival rate (Table bub-3(RNAi) embryos exposed to anoxia also had a reduced survival rate in comparison to wild-type animals suggesting that bub-3 functions as a spindle checkpoint protein in developing embryos. The hcp-1(RNAi) embryos were not sensitive to anoxia (Table hcp-2(RNAi) or hcp-2(ok1757) embryos (data not shown). These results suggest that hcp-1 and hcp-2 are not required for anoxia induced metaphase arrest and do not function as a spindle checkpoint protein.Our data suggest that the development of gression ,29. Unlint genes ,22. Simihcp-1 and san-1 gene products in developing embryos we determined if chromosome loss was occurring in the san-1(ok1580);hcp-1(RNAi) embryos. We analyzed chromosome structure in mitotic blastomeres from wild-type, hcp-1(RNAi), san-1(ok1580), and san-1(ok1580);hcp-1(RNAi) embryos collected, fixed and stained as previously described [san-1(ok1580), and hcp-1(RNAi) embryos ;hcp-1(RNAi) embryos had a significant increase in abnormal nuclei   Figure . These a) Figure , arrows.hcp-1(RNAi)  embryos ;hcp-1(RNAi) embryos with greater than 30 blastomeres, the nuclear pore complex, detected by mAb414, were in small aggregates surrounding the metaphase plates  ;hcp-1(RNAi) embryos.In young wild-type embryos (less than 30 blastomeres) the nuclear pore complexes detected by mAb 414 do not diminish during mitosis . In cont) Figure , and sans Figure . Howeversan-1(ok1580);hcp-1(RNAi) embryos we analyzed chromosome, kinetochore and spindle microtubule structure using indirect immunofluorescence. We first stained embryos with MPM-2, an antibody that recognizes mitotic proteins localized to the kinetochore and centrosomes [san-1(ok1580) and hcp-1(RNAi) embryos was similar to that of wild-type embryos ;hcp-1(RNAi) embryos contained both normal and abnormal kinetochore structure, as determined by the presence or absence of the two-line formation along the sister chromatids  and hcp-1(RNAi) embryos were similar to that of wild-type embryos ;hcp-1(RNAi) blastomeres with normally formed metaphase plates had normal centromere and microtubule structure ;hcp-1(RNAi) blastomeres with abnormal chromosome structure contained HCP-3 localized with chromosomes  and spindle microtubules, respectively ,26. HCP-s Figure . The sane Figure . The sans Figure ; yet in s Figure . In thess Figure . Embryossan-1(ok1580);hcp-1(RNAi) animals, it is likely that the blastomeres were transitioning through mitosis abnormally. To determine if this occurred in vivo we produced a san-1(ok1580);tbg-1::GFP;pie-1::GFP::H2B strain  for live cell imaging using a spinning disc confocal microscope. The san-1(ok1580);hcp-1(RNAi);tbg-1::GFP;pie-1::GFP::H2B embryos contained blastomeres with abnormal mitotic progression. Analysis of a typical san-1(ok1580);hcp-1(RNAi) embryo showed one blastomere which progressed to anaphase with normal chromosome segregation, and another blastomere with abnormal chromosome segregation leading to anaphase bridging ;hcp-1(RNAi) embryos.Given the presence of anaphase bridging, lagging chromosomes, and abnormal chromosome structure observed in the san-1(ok1580);hcp-1(RNAi) embryos abnormally segregate chromosomes which likely leads to developmental defects and lethality. The hcp-1(RNAi) embryos have not been shown to have chromosome segregation issues that lead to increased lethality. One possible reason for the synthetic lethal interaction between san-1 and hcp-1 is that loss of san-1 amplifies a subtle hcp-1 defect. Given the defect in progression through mitosis observed with mAb 414, timing of mitotic progression is possibly perturbed when hcp-1 is absent and loss of san-1 increases this defect. Therefore, we conducted live cell imaging to compare mitotic progression in hcp-1(RNAi);tbg-1::GFP;pie-1::GFP::H2B and control animals. We analyzed mitosis in the AB and P cell blastomeres of a 2-cell embryo. We determined the time it took to transition from nuclear envelope break down (NEB) to formation of the metaphase plate, metaphase to anaphase onset and NEB to anaphase onset in the P cell of a wild-type and hcp-1(RNAi) 2-cell embryo. We also determined the time between NEB of the AB cell and NEB of the P cell. We found that the mitotic progression of hcp-1(RNAi) embryos analyzed at 21°C did not have any chromosomal defects and the time it took to progress through mitosis was not significantly different in comparison to control embryos  animals require a functional spindle checkpoint for normal development.Thus far, we have shown that the san-1ok150;hcp-1. We determined that san-1(ok1580);hcp-1(RNAi), san-1(ok1580);bub-3(RNAi), san-1(ok1580);mdf-1(RNAi), and