Aedes aegypti, modulating arbovirus infection of mosquitoes. Sindbis virus  is an arbovirus that infects Ae. aegypti in the laboratory. SINV strain TR339 encounters a midgut escape barrier (MEB) during infection of Ae. aegypti. The nature of this barrier is not well understood. To investigate the role of the midgut as the central organ determining vector competence for arboviruses, we generated transgenic mosquitoes in which the RNAi pathway was impaired in midgut tissue of bloodfed females. We used these mosquitoes to reveal effects of RNAi impairment in the midgut on SINV replication, midgut infection and dissemination efficiencies, and mosquito longevity.The RNA interference (RNAi) pathway acts as an innate antiviral immune response in Aa-dcr2 gene. In midgut tissue of the transgenic Carb/dcr16 line, Aa-dcr2 expression was reduced ~50% between 1-7 days post-bloodmeal (pbm) when compared to the recipient mosquito strain. After infection with SINV-TR339EGFP, Aa-dcr2 expression levels were enhanced in both mosquito strains. In the RNAi pathway impaired mosquito strain SINV titers and midgut infection rates were significantly higher at 7 days pbm. There was also a strong tendency for increased virus dissemination rates among the transgenic mosquitoes. Between 7-14 days pbm, SINV was diminished in midgut tissue of the transgenic mosquitoes. Transgenic impairment of the RNAi pathway and/or SINV infection did not affect longevity of the mosquitoes.As a novel tool for studying arbovirus-mosquito interactions, we engineered a transgenic mosquito line with an impaired RNAi pathway in the midgut of bloodfed females by silencing expression of the Ae. aegypti, the recombinant SINV-TR339EGFP was confronted with both MEB and a midgut infection barrier (MIB). Impairment of the RNAi pathway in the midgut strongly reduced both midgut barriers for the virus. This confirms that the endogenous RNAi pathway of Ae. aegypti modulates vector competence for SINV in the midgut. The RNAi pathway acts as a gatekeeper to the incoming virus by affecting infection rate of the midgut, intensity of infection, and dissemination from the midgut to secondary tissues.We showed that RNAi impaired transgenic mosquitoes are a useful tool for studying arbovirus-mosquito interactions at the molecular level. Following ingestion by  The RNA interference (RNAi) pathway is an innate immune pathway of invertebrates such as nematodes, trypanosomes, hydra, planaria, and insects . In mosqTogaviridae; genus: Alphavirus) is an arbovirus with a positive sense single-stranded RNA genome. A dsRNA intermediate is formed during replication, which triggers the RNAi pathway causing homology-dependent destruction of viral RNA . The plates were incubated at 37°C for 4 days. Cells were then stained with MTT  , incubated at 37°C for 24 h and the number of plaques was counted for each sample. Virus titers of individual mosquitoes were calculated as pfu/ml.SINV-TR339EGFP virus stocks were generated from an infectious cDNA clone that contained the EGFP marker gene under control of a duplicated sub-genomic promoter located upstream of the coding sequence for the structural genes . BrieflySeven day-old Carb/dcr16 and HWE females were either fed with a non-infectious bloodmeal or with a bloodmeal containing SINV-TR339EGFP. After bloodfeeding, 50 mosquitoes of each treatment were put into 470 ml cardboard containers and provided with sugar and water. A control consisting of females that were sugarfed only was included in the experiment. For a period of 28 days after bloodfeeding the daily number of surviving mosquitoes in each container was recorded.dcr2 ratios and SINV-TR339EGFP infection levels were normalized using a log10 transformation. Aa-dcr2 ratios, virus infection levels, and virus infection/dissemination rates were then analyzed using the least-squares means test followed by pair-wise comparisons with the Tukey-Kramer test.Statistical analyses were performed using SAS Statistical Analysis Software . The MIXED procedure was used for restricted maximum likelihood parameter estimation with incomplete data. Aa-CCHK designed and performed the qRT-PCR assays, virus challenge and survival experiments, analyzed the data and wrote the manuscript. JP assisted with sample preparations, qRT-PCR assays, mosquito rearing and virus challenge experiments. ISV performed the Northern blot. KEO conceived the study, analyzed the data and edited the manuscript. AWEF conceived the study, generated the IR effector construct and the transgenic mosquitoes, performed the Genome Walking experiment, analyzed the data and edited the manuscript. All authors read and approved the final manuscript.


The stability of ophthalmic preparations in multidose containers is influenced by the preservative as well as the stability of the active ingredient. Unstable drugs may require refrigeration to preserve their active ingredient level and they are more likely to degrade over time, therefore becoming more susceptible to degradation based on patient mishandling. The purpose of this study was to determine the degree of molecular degradation that occurs in bimatoprost and latanoprost in a patient-use setting.This was an open-label, laboratory evaluation of the relative stability of bimatoprost and latanoprost. Patients presently using bimatoprost (n = 31) or latanoprost (n = 34) were identified at 2 clinical sites in Brazil. Patients were instructed to use and store their drops as usual and return all used medication bottles between day 28 and day 34 after opening.Bimatoprost demonstrated no degradation, but latanoprost degraded at various levels. The mean age of bimatoprost was 43.0 ± 3.4 days and the mean age of latanoprost was 43.9 ± 2.8 days (P = .072). The mean percentage of labeled concentration was 103.7% in the bimatoprost bottles and 88.1% in the latanoprost bottles (P < 001).This study showed that bimatoprost maintained ≥100% concentration throughout the study period while latanoprost did not. The stability of ophthalmic preparations in multidose containers is influenced by the preservative as well as the stability of the active ingredient . UnstablNumerous clinical studies show that the synthetic prostamide analog, bimatoprost, is a highly efficacious IOP lowering agent -4. BimatWhether temperature and humidity conditions found in actual practice environments, which vary across geographic locations and seasons, induce the degree of drug degradation found under laboratory conditions is not known. The purpose of this study was to determine whether storage and use practices of bimatoprost and latanoprost IOP-lowering medications affect the degradation rate of the active molecules of these drugs. Sao Paolo, Brazil, where the medications were used, is located in an equatorial climate zone characterized by a hot, dry winter season . SimilarThis was an open-label, laboratory evaluation of the relative stability of bimatoprost and latanoprost. Patients presently using bimatoprost (n = 31) or latanoprost (n = 34) were identified at two clinical sites in Sao Paulo, Brazil. After granting informed consent, all patients were instructed to store and use their eye drops at room temperature and to return all used medication bottles between day 28 and day 34 after opening. The bottles were collected between May and July 2002.Once patients returned the used bottles, they were collected and stored at room temperature for subsequent shipment to a central independent laboratory . During shipping (next-day air), the bottles were stored in insulative packaging with ice packs. The bottles arrived at the testing facility between day 35 and day 42. Shipment and arrival of bottles was scheduled so that all bottles were tested on approximately day 42 after opening.® C18 reverse-phase column, UV detection at 210 nm, and a mobile phase of 72/18/10  containing 0.03% (w/v) trifluoroacetic acid. Recovery studies were conducted for commercially available bimatoprost 0.03% preserved ophthalmic solution. Studies performed at 80%, 100%, and 125% of the label claim yielded recoveries of 100.5%, 100.6%, and 100.2% for peak areas and 101.5%, 100.6%, and 100.4% using peak heights, respectively. Linearity for bimatoprost was demonstrated from 0.002% to 0.009% (w/v) bimatoprost after dilution . Correlation coefficients (r) of 0.9999 by peak areas and 0.9992 by peak heights were obtained and a single point standard was used for calculations using peak areas and peak heights.All methods were validated by the testing facility, and analyses were carried out in accordance with good laboratory practices. High performance liquid chromatography (HPLC) was used to separate the molecules of interest from degradation byproducts and synthetic impurities, and for quantitation. The method for bimatoprost (AGN 192024) used a Waters SymmetryFor latanoprost, the method employed a Waters 600S equipped with a Waters 626 pump, a Waters 486 detector, and a Waters 717 Plus autosampler or equivalent, UV detection at 205 nm and mobile phase of 6/1/13  containing 0.05% (w/v) trifluoroacetic acid. Latanoprost stock solution with an acceptable concentration between 0.45 and 0.55 μg/mL was used in recovery studies; representing a range from 90% to 