san-1(ok1580);mdf-2(RNAi) had a reduced ability to survive. The genetic interaction between san-1, mdf-1, and mdf-2 is expected, since these genes are thought to function in the spindle checkpoint pathway in mitotic and meiotic cells [C. elegans bub-3 gene product has high homology to BUB-3 from other systems and bub-3(RNAi) embryos are more sensitive to the microtubule disrupting process of anoxia exposure, supporting the idea that the Y54G9A.6 gene encodes the bub-3 spindle checkpoint gene.The use of synthetic lethal screens is a genetic tool to elucidate genes that interact in similar cellular functions or pathways. Thus, when a genetic mutant does not have a severe phenotype the use of a synthetic lethal screen to identify double mutants that have a phenotype distinct or more severe then the phenotype(s) observed for the individual single mutants often will aid in further understanding the function of the gene products. To analyze ic cells -21. The hcp-1/2 and the spindle checkpoint genes is likely to be more complex. The viability of san-1(ok1580);hcp-1(RNAi) and mdf-2(RNAi);hcp-1(RNAi) animals was severely compromised, suggesting that HCP-1 interacts with MDF-2 and SAN-1. However, the bub-3(RNAi);hcp-1(RNAi) animals did not have a significant decrease in survival rate. This could be due to BUB-3 and HCP-1 not involved together in a specific function. The san-1(ok1580);hcp-2(ok1757) animals have embryonic and larvae lethal phenotypes. We isolated a san-1(ok1580);hcp-2(ok1757) double mutant and determined that it had more severe phenotypes than that observed in the single mutants; this suggests that hcp-2 and san-1 genetically interact. However, the phenotype of the san-1(ok1580);hcp-2(ok1757) double mutants was not as severe as that observed san-1(ok1580);hcp-1(RNAi) animals, suggesting that HCP-1 and HCP-2 are not be completely redundant. HCP-1 and HCP-2 are 54% similar mostly at their N- and C-termini [The genetic interaction between -termini . Althoug-termini . Thus, isan-1(ok1580);hcp-1(RNAi) animals had severe developmental defects including embryo and larvae lethality. The viability defects observed are likely due to abnormal chromosome segregation, which will in turn compromise cellular structure and function. The san-1(ok1580);bub-3(RNAi) and san-1(ok1580);mdf-2(RNAi) animals had gonad defects. We and others have shown that san-1(ok1580) and mdf-2(RNAi) animals have low level gonad defects [san-1(ok1580) hermaphrodites and others have shown that mdf-2(av14) mutants have a reduced brood size, further supporting the role the checkpoint genes have in germline function [hcp-1(RNAi);hcp-2(RNAi) animals also have meiotic defects, supporting the idea that hcp-1 has a role in meiosis [The majority of  defects ,19; yet  meiosis . The exasan-1(ok1580);hcp-1(RNAi) embryos have severe chromosome segregation defects, and on average 55.3% of the embryos die and approximately 0.9% of the animals reach adulthood. Thus, in san-1(ok1580);hcp-1(RNAi) embryos, embryogenesis can progress, but the ability to produce viable adults is severely compromised. We used myo-2::GFP to analyze the structure of the developing pharynx in the san-1(ok1580);hcp-1(RNAi) embryos and found that there was not only abnormal pharynx morphology but we often observed a reduction in the myo-2::GFP in the embryos. It is not known if the reduction in myo-2::GFP in the san-1(ok1580);hcp-1(RNAi);myo-2::GFP animals is due to abnormal differentiation, the loss of myo-2::GFP DNA or abnormal regulation of the myo-2::GFP. An increase in genome loss due to chromosome segregation issues could result in either of these possibilities.The san-1(ok1580);hcp-1(RNAi) embryos have chromosome segregation defects leading to abnormal nuclei, anaphase bridging and lagging chromosomes. We determined that the nuclear pore complexes, detected by mAb414, formed small aggregates surrounding the metaphase plates in san-1(ok1580);hcp-1(RNAi) embryos, indicating that the nuclear membrane pore complexes are not completely breaking down. This may indicate that SAN-1 and HCP-1 influences additional mitotic events in addition to chromosome segregation. The centromeric protein HCP-3 was observed to have a normal or an abnormal localization pattern in the san-1(ok1580);hcp-1(RNAi) blastomeres. The abnormal localization of HCP-3 can be interpreted in several ways. First, it could be due to SAN-1 and HCP-1 directly regulating the localization of HCP-3 to the region that marks the centromere, however this seems unlikely since the kinetochore assembly pathway clearly places HCP-3 upstream of HCP-1 and SAN-1. Alternatively, the abnormal HCP-3 localization could