110% of the labelled concentration of samples after 100-fold dilution. A Free Acid Standard, Latanoprost Standard, Latanoprost and Free Acid Mixed LOQ Standard, and Latanoprost and Free Acid Mixed Standard were run against the diluted samples to determine the latanoprost concentration. A bracketing standard was injected after every 6-sample injection. The area of the latanoprost and free acid peaks within this bracketing standard were within 5% of the average area for the initial five injections of the latanoprost and free acid peaks. The relative standard deviation (RSD) for the 5 injections of the latanoprost and free acid mixed standard were ≤2% for each peak. Latanoprost concentration was determined using the following formula:Latanoprost concentration (μg/mL) = (average area latanoprost in sample)*/(average area latanoprost in standard).The primary endpoint, percentage of labeled concentration in the bottles at the time of testing, was calculated as: (concentration of intact drug in bottle/concentration of the drug indicated on the label) × 100. Percent concentrations then were recorded for each bottle of bimatoprost and latanoprost.Between-group comparisons were made using paired t-tests. The a priori level of significance for all tests was 0.05. The percentage of label claim was calculated as concentration remaining in the bottle/label claim × 100.The bottles were collected between May and July 2002, which corresponds to the end of fall and the beginning of the dry winter season in Sao Paulo. The average ambient temperature ranged between 14°C and 24°C.P = .072). Bimatoprost bottles retained a significantly greater percentage of the labeled active drug concentration after patients completed their course of therapy than did the latanoprost bottles. The mean percentage (± SD) of labeled concentration was 103.7% ± 1.3% in the bimatoprost bottles compared with 88.1% ± 10.8% in the latanoprost bottles (P < .001) .There was no significant between-group difference in the mean age of the bottles on the test date. The mean bottle age was 43.0 ± 3.4 days with bimatoprost and 43.9 ± 2.8 days with latanoprost (This study demonstrated that bimatoprost maintained its labeled concentration of active drug, while the latanoprost active drug concentration degraded over the course of normal patient use in Sao Paolo, Brazil.In the present study, the active drug concentration in all bimatoprost bottles tested was at least 100% of the labeled concentration more than 40 days after they were originally opened. Conversely, with latanoprost, only 8.8% of the bottles tested had at least 100% of the labeled active drug concentration. This result differs from a patient-use stability study conducted in Los Angeles, California, USA, where little degradation of latanoprost was found , and varOne limitation of the study was that baseline concentrations of the drugs were not measured prior to patient use. However, regulatory agencies and pharmaceutical manufacturing specifications require that actual drug concentrations be within a narrow range approximating the labelled strength. Approximately 10% more or less than the labelled concentration generally defines this tolerance, but it may be tighter depending on the characteristics of the drug . The labWhether the degree of drug degradation that occurs under normal-use conditions is clinically relevant is unclear. Clinical trials over 1 to 6 months have shown that bimatoprost produces IOP reduction greater than or comparable to latanoprost in various patient populations ,13-17, aLoss of drug activity may reduce IOP-lowering efficacy and cause wide fluctuations in a patient's IOP, which may be an independent risk factor for progressive disease ,21. BothPackage inserts indicate that bimatoprost should be stored between 2°C and 25°C, and no shelf life limitation is indicated . LatanopPatients on ocular hypotensive drugs that consistently lower IOP are less likely to progress than patients whose medication levels decrease over time. In this study, latanoprost formulations degraded while bimatoprost did not, suggesting that patients are more likely to receive the prescribed dose of bimatoprost after opening. Patients that receive the labelled dose, which has been clinically shown to achieve maximal IOP lowering from that medication, are more likely to halt glaucomatous progression.MDP, NK and CCU declare that they have no competing interests. JGW is an employee of and owns stock in Allergan, Inc.MDP, NK, CCU collected the study medication bottles. MDP and JGW conceived of the study. All authors participated in the study design and coordination, and read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


The prevalence of vesicoureteral reflux (VUR) has been estimated as. 4 to 1.8% among the pediatric population. In children with urinary tract infection the prevalence is typically from 30-50% with higher incidence occurring in infancy. When correction of VUR is determined to be necessary, traditionally open ureteral reimplantation by a variety of techniques has been the mainstay of treatment. This approach is justified because surgical correction affords a very high success rate of 99% in experienced hands and a low complication rate. In that context the purpose of this review article is to highlight the use of laparoscopy and robot-assisted techniques to perform ureteric reimplantation for the management of pediatric VUR. A detailed review of recent literature on the subject is performed to find out various aspects of minimally invasive surgery in the treatment of VUR, highlighting evolution of management approaches, operative steps, complications, results and the current status in clinical practice. We also share our experience on the subject. Vesicoureteral reflux (VUR), the most common urological abnormality in children, is considered as a significant factor for the development of urinary tract infection (UTI), progressive renal damage or scarring and end stage renal failure.–6 SurgicIn the last decade there has been a shift to perform ureteric reimplantation using the laparoscopic approach in order to provide all advantages of a minimally invasive technique and long-term results similar to traditional open ureteric reimplantation.et al., reported their experience in 22 children who underwent percutaneous endoscopic trigonoplasty.[The first laparoscopic extravesical  approach was used to perform modified Lich-Gregoir reimplantation. The Lich-Gregoir method, which evolved in the 1960s, is the most popular extravesical procedure and the first laparoscopic extravesical approach was performed by using the modified Lich-Gregoir technique. In 1993 and 1994, several groups reported experimental efforts with extravesical laparoscopic reimplantation in the porcine model 4,5 where animals first had reflux created by endoscopically incising the submucosal tunnel. Using a transperitoneal approach, on the posterior wall of the bladder a detrusor incision was created proximal to the ureteral hiatus, the ureter was positioned within the trough and the detrusor closed over it with either staples or absorbable sutures. The early results were promising although investigators reported it to be technically challenging. Shortly after the initial reports of extravesical ureteral reimplantation, experience with a combined transvesical and transurethral endoscopic approach to ureteral reimplantation was reported by two groups.12 In 199noplasty.2 into the retroperitoneal space and subsequent pneumoretroperitoneum and bladder collapse. The ureters were then reimplanted in a cross-trigonal fashion after the detrusor hiatus had been reapproximated with interrupted polyglactin sutures. The length of the submucosal tunnel was limited and the procedure probably achieved a 2 to 2.5 cm ureteral reimplantation on each side. At one year follow-up, 10 of 12 ureters managed in this fashion showed no reflux.Following this initial disappointing resolution rate with trigonoplasty, members of the same group performed transvesical mobilization of the ureters again using two suprapubic transvesical ports and a cystoscope. Using siIn an effort to improve on their results, Okamura and associates developed a new procedure called endoscopic trigonoplasty II.16 This wIn 2003 laparoscopic Cohen procedure using a pneumovesical approach was first described in an animal model. We devel2 pneumovesicum and it could also serve as an additional suction irrigation device during subsequent dissection and ureteric reimplantation. A 5-mm 30-degree scope is used to provide intravesical vision. Two more 3-5 mm working ports are then inserted along the interspinous skin crease on either side of the lower lateral wall of the distended bladder under vesicoscopic guidance [The patient is positioned supine with the legs separated apart for cystoscopy and bladder catheterization intraoperatively. For small infants the surgeon can stand and operate over the patient's head whereas for older children, the surgeon usually stands on the patient's left side. The video column is placed between the patient's legs at the end of the table. The port placement is preceded by transurethral cystoscopy to allow placement of the first camera port under cystoscopic guidance. The bladder is first distended with saline and a 2-0 monofilament traction suture is passed percutaneously at the bladder dome under cystoscopic vision, through both the abdominal and bladder walls. This helps to keep the bladder wall from falling away when the first camera port site incision is made and during insertion of the cannula. A 5-mm Step port  is then inserted under cystoscopic vision. A urethral catheter is then inserted to drain the bladder and start carbon dioxide insufflation to 10-12mmHg pressure. The urethral catheter is used to occlude the internal urethral meatus to secure COguidance . A 3–4 cguidance . IntraveThe ureter is mobilized by first circumscribing it around the ureteral orifice using hook electrocautery . With trWe have not faced any major complications with this technique. In the early part of the series when the cannulas were not secured to the bladder wall, displacement of the port outside the bladder wall occurred. This resulted in gas leakage into the extravesical space, with compromise of the intravesical space and endoscopic vision. It is usually possible to reintroduce the ports but securing the ports perfectly is the key to the success of this technique. We have Our operative success for laparoscopic Cohen performed in 60 children (46 girls and 14 boys) with an average age 1.81 years (range one month-7.24 years) and a mean follow-up of two years (range 1.1–5 years) has been 97.6%. These results are encouraging and endoscopic intravesical ureteric mobilization and cross-trigonal ureteral reimplantation can be safely performed with routine pediatric laparoscopic surgical techniques and instruments under carbon dioxide insufflation of the bladder.19The extravesical approach can be performed unilaterally or bilaterally, applying Lich-Gregoir technique. The patient is in supine position for an extravesical laparoscopic ureteral reimplantation. The bladder is first inspected with cystoscopy. A 3-Fr ureteral catheter may be placed at this time to help laparoscopic identification although we do not routinely use it. After scopy the bladder is drained by a catheter. An infraumbilical incision is made to place a 5-mm trocar for vision by open method. The other two trocars are placed along the lateral border of the rectus. Ports are fixed to the abdominal wall using a stitch which is also used to close the fascia. The ureter is normally seen well at the pelvic brim and can be followed to its insertion into the bladder. The technique follows the same steps as the open Lich-Gregoir procedure. It starts by dissecting the ureter after opening the peritoneum just over the posterior bladder wall. In females the ureter can be found in the anterior relation to the uterus. The ureter is freed from the surrounding tissue keeping its vessels intact. Approximately 4-5 cm is dissected to permit mobility and to prevent kinking as the bladder tunnel is created for the ureter. It is important to take care not to damage the Vas during the dissection around the UV junction. Adequate exposure of the posterior bladder wall is a key factor in this operation. A hitch stitch through the posterior bladder wall can be used to improve the exposure of the ureteral hiatus, attaching to the abdominal wall or through it.Once the ureter is free, the size of the tunnel is estimated after the bladder is partially distended. The ureter should course slightly laterally to avoid kinking of the ureter. A tunnel is adequately dissected to obtain a 5:1 ratio of length to width; the detrusor muscle is divided full thickness using a cautery hook while keeping the mucosa intact. Any perforations of the mucosa are closed using a 6-0 chromic suture. The ureter is positioned in the tunnel so as to avoid any kinking or excessive compression of the ureter to prevent obstruction. Closure may be from proximal end of the incision to distal or in the reverse fashion. In the latter, the ureter is well visualized while in the former needle needs to be passed under the ureter each time. The peritoneum is closed in a running fashion and a catheter is left in place, for one day. The indications for using the MIS extravesical Lich-Gregor approach are the same as for the open surgical technique although some investigators do not find the extravesical procedure appropriate for patients with megaureters requiring tapering.The common complication is accidental bladder mucosal perforations during the dissection of the detrusor muscle trough for the submucosal tunnel. The bladder mucosa perforations can be prevented by not over distending the bladder and use of blunt instruments like the suction tip to do the dissection of the mucosa from the detrusor muscle. Another limitation of this approach is transgressing the peritoneal cavity and difficult retraction of the bladder for the want of good exposure.et al., in 2004 have taken a similar approach and have documented excellent results and recovery in the post pubertal patients.[Lakshmanan and Fung reported extravesical laparoscopic reimplant in 99 ureters in 66 children aged four to 18 years.[patients. They alsThis technology has provided an opportunity to apply new techniques to practice MIS. From our own experience we believe that one of the main advantages of the robotic system, especially in children, is of offering surgeons inexperienced in laparoscopy the benefits of high dexterity in minimally invasive procedures and for the experienced laparoscopic surgeon to improve their capacity. Robot -a23The robot-assisted extravesical approach for treating VUR can be performed unilaterally or bilaterally, following the same steps as have been described in the laparoscopic section. Typically, the patient is in the supine position and legs placed apart. An open technique is used to place the first trocar, the 12mm camera port, in the umbilicus. The working ports, 8mm, are positioned in the mid-clavicular line bilaterally, about 1cm below the umbilical line. Ports are fixed and secured firmly to the abdominal wall using a stitch which is also used to close the fascia later. The robot is docked over the child's feet end to perform the surgery. For the robotic Cohen procedure the patient and robot docking position is the same as described for the extravesical approach. The procedure steps are the same as described earlier in the laparoscopic pneumovesicum reimplantation technique. While doing a robot-assisted procedure the main difference is the use of 12mm camera port and two 8mm working ports. Our experience of conventional lap for this technique has been quite different. The working space after all the three robotic ports have been placed inside the bladder is quite limiting as against what we usually experience with 5mm ports and 3mm instruments doing this surgery. There isn't enough data doing this technique by using robotic assistance. In our eDisadvantages of this robotic system include the lack of tactile sensation and thus visualization of anatomic landmarks is the key to successfully completing the operation. The unscrubbed surgeon is away from the operating table and therefore must depend on an experienced scrubbed assistant. Active communication between the primary surgeon, first assistant and staff is imperative. Although the learning curve for the surgeon may be short there is a substantial learning curve for the ancillary staff. Finally, the cost of the da Vinci robot is always a consideration. An initial investment of over $1,000,000 and subsequent running costs of $80,000 to $100,000 a year may not make this procedure feasible at many centers.Robotic surgery in pediatric urology is an evolving technique. The computer-assisted system has introduced smaller instruments (5mm) which are available although do not provide any added advantage in efficiency, primarily due to their design and the monocular lens system. Another improvement in design is the addition of a fourth arm which can be applied as a retractor and developing a smaller robot with better maneuverability while docking. As the technology continues to get better, the efficiency of the robotic system is likely to improve and offer the means to overcome impediment in children. Animal studies demonstrate robotic assistance can increase the applicability of minimally invasive surgery to complex procedures in children and neonates; however,To further determine the role of robotics in performing ureteric reimplantation in children, rigorous prospective research is needed that combines surgical and technical outcomes with overall subjective or cosmetic outcome and economic analysis.Laparoscopic surgery has gradually made its place in surgically dealing VUR. Laparoscopic and robot-assisted extravesical and intravesical surgeries have shown good early results.1921 It h1910The future of transvesical and extravesical minimally invasive procedures in children is promising and should be most interesting to follow over the next five to 10 years. Progress in the area of transvesical and extravesical surgery for treating vesicoureteral reflux in children will be made as more and more surgeons take up these procedures by laparoscopy and also by robot-assisted procedure with future innovations in robotic instruments.