be due to chromosome segregation defects causing loss of chromatin that marks the centromere. Finally, it is possible that the segregation defects result in chromatin fragments that HCP-3 associates with. Further studies would be required to determine the mechanism leading to abnormal HCP-3 localization.Closer examination of the chromosomes, nuclear pore proteins, centromere, kinetochore, and microtubules indicates that the san-1(ok1580);hcp-1(RNAi) embryos, the kinetochore proteins recognized by the MPM-2 antibody were not associated with the chromosomes when the chromosome structure was aberrant. Furthermore, the microtubule structure was altered in blastomeres with abnormal chromosome structure. Together, these data suggests that in the san-1(ok1580);hcp-1(RNAi) blastomeres the kinetochore and microtubule interaction is sometimes, but not always, compromised. Given the increase in anaphase bridges, lagging chromosomes and abnormal mitotic nuclei, the microtubule and kinetochore interaction in san-1(ok1580);hcp-1(RNAi) animals is likely not sufficient for proper chromosome segregation.In C. elegans hcp-1(RNAi);hcp-2(RNAi) blastomeres take longer to progress through mitosis [hcp-1(RNAi) blastomeres of embryos analyzed at 21°C did not take longer to progress from prometaphase to metaphase, in comparison to control blastomeres, suggesting that mitotic timing of hcp-1(RNAi) blastomeres is normal as long as hcp-2 gene product is present.Others have shown using mammalian cell culture (HeLa cells) that there is a prolonged mitosis in CENP-F (RNAi) cells and this prolonged mitosis is dependent upon the spindle checkpoint gene BUBR1 . In C. e mitosis . Our dathcp-1(RNAi) animals the spindle checkpoint is being activated to ensure proper chromosome segregation and genome defects will occur without normal spindle checkpoint function; specifically SAN-1 and MDF-2 function are required. Given that the involvement of the spindle checkpoint in tumor progression, it is of interest to fully understand the genes products that interact with the spindle checkpoint pathway to ensure genome fidelity and maintenance.CENP-F is required for chromosome alignment  and silesan-1 (mad-3 homologue). We show that the spindle checkpoint genes, san-1 and mdf-2, not only interact with one another but also interact with the CENP-F homologue hcp-1. The bub-3 spindle checkpoint gene does not genetically interact with hcp-1; suggesting that hcp-1 only interacts with a subset of the spindle checkpoint.In this report we identified genes that genetically interact with the spindle checkpoint gene san-1(ok1580);hcp-1(RNAi) embryos demonstrates that C. elegans embryos require san-1 and hcp-1 for proper chromosome segregation. Furthermore, gonad morphological defects were observed in san-1(ok1580);mdf-2(RNAi) and san-1(ok1580);bub-3(RNAi) animals further supporting a functional role of the spindle checkpoints in meiosis. We provide evidence to suggest that the hcp-1 and hcp-2 gene products may have overlapping but also distinct functions in C. elegans. Together, our data suggest that in the C. elegans embryo, HCP-1 functions in proper alignment and segregation of chromosomes and these functions are dependent upon elements of the spindle checkpoint pathway but not all of the spindle checkpoint proteins. Given the important role the spindle checkpoint and kinetochore proteins have in chromosome segregation and cell cycle progression, it is of great interest to characterize these gene products in developing organisms such as C. elegans.Detailed phenotype analysis of the E. coli (OP50) and raised at 20°C as described . The following strains were obtained from the Caenorhabditis elegans Genetics Center: RB1391(san-1(ok1580)), PD4790 (myo-2::GFP), TH32 , and RB1492 (hcp-2(ok1757)). The Caenorhabditis elegans Gene Knockout Consortium  produced the san-1(ok1580) and hcp-2(ok1757) deletion alleles. The hcp-2(ok1757) mutant (strain RB1492) was back-crossed to wild-type animals three times (strain PM117). The san-1(ok1580) allele was crossed, using standard genetic techniques, to produce the following strains: PM107 (san-1(ok1580);myo-2::GFP), PM115 (san-1(ok1580);tbg-1::GFP;pie-1::GFP::H2B), and PM116 (san-1(ok1580);hcp-2(ok1757)). The san-1(ok1580) allele was backcrossed into wild-type background once. The san-1(ok1580) and hcp-2(ok1757) alleles were genotyped by single worm PCR with the following primers: san-1 forward primer (CGC TTA AAG CTT GAT CAA CTT CTCG), san-1 reverse primer (GCT AGT GAT TTC TCC TCC GTT TTC TCA), hcp-2 forward primer (ACT CTG AAG TCG GAA CAT GAA ATT), and hcp-2 reverse primer (TGA AGA GCC TTC TGT GCA