Genetic screens, where the effects of modifying gene function on cell behaviour are assessed in a systematic fashion, have for some time provided useful information to those interested in disease pathogenesis and treatment. Genetic screens exploiting the phenomenon of RNA interference (RNAi) are now becoming commonplace. This article explains the different RNAi screen formats and describes some of the applications of RNAi screening that may be pertinent to the research pathologist. Put simply, experimental RNAi allows the research scientist to assess the function of a gene or protein by silencing its expression using synthetic RNAs or plasmids (an effect often referred to as “knockdown”). This technique exploits a physiological mechanism that represses gene expression by causing the degradation of protein-coding messenger RNA (mRNA) transcripts. In mammalian cells, physiological RNAi is primarily mediated by non-protein-coding RNA transcripts, known as microRNAs (miRNAs) . miRNAs Caenorhabditis elegans. In this context, dsRNA is processed into siRNA by an enzyme, Dicer.2In 1998, Mello and Fire demonstrated that potent gene silencing could be experimentally induced by injecting double-stranded (ds)RNA into the nematode The experimental use of synthetic siRNAs and shRNA plasmids has profoundly changed the way in which the loss of function of particular genes or proteins is studied. Previously, techniques that were either more time consuming, such as gene targeting , or capricious, such as antisense RNA, were used. Alternatively specific inhibitors of a particular protein could be used, but these are limited in scope. Now RNAi reagents can be purchased and trivially used to silence almost any gene in the genome.www.dharmacon.com), but if long-term silencing is required, shRNA expression constructs are more often the solution. These constructs express miRNAs or shRNAs that are processed by the target cell’s own RNA-processing machinery into siRNA, which in turn mediate gene silencing. These constructs come in a variety of formats and can be purchased either as simple plasmids or as viral constructs that are easier to deliver to a wider range of cell lines. shRNA vector-based reagents can provide long-term, stable gene silencing, as the vectors used are able to integrate into genomic DNA and are thus copied along with cellular DNA. Although the inherent instability of some of the initial viral systems used to express shRNA limited their utility, this problem has now been addressed in the most recent versions that are available from commercial suppliers. One of the more recent advances in shRNA construct design has been the use of vectors that express miRNAs instead of shRNAs. It is proposed that, as this process more closely mirrors the mechanism by which endogenous mammalian miRNAs mediate gene silencing, it leads to an improvement in silencing efficiency.in vivo RNAi screens in whole animals, as opposed to in vitro screens in cultured cells. Already, individual genes can be targeted in mice using RNAi,7As described above, the types of RNAi reagents fall into two main categories, siRNAs and shRNA expression constructs. et alsiRNAs and shRNA expression constructs are now available as libraries that contain reagents targeting any number of genes. Depending on the application, RNAi reagents targeting a single gene, a particular gene subset, for example protein kinases, or indeed the entire genome may be purchased. The commercial availability of such reagents now means that a number of genes can be screened simultaneously, and relatively straightforwardly, for their involvement in a chosen biological phenotype. The format of how such a screen is performed is largely determined by the choice of RNAi reagent. Both shRNAs and siRNAs are well suited to “one gene per well” screens  where ceThe scale of whole-genome screens that involve the use of hundreds of multi-well plates can often be daunting. Given this, an alternative approach is to use shRNA-expression constructs in “pooled” screens . Here, pRNAi, RNA interference. The process by which RNAs that encode proteins (messenger RNAs (mRNAs)) are degraded in a sequence-specific manner by binding to a short interferring RNA.siRNA, short interfering RNA. The RNA species that binds mRNA in the RNA-induced silencing complex (RISC). The mRNA/siRNA interaction activates the nuclease activity of RISC, leading to degradation of the mRNA.shRNA, short hairpin RNA. A siRNA precursor, encompassing a “stem” and a “loop” structure formed by base pairing between short regions of the RNA sequence (these form the “stem”) separated by a short sequence that does not form base pairs (which forms the “loop”).microRNA (miRNA), a non-protein-coding RNA species. One of the known functions of miRNAs in mammalian cells is in RNA interferencepooled/non-pooled screens. In non-pooled screens, RNAi reagents targeting one gene are introduced into a single-cell population. Non-pooled screens are normally carried out in multi-well plates, with each well representing RNAi reagents targeting one gene. In pooled screens, RNAi reagents targeting a number of genes are introduced into the cell population at the same time.Infection, the process by which DNA, packed within a viral coat, is introduced into cells. DNA-encoding miRNAs or shRNAs can be packaged as virus and infected into cells.Transfection, the process by which “naked” or non-virally packaged DNA is introduced into cells. Normally this is achieved by binding DNA to a lipid; the resultant lipid sphere binds to and merges with the target cell’s plasma membrane, releasing the DNA into cells.C elegans and Drosophila melanogaster to take up long dsRNAs by feeding or soaking methods, eliminating the requirement for expensive transfection reagents. Recent studies indicate that many results from RNAi screens in model organisms translate to humans.The commercial availability and relative ease of use of RNAi libraries now means that most research labs are able to perform an RNAi screen, be it genome wide or targeting a smaller gene subset. Given this, it is worth describing the major issues that should be considered before embarking upon such an enterprise. As with any well thought out experiment, the screen assay to be measured must carefully reflect the clinical, pharmacological or biological question. The assay system must also be robust, reproducible and affordable. Most laboratories use human tumour cell lines for RNAi screens, as these can be routinely grown and transfected but, depending on the phenotype examined, the use of a non-human model system might be considered. The biggest advantage is cost, which can be significantly lower in model organisms because of the ability of Positive controls can be used to define the magnitude of change that is thought to constitute a meaningful biological effect and verify that the screen is returning valid biological data. They also provide biologically relevant thresholds for identifying positive hits from a screen. Comparison of positive and negative controls also allows the screening window coefficient or Z factorOnce decisions have been made on the factors described above, a typical screening strategy is as follows. Firstly, experimental parameters need to be optimised. Probably, the best place to start is to ensure that high-efficiency transfection or infection can be achieved in the format (multi-well plate or other) that is to be used for the screen. Ideally, one is looking for transfection/infection conditions that enable efficient delivery of the RNAi reagent without causing excessive, non-specific toxicity. One simple trick for assessing RNAi delivery parameters is to transfect or infect cells with RNAi reagents that silence genes that are essential for cell viability and compare cell death with control transfected cells. In human tumour cells, RNAi reagents targeting Polo-like kinase 1 are often used for this purpose, as knocking down this kinase consistently kills most cancer cells.et al.It is also absolutely vital to consider the analysis of screen data before embarking upon the screen itself. Perhaps one of the best methods of screen analysis has been described by Boutros Once the screen and data analysis are performed, it is often common to perform a secondary screen using only the control RNAi reagents and those silencing the “hits” identified in the screen. Subsequent to this technical validation, it is necessary to eliminate the possibility of off-target effects, ie, effects that are due to silencing of genes other than the intended targets. Although silencing of genes via RNAi requires high complementarity to the target mRNA, off-target effects may require considerably less, in a similar fashion to the microRNA machinery. A 7–8-nucleotide “seed” region at the 5′ end of the anti-sense strand is most critical in generating off-target effects, particularly when this seed region occurs in the 3′ untranslated region of a potential off-target gene.www.ingenuity.com). However, it must be noted that this approach is only as good as the quality of the data in the pathway or interaction database that is used. Furthermore, this approach to validation is perhaps only appropriate for whole-genome RNAi screens, as particular gene subset screens may already be biased towards particular pathways.Validating individual hits from a screen can often be a more time-consuming and expensive enterprise than the screen itself. Given this, one may choose to use a more rapid approach that exploits the improvements in bioinformatic analysis that are now available. For example, one can now take the list of “hit” genes from a RNAi screen and subject this list to some form of pathway analysis; where a number of hits from the screen fall into the same biological pathway, or indeed have a well-established physical or function interaction, one has some confidence that the effects are real. To achieve this, one has the option of using many of the pathway analysis tools that are publicly available or even the commercial tools such as Ingenuity (RNAi screens are now becoming part of everyday technology used in the research laboratory.There are a number of ways to use this technology based on the type of question asked and the scale of the undertaking.RNAi technology has the potential to dissect basic biological phenotypes, as well as providing insight into clinically relevant questions.As the reader will see below, RNAi screens represent more of a research tool rather than an application that could be used in the diagnostic laboratory. Nevertheless, individual RNAi reagents, rather than RNAi libraries and screens, do serve one obvious application for use in the diagnostic laboratory. The ability of RNAi reagents to knockdown the expression of specific proteins makes them ideal for the validation of antibodies that are to be used in diagnostic tests. For example, cells transfected with siRNAs targeting one gene/protein, when compared with control-transfected cells, serve as ideal controls for identifying the optimal conditions for the use of an antibody.Where RNAi screens are involved, the interest to the pathologist is their utility in the dissection of basic cellular pathways, but also in more translational areas such as identifying novel therapeutic approaches and mechanisms of drug resistance. To illustrate this, we present a number of “case reports” describing a selection of the more pertinent RNAi screens.Flaviviruses, such as West Nile virus, represent a significant risk to human health. To understand what determines whether a cell can be infected with West Nile virus, Krishnan and colleagues5q-syndrome, a subtype of myelodysplastic syndrome, is characterised by a defect in erythroid differentiation and, as the name suggests, is thought to be caused by haploinsufficiency of one gene or a number of genes located within a 1.5-megabase region of chromosome 5q. To identify the causative defects in this syndrome, Ebert and colleaguesPIK3CA, conferring resistance to trastuzumab. Significantly, this RNAi screen suggests that analysis of the PI3K pathway may provide biomarkers to predict the likelihood of patient response to trastuzumab treatment.Many RNAi screens have been carried out with the aim of exploring the mechanism of resistance to chemotherapeutic drugs. For example, we recently used an RNAi screen to identify novel determinants of response to the commonly used breast cancer therapy, tamoxifen.Although RNAi technology is still evolving, it is also now suitably mature that RNAi screens are now becoming part of the everyday armoury of the research laboratory. Hopefully, we have shown you that there are a number of different ways to use this technology, primarily based on the type of question one has and the scale of the undertaking that one wishes to perform. Nevertheless, it is certainly becoming clear that this technology has the potential to allow the pathologist to not only dissect basic biological issues but also questions of a more translational bent.


One must demand an accurate, safe, radiation-free, and noninvasive method for reflux examination as the ideal possibility for reflux screening. Of course the available different imaging modalities are far from this ideal situation, but minimal radiation exposure is indeed a permanent objective. Additionally since all of these studies might be quite stressful to the child and the family, a specially designed and equipped environment is obligatory for the comfort of all involved. An absolute ideal modality in the diagnosis of VUR would be the definition of a certain marker in serum or urine that could identify children with VUR without the need for any interventional screening modality. Therefore more and more efforts have to be made in the future to investigate different markers for this purpose. Since reflux is one of the most frequent congenital conditions pediatric urologist have to deal with potential risks that might lead to renal insufficiency, noninvasive and radiation-free modalities should become the methods of choice, hopefully in the near future. Differentimaging modalities for evaluating vesicoureteral reflux (VUR) are nowadaysavailable and more and more attention is paid to concerns about radiationsafety, especially when multiple imaging studies are necessary with repeatexposition to ionizing radiation during a conservative follow up. Minimalradiation exposure is now a permanent objective.Experienceand logistic availability are often responsible for choosing the one or theother imaging study. Moreover, patient age, gender, race, and parentalpreference and anxiety about invasiveness and radiation exposure play anadditional role as well.Inthe past years, different innovative techniques like low-dose fluoroscopy havedefinitively decreased radiation dose to the patient. On the other hand,diagnostic quality images are mandatory for appropriate