AA). DNA sequence analysis to verify the deleted region of the san-1(ok1580) and hcp-2(ok1757) strains was conducted using standard molecular techniques.The wild-type Bristol strain (N2) and mutant strains were cultured on NGM plates seeded with Saccharomyces cerevisiae was used to conduct BLASTp against the C. elegans, Homo sapiens and D. melanogastor genome to identify the most identical protein for each species. The primary amino acid sequences were aligned using ClustalW and processed using the LaserGene sequence analysis software package (DNASTAR). Clustal analysis between the C. elegans Y54G9A.6 gene product (referred to as BUB-3) and the S. cerevisiae, H. sapiens and D. melanogastor BUB-3 proteins was conducted to determine percent identity between the proteins.The BUB-3 protein from the yeast E. coli (OP50). To assay for the high incidence of males (him) phenotype several hermaphrodites, grown in the absence of males, were allowed to lay eggs for several hours and the percent of male progeny was determined (for wild-type and san-1(ok1580), n = 244, n = 325, respectively). The san-1(ok1580) animals were assayed for larval arrest and slow growth phenotypes (for wild-type and san-1(ok1580), n = 210, n = 218, respectively). To determine if larval arrest or slow growth was observed we collected a synchronized population of L1 larvae and placed on OP50 NGM plates at 20°C for three to five days. We quantified the number of animals that developed to adulthood within three days, animals that developed to adulthood within three to five days (slow growth) and those that remained arrested as larvae . To assay for adult death phenotype, animals were grown for the first 5 days of adulthood and analyzed every day for death or survivorship (for wild-type and san-1(ok1580), n = 210, n = 207, respectively). Hermaphrodites were moved daily to fresh plates and lost worms were eliminated from analysis. The average brood size was determined by quantifying the number of offspring individual hermaphrodites produced (for wild-type and san-1(ok1580), n = 5, n = 10, respectively). To quantify the number of eggs within the uterus we collected 1-day old adults, placed on an agar pad and visualized using a Zeiss compound microscope; the number of eggs within the uterus was determined (for wild-type and san-1(ok1580), n = 16). To assay for protruding vulva phenotype we collected 1-day old adults and counted the number of adult animals that had a protruding vulva (for wild-type and san-1(ok1580), n = 210, n = 207, respectively). The p values (P) were determined using standard student t test.For all experiments animals were kept at 20°C and grown on san-1(ok1580)) were grown to adulthood on NGM plates supplemented with 200 μg/ml ampicillin, 12.5 μg/ml tetracycline and 1 mM IPTG, seeded with a bacterial strain expressing dsRNA specific for mdf-2, mdf-1, bub-3, hcp-1, hcp-2 or control food [hcp-1 and hcp-2. Briefly, plasmid vectors for feeding RNAi were constructed by cloning either a 1.1 kb Spe I – Dra I fragment from the hcp-1 cDNA LM46-3 or a 1.6 kb Sal I fragment from the hcp-2 cDNA, yk19h10 into pPD129.36, which allows IPTG induction of double stranded RNA. Respective feeding vectors were transformed into the HTH115(BL23) E. coli strain and fed to animals, consistent with previously described RNAi methodology [E. coli strain, which has a plasmid with no insert, on identical NGM IPTG plates to account for any differences attributable to the vector. We conducted RT-PCR experiments to analyze the efficiency of RNAi by quantifying the level of bub-3 transcript in wild-type and bub-3(RNAi) animals. Methodology is as previously described [bub-3 are as follow: 5'TGGGATCCTTTCAATCGGAAGC3' and 5'TTATTTCGGTCTGCTCCGGA3'. The P value was determined using the Mann-Whitney U Test.RNA interference (RNAi) was used to decrease the expression of specific genes. For all experiments, the methodology was similar to that previously described . Briefly, a synchronous population of L1 larvae  animals were grown, from the L1 larval stage to the 1 day old adult stage, on RNAi food . The adults were placed on a fresh RNAi plate and allowed to lay eggs for 1–2 hours. The adults were removed and the embryos were placed into anoxia, for 1 day, using the BioBag type A environmental chamber. The embryos were allowed to recover in air and assayed 24 hours later for the ability to hatch and three days later for the ability to reach adulthood. Four independent experiments with at least a total of 200 embryos were analyzed.To assay whether specific genes are required for spindle checkpoint activity we exposed embryos to anoxia as previously described ,29. Briesan-1(ok1580), or san-1(ok1580);myo-2::GFP) were grown to adulthood on appropriate RNAi plates . To analyze the developing pharynx in embryos, both the san-1(ok1580);myo-2::GFP and the san-1(ok1580);hcp-1(RNAi);myo-2::GFP adult animals were dissected and the embryos were placed onto 2.5% agarose pads for microscopy analysis. To analyze the morphology of control, mdf-2(RNAi), hcp-1(RNAi), san-1(ok1580), san-1(ok1580);hcp-1(RNAi), san-1(ok1580);mdf-2(RNAi), and san-1(ok1580);bub-3(RNAi) animals, RNAi was conducted as stated above and the adult animals were placed on fresh RNAi plates and allowed to lay eggs for several hours. The embryos developed for three days at 20°C before DIC microscopy analysis. For all experiments the animals were placed on a 2.5% agarose pad and visualized using a motorized Zeiss Axioskop Fluorescent microscope and imaged using Openlab 3.17. At least three independent experiments were conducted.The L1 larvae  animals were grown on appropriate RNAi food from L1 stage to adulthood. The adults were collected and dissected to release embryos. Embryos were collected, fixed and stained as previously described [Wild-type and escribed ,29. Brieescribed ; the mAbescribed ; mAb MPMescribed ; anti-HCescribed ; and YL1tbg-1::GFP, pie-1::GFP::H2B) or PM115 (san-1(ok1580);tbg-1::GFP;pie-1::GFP::H2B) strain. L1 larvae were grown on RNAi control or hcp-1 RNAi food to adulthood at 20°C. 1-day old gravid adults were dissected and embryos of the appropriate stage were collected by mouth pipet and placed on a 2.5% agarose pad. The temperature of the room in which the embryos were analyzed was at 21°C. Mitotic progression was quantified for 2-cell embryos using a spinning disc confocal microscope. To examine mitotic progression, the time it took, in seconds, to transition from the onset of prometaphase (as seen by nuclear envelope breakdown and condensed chromosomes) to the formation of the metaphase plate, and from the formation of the metaphase plate to the onset of anaphase was determined. Seven embryos were analyzed, using the Simple PCI Version 6 software program. The p-value (P) was determined using a standard student t-test. Movies, to demonstrate mitotic progression in the san-1(ok1580);tbg-1::GFP;pie-1::GFP::H2B embryos, were obtained by analyzing live, young embryos using a spinning disc confocal microscope. Images were collected, processed using NIH Image, and imported into Quick Time for display.To analyze mitotic progression the chromosomes and centriole were used as markers, using the TH32 (The author(s) declare that they have no competing interests.VAH conducted the indirect immunofluorescent assays and confocal analysis, produced and analyzed genetic crosses, assisted with experimental design and data interpretation, generated images, and edited the manuscript. AMS conducted genetic crosses, assisted with RNAi viability assays, conducted RT-PCR reactions and edited the manuscript. LLM conducted bioinformatics analysis, conceived experimental design, assisted with interpretation of data, provided necessary reagents for the study, generated images, and edited the manuscript. PAP conceived experimental design, conducted RNAi viability assays, analyzed phenotypes of mutants and RNAi animals, conducted genetic crosses, analyzed live cell images, generated images, and wrote the manuscript. All authors read and approved the final manuscript.hcp-1 in C. elegans [In the process of revision of this manuscript others also demonstrated the genetic interaction between the spindle checkpoint genes and  elegans .C. elegans encodes a protein (BUB-3) with homology to the spindle checkpoint protein BUB3. The amino acid identity between the C. elegans putative BUB-3 protein and other BUB3 proteins is 44% for H. sapiens, 43% for D. melanogastor and 19% for S. cerevisiae. Identical amino acids have a black background shade.The Y54G9A.6 gene encodes the putative BUB-3 protein. A multiple sequence alignment using ClustalW indicates that the Y54G9A.6 gene in Click here for filesan-1(ok1580);hcp-1(RNAi);tbg-1::GFP;pie-1::GFP::H2B embryos. A spinning disc confocal microscope to analyze chromosome segregation in the san-1(ok1580);hcp-1(RNAi) animals. Images were collected, processed using NIH Image and imported into Quick Time for display. In this embryo there are two blastomeres that progress through metaphase. One of the blastomeres displays normal mitotic progression (top metaphase blastomere) whereas another blastomere displays abnormal chromosome segregation (bottom metaphase blastomere) leading to anaphase bridging.Live cell imaging of Click here for file