diagnosis andtreatment. Ultrasonography, nuclear medicine, and magnetic resonance arepreferred to intravenous urography and computed tomography whenever possible.In general, voiding cystourethrography has frequent indications in pediatricurology and efforts are made to replace it by radionuclide cystograms orsonocystograms in order to reduce the exposure to ionizing radiation.Furthermore,there is also no consensus about the timing of evaluation for possible refluxas well as in the conservative follow up or after intervention.Allthese three modalities can be used to demonstrate the presence or absence ofVUR. Voiding cystourethrograpy (VCUG) as well as direct radionuclidecystography (RNC) are invasive diagnostic tools with the need for patientpreparation and catheterization and the exposition of the patient to ionizingradiation although VCUG is a fluoroscopic examination and RNC a nuclearmedicine study, respectively. Additionally, an indirect RNC with theintravenous administration of technetium-99m-labeled diehtylenetriaminepentaacetic acid is a possible tool with the assumption of a possible VUR whenradioisotope counts increase in the renal areas after voiding. But the falsenegative rates may vary between 22 to 51% [Eventhough being invasive as well since catheterization is mandatory, the bigadvantage of performing a sonocystography is the prevention of any ionizingradiation to the patient at all.Fora conventional VCUG, a water soluble contrast medium is instilled into thebladder after preparation and transurethral sterile catheterization. An optionis the suprapubic administration of the contrast, but still invasive. Differentfluoroscopic images are taken to demonstrate the presence or absence ofvesicoureteral reflux.Thesame procedure (preparation and catheterization) is necessary for performing anRNC. Usually technetium 99m pertechnetate is the radiopharmacon of choice to beinstilled into the bladder. The radioactive emissions are continuously recordedwith a gamma camera.Whencomparing these two wide spread diagnostic modalities, the big advantages of aclassical VCUG are the provision of high-resolution images with a clearevaluation of the bladder wall, the urethra especially in males ,any sigAlthoughrecent improvements introducing low-dose fluoroscopy techniques and pulsefluoroscopy with the add of digital enhancing modalities have decreased theradiation dose to the patients dramatically –5,stillOnthe other hand, a direct RNC allows continuous monitoring for VUR throughoutthe whole examination time without any additional radiation introduced.Therefore, some authors prefer RNC to be more sensitive in the diagnosis of VUR althoughThemain advantage of RNC over fluoroscopic VCUG is definitively decreasedradiation exposure of the patient. The average effective radiation dose of aVCUG using low-dose fluoroscopy is around 3 mrem, compared to 0.5 mrem for anRNC. Of course the average effective dose of the VCUG is variable and dependson the patient size, operator, and machinery . The senSonocystographymay be used as a very sensitive tool in the detection of a possible VURespecially since the intervention of various ultrasound echo enhancing agents. First, Onemust demand an accurate, safe, radiation-free, and noninvasive method forreflux examination as the ideal possibility for reflux screening. Additionally,since all of these studies might be quite stressful to the child and the familya specially designed and equipped environment is obligatory for the comfort ofall involved. Preparation and education of the families help to reducediscomfort. If needed, sedation with the use of midazolam can be beneficialwithout any negative influence on the outcome of the examination .Contrastenhanced ultrasound allows an accurate and safe diagnosis and is in addition toVCUG and RNC radiation free as well; but unfortunately, still an invasiveprocedure with the insertion of a catheter. A future prospective might be anexogenous bubble generation to fulfil one of the most important criteria inreflux diagnosis: being noninvasive. Efforts are already being made to achievethis goal. Till then nuclear medicine studies and contrast studies will remainessential for the evaluation of VUR.2, urinary β2-microglobulin,urinary interleukin levels, and serum endothelium leukocyte adhesion moleculehave been shown to be elevated in patients with VUR compared to controls. Sofar, none of these methods can localize which kidney is affected by reflux norcan they assess the grade but they probably offer the potential advantage ofrapidly screening for VUR.Anabsolute ideal modality in the diagnosis of VUR would be the definition of acertain marker in serum or urine that could identify children with VUR. Basicresearch is going on to investigate different markers that have been found tobe elevated in children with VUR . Measureβ-hexosaminidase, has been shown to be higherin patients with VUR and renal scarring [Anothermarker, scarring . Tamm-Hoscarring . Interesscarring . Stilla


PGC-1α deficiency in an atherosclerotic mouse model.Atherosclerosis is a chronic inflammatory disease that evolves from the interaction of activated endothelial cells, macrophages, lymphocytes and modified lipoproteins (LDLs). In the last years many molecules with crucial metabolic functions have been shown to prevent important steps in the progression of atherogenesis, including peroxisome proliferator activated receptors (PPARs) and the class III histone deacetylase (HDAC) SIRT1. The PPARγ coactivator 1 alpha (Ppargc1a or PGC-1α) was identified as an important transcriptional cofactor of PPARγ and is activated by SIRT1. The aim of this study was to analyze total −/− PGC-1α−/−ApoE and −/− PGC-1α+/+ApoE mice kept on a high cholesterol diet. Despite having more macrophages and a higher ICAM-1 expression in plaques, −/− PGC-1α−/−ApoE did not display more or larger atherosclerotic plaques than their −/− PGC-1α+/+ApoE littermates. In line with the previously published phenotype of −/−PGC-1α mice, −/− PGC-1α−/−ApoE mice had marked reduced body, liver and epididymal white adipose tissue (WAT) weight. VLDL/LDL-cholesterol and triglyceride contents were also reduced. Aortic expression of PPARα and PPARγ, two crucial regulators for adipocyte differentitation and glucose and lipid metabolism, as well as the expression of some PPAR target genes was significantly reduced in −/− PGC-1α−/−ApoE mice. Importantly, the epididymal WAT and aortic expression of IL-18 and IL-18 plasma levels, a pro-atherosclerotic cytokine, was markedly reduced in −/− PGC-1α−/−ApoE mice.To investigate if total PGC-1α deficiency affects atherosclerosis, we compared −/− PGC-1α−/−ApoE mice, similar as −/−PGC-1α mice exhibit markedly reduced total body and visceral fat weight. Since inflammation of visceral fat is a crucial trigger of atherogenesis, decreased visceral fat in PGC-1α-deficient mice may explain why these mice do not develop enhanced atherosclerosis. Atherosclerosis is a chronic inflammatory disease that results from interaction between activated endothelial cells, modified low-density lipoproteins (LDL), monocyte-derived macrophages, T cells, and the vessel wall. Activated endothelial cells express adhesion molecules that attract and recruit blood monocytes and lymphocytes. Upon binding to the endothelial layer, these monocytes transmigrate into the subintimal space, and differentiate into macrophages. Plaque macrophages interact with lymphatic cells, mainly T cells, ingest modified LDL via scavenger receptors and become foam cells, thereby promoting plaque formation PGC-1α was the first described member of the small PGC-1 family of coactivators The phenotype of PGC-1α knock-out mice underlines the central role of this transcription cofactor in homeostatic control of metabolism: they are leaner than wild-type (WT) littermates, have markedly reduced body fat content, and are resistant to diet-induced obesity, hence protected from developing insulin resistance and impaired glucose tolerance in vitro has been shown to prevent reactive oxygen species (ROS) production and NAD(P)H oxidase activity, with subsequently reduced NF-κB activity and lower expression levels of MCP-1 and VCAM-1 in vitro and improved endothelial dysfunction in aortic rings ex vivoOverexpression of PGC-1α in human aortic smooth muscle and endothelial cells The following studies suggest a link between PGC-1α and atherogenesis at the clinical level: Xie et al. reported a correlation between PGC-1α polymorphism and hypertension PGC-1α deficiency on atherogenesis by comparing −/− PGC-1α−/−ApoE and −/− PGC-1α+/+ApoE mice.Thus, we investigated the effects of −/−PGC-1α with −/−ApoE mice, and compared 20-week old male −/− PGC-1α−/−ApoE and −/− PGC-1α+/+ApoE mice that were kept on a high-cholesterol diet for 12 weeks. Histomorphometry of thoraco-abdominal aortae stained with Oil-Red O (ORO) revealed no difference in atherosclerotic plaque area between −/− PGC-1α−/−ApoE and −/− PGC-1α+/+ApoE mice , Cebpa (C/EBP-α), Fabp4 (aP2), Fasn (Fatty acid synthase), Fatp1 (Fatty acid transport protein 1), Lipe (Hormone-sensitive lipase), Lpl (Lipoprotein lipase), LXR-α (Liver X receptor α), Pck1 (Phosphoenolpyruvate carboxykinase 1), and Ucp1 (Uncoupling protein 1). Expression of Cebpa, Fabp4, Pck1, and Ucp1 was significantly lower in −/− PGC-1α−/−ApoE compared to −/− PGC-1α+/+ApoE mice, while the expression of Fasn showed the same trend and mRNA levels of Adipoq, Fatp1, Lipe, Lpl, and LXR-α did not differ (PPARα and PPARγ expression and function may at least in part be suppressed in −/− PGC-1α−/−ApoE mice.Peroxisome proliferator activated receptors (PPARs) are important regulators of adipocyte differentiation as well as lipid metabolism and inflammation and their transcription is regulated by PGC-1α −/− mice , whereas−/− mice . To examt differ . These d−/− PGC-1α−/−ApoE and −/− PGC-1α+/+ApoE mice. While expression of Adipoq, Nampt (Nicotinamide phosphoribosyltransferase), Retn (Resistin), IL-6, IL-10, TGF-β, MCP-1, IFN-γ, Agt (Angiotensinogen), 11β-HSD1 (11-beta-hydroxysteroid dehydrogenase 1), TNFα, and Lpl was only mildly reduced or unchanged, the expression of leptin, Rarres2 (chemerin), Serpine1 (PAI-1), and IL-18 was lower, and expression of complement factor D (Cfd or adipsin) higher in −/− PGC-1α−/−ApoE compared to −/− PGC-1α+/+ApoE epididymal WAT  in atherosclerotic lesions IL-18 and SR-PSOX/CXCL16 mRNA levels were reduced in −/− PGC-1α−/−ApoE mice, while IFN-γ expression did not differ between the two genotypes , Serpine1 (PAI-1), and IL-18 in visceral adipose tissue could be sufficient to avoid increased atherogenesis. Rarres2 is associated with white adipose tissue inflammation and promotes mobilization and chemotaxis of dendritic cells and macrophages Reduced expression of PAI-1-deficient mice showed attenuated neointima formation after perivascular cuff-induced injury PAI-1 overexpression prevented the development of abdominal aortic aneurysm PAI-1 is an anti-fibrinolytic enzyme and has beneficial and deleterious effects in atherogenesis. For example, −/− PGC-1α−/−ApoE mice was observed for IL-18. IL-18 is a pro-atherogenic cytokine: Overexpression of IL-18 binding protein and direct injection of recombinant IL-18 accelerate atherogenesis, whereas IL-18 deficiency diminishes plaque formation in −/−ApoE mice −/− PGC-1α−/−ApoE mice. It is conceivable that the lower expression of IL-18 alone is sufficient to avoid an acceleration of atherogenesis in our −/− PGC-1α−/−ApoE mouse model.The lowest expression of the tested cytokines in the visceral WAT of −/− PGC-1α−/−ApoE mice. However, protein levels of the soluble form of SR-PSOX/CXCL16 in plasma did not differ between −/− PGC-1α−/−ApoE and −/− PGC-1α+/+ApoE mice, suggesting that the proteolytic cleavage of this chemokine is not affected in −/− PGC-1α−/−ApoE mice.Interestingly, IL-18-mediated increase of atherosclerosis is accompanied by elevation of SR-PSOX/CXCL16 expression Cfd encodes adipsin, the mouse homolog of human complement factor D, which is a serine protease that cleaves factor B in the alternative complement pathway, and it is secreted at high levels in adipose tissue adipsin was higher in −/− PGC-1α−/−ApoE compared to −/− PGC-1α+/+ApoE mice. Expression of adipsin and other components of the alternative complement pathway correlate with atherosclerosis −/− PGC-1α−/−ApoE provides a pro-atherogenic contribution.−/− PGC-1α−/−ApoE mice. Further studies using tissue-specific PGC-1α knockout or overexpression will be necessary to address these questions in more detail.Atherosclerosis is a disease combining the complexity of lipid/lipoprotein and inflammatory/immune disorders −/−ApoE C57BL/6 −/−PGC-1α C57BL/6 −/− PGC-1α−/−ApoE mice and −/− PGC-1α+/+ApoE littermates. Of those, male mice were fed a high-cholesterol diet  for 12 weeks starting at the age of 8 weeks. Mice were weighted before being sacrificed, and biopsies of aortae, heart, liver, spleen, brown and white adipose tissue, and pancreas frozen in liquid nitrogen or OCT  for later analyses.All animal procedures were approved by the local animal committee  and performed in accordance with our institutional guidelines.en face analysis. Collagen, fibrous cap thickness, and necrotic core size were analyzed on Elastica van Gieson (EVG)-stained cryosections of the aortic sinus as described 5 mm serial cryosections from the aortic sinus were stained with rat anti-CD68, rat anti-CD3 (Abcam), rat anti-VCAM-1 (BD Biosciences), rat anti-ICAM-1 (Serotec), or oil-red O (ORO). Thoraco-abdominal aortae were fixed with 4% paraformaldehyde and plaques stained with ORO for Total RNA isolated from proximal aortae was extracted with TRIZOL (Invitrogen), reverse transcribed with Ready-To-Go You-Prime First-Strand Beads , and the cDNA (n≥9 per genotype) quantified by qPCR using SYBR Green JumpStart Taq ReadyMix (Sigma-Aldrich). Primer sequences can be found in the supplemental Quantification of IL-18 and CXCL16 in plasma of mice where performed with Mouse IL-18 Platinum ELISA kit (Bender MedSystems) and Mouse CXCL16 ELISA kit (RayBiotech) according to the manufacturers instructions. Plasma was diluted 1∶2 for the IL-18, and 1∶32 for the CXCL16 ELISA assay.Total plasma cholesterol and triglycerides were quantified using Infinity Cholesterol TR13421 and Infinity Triglycerides TR22421 (Thermo Electron Cooperation), respectively. The lipid distribution in plasma lipoprotein fractions was assessed by fast-performance liquid chromatography gel filtration with a Tricorn Superose 6 10/300 GL column  en face ORO quantification was analyzed using a non-parametric Mann-Whitney U t-test. Statistical significance of differences of all other experiments was calculated using an unpaired Student's t-test. Significance was accepted at the level of p<0.05.Data are presented as mean ± SEM. The Table S1Primer sequences.(0.08 MB PDF)Click here for additional data file.


Vertebrate brains are composed of two hemispheres that receive input, compute, and interact to form a unified response. How the partially different processes of both hemispheres are integrated to create a single output is largely unknown. In some cases one hemisphere takes charge of the response selection – a process known as metacontrol. Thus far, this phenomenon has only been shown in a handful of studies with primates, mostly conducted in humans. Metacontrol, however, is even more relevant for animals like birds with laterally placed eyes and complete chiasmatic decussation since visual input to the hemispheres is largely different.Columba livia) were trained with a color discrimination task. Each hemisphere was trained with a different color pair and therefore had a different experience. Subsequently, the pigeons were binocularly examined with two additional stimuli that combined the positive color of one hemisphere with a negative color that had been shown to the other, omitting the availability of a coherent solution and confronting the pigeons with a conflicting situation. Some of the pigeons responded to both stimuli, indicating that none of the hemispheres dominated the overall preference. Some birds, however, responded primarily to one of the conflicting stimuli, showing that they based their choice on the left- or right-monocularly learned color pair, indicating hemispheric metacontrol.Homing pigeons (We could demonstrate for the first time that metacontrol is a widespread phenomenon that also exists in birds, and thus in principle requires no corpus callosum. Our results are closely similar to those in humans: monocular performance was higher than binocular one and animals displayed different modes of hemispheric dominance. Thus, metacontrol is a dynamic and widely distributed process that possibly constitutes a requirement for all animals with a bipartite brain to confront the problem of choosing between two hemisphere-bound behavioral options. Since the pioneering study of Broca in the nineteenth century, it is widely known that each of the two cerebral hemispheres processes and computes information differently. As outlined below, several studies report that this asymmetrical organization can be accompanied by unilateral control over a task. In this case, the performances under bilateral viewing are similar to the performances of a single hemisphere, and different from the performances of the other half-brain. The choice mechanism that determines which hemisphere will dominate the task is known as metacontrol Both left hemisphere (LH) metacontrol, meaning left hemisphere control over the task, and right hemisphere (RH) metacontrol were observed in humans Metacontrol has been shown in healthy humans To our knowledge, apart from humans, metacontrol was examined only in monkeys Columba livia) in a simple color discrimination task that these birds master quickly, and for which there are little or no hemispheric differences Both humans and macaques have frontally placed eyes that have a single fovea. Foveated objects are perceived by both hemispheres. Since primates produce a very high amount of eye movements, both half-brains see the majority of objects in the front of the animal. Conflicting and response-demanding input into the hemispheres is therefore not a major problem as long as stimuli are foveated. This is radically different for most birds. All birds have a virtually complete crossing of their optic nerves, transmitting visual input to the contralateral hemisphere 14 pigeons were the subjects of this study. Five were naïve and the rest had participated in former, unrelated, experiments. The birds were housed individually in a room with other conspecifics and placed on a 12/12h light/dark cycle. They were kept at 80–90% of their free feeding weight. Food was provided during the experiment and after experimental sessions. Water was freely available in their home cages throughout the experimental period. The pigeons were trained on average 6 times a week.The experiment was conducted according to the specifications of the German law for the prevention of cruelty to animals and hence, the European Communities Council Directive of 24 November 1986.2, Philips), with a resolution of 1024×768 Pixels. Pecking correctly on the pecking key reinforced the pigeons with the activation of the feeder. Experimental sessions and data collection were controlled by a Pentium PC running MATLAB  and a partial pre-version of Biopsy Toolbox The experiment was conducted in a 33(w)×34.5(d)×36(h) cm custom made Skinner box. The box was equipped with a house light on the side panel, a centered feeder containing mixed grains , a feeder light located above the feeder that was lit simultaneously with the feeder activation. Additionally, a centrally located transparent pecking key was located on the front panel, with its upper right corner being located 14(w)×7.5(h) cm from the upper right corner of the Skinner box. Through the pecking key, the pigeons viewed the 5(w)×2.8(h) cm stimuli that were presented on a TFT LCD monitor (Brilliance 150PThe stimuli used were 5(w)×2.8(h) cm rectangles. Training stimuli were half colored: the Red and Green stimuli were colored in their upper half, while the Cyan and Magenta stimuli were colored in their lower half . For sevTest stimuli were a combination of a Go training color with a NoGo belonging to the other color pair, i.e. Red-Magenta or Green-Cyan . The tesThe five naïve pigeons were autoshaped to peck on a lighted pecking key (white square) in a standard autoshaping procedure containing 40 trials. The white square was presented for 5 seconds followed by 3 seconds of food access. After the pigeons started to respond to the pecking key, they were trained with a continuous reinforcement schedule. Subsequently, the pigeons were progressively trained with variable ratio , fixed interval  and variable interval  schedules. Each schedule proceeded until the pigeons responded correctly to more than 85% of the trials in two consecutive sessions. Each session contained 40 trials. Afterwards, they were monocularly trained in a VI20 schedule, in order to make them familiar with wearing and working with an eye cap. The other nine pigeons were already familiar with the Skinner box and the eye caps.Monocular viewing was made possible using eye caps. A velcro ring was fixed to the skin around the eyes using non-toxic glue. A cap could be attached to the ring, blocking the view of this eye and thus the contralateral hemisphere. The pigeons were adapted to the caps prior to the monocular testing sessions by wearing them in their home cages. The animals wore a cap for about 25 minutes before each testing session.A Go-NoGo task was used to teach the pigeons the discrimination. The schedule used was similar to the one used by Yamazaki et al. As the pigeon optic nerve decussates virtually completely at the optic crossing, each hemisphere can be tested separately by occluding one eye Four pigeons were trained in a Red/Green (Go/NoGo) color discrimination with the left hemisphere and a Cyan/Magenta discrimination with the right hemisphere.Four pigeons were trained in a Green/Red discrimination with the LH and a Magenta/Cyan discrimination with the RH.Three pigeons were trained in a Cyan/Magenta discrimination with the LH and a Red/Green discrimination with the RH.Three pigeons were trained in a Magenta/Cyan discrimination with the LH and a Green/Red discrimination with the RH.Each of the two hemispheres was tested alternately.The discrimination criterion was rho≥.9 in two out of three consecutive sessions, for both hemispheres.The test stimuli were either Go-color learned by the left hemisphere combined with a NoGo-color trained by the right hemisphere (LH-Go & RH-NoGo), or a Go-color trained by the right hemisphere combined with a NoGo-color trained by the left hemisphere (LH-NoGo & RH-Go).The binocularly seeing test sessions contained six stimuli: the four monocularly-learned stimuli: LH-Go (the Go-color learned by the LH), LH-NoGo (the NoGo-color learned by the LH), RH-Go and RH-NoGo, as well as the two critical test stimuli: LH-Go combined with RH-NoGo and RH-Go combined with LH-NoGo ((LH-Go & RH-NoGo) and (LH-NoGo & RH-Go), respectively). Each of the six stimuli appeared 8 times. The stimuli were presented in a random order that was changed among the pigeons. Test stimuli were not reinforced.The rho value was used to index performances Laterality index indicated if binocularly there was a performance difference between the LH-learned and the RH-learned color information. It was measured using the rho values obtained from the binocular discrimination of the monocularly–learned color pairs.The The laterality index was calculated by the following formula:Dominance index indicated the type of hemispheric interaction during the conflicting situation. The dominance index was computed by the following formula, using the rho values calculated from the performances with the test stimuli:rho(LHtest) is the rho value for the number of times the pigeon pecked on the test stimulus containing the Go color learned by the LH: (LH-Go & RH-NoGo) relative to the number of pecks on the other test stimulus containing the Go color learned by the RH: (RH-Go & LH-NoGo).The rho(RHtest) is the rho value for the number of times the pigeon pecked on the test stimulus containing the Go color learned by the RH (RH-Go & LH-NoGo) relative to the number of pecks on the test stimulus containing the Go color learned by the LH (LH-Go & RH-NoGo).Bootstrap analysis was further performed to determine the likelihood of receiving the obtained dominance index values. The analysis was done for every animal separately by randomly assigning the pecks in the 16 test trials into Go and NoGo groups, for 1000 times. Following the reassignment, the distribution of dominance index value was computed, and a Z-score was used to calculate the probability of the obtained index.One sample t-test was used to calculate if the laterality index and the dominance index differ from zero. Using paired t-tests we compared the performances of the two hemispheres. A 2×2 repeated measures ANOVA with the factors session (last monocular session vs. binocular session) and Hemisphere (RH vs. LH) analyzed the performances with the monocularly-learned stimuli. Pearson correlations were further used.Means values are reported in the format of mean±SEM.p = .362). Since reaching the discrimination criterion and moving to the test session depended on both hemispheres, in some pigeons the hemispheres were overtrained. Thus, overall, with the left hemisphere the pigeons were trained on average for 13.1±2.6 sessions, and had 13±2.6 sessions with the right hemisphere. The average performances in the last training session were rho = .971±.005 and rho = .975±.007 with the left- and the right-hemisphere, respectively. The hemispheres did not differ in their performances in the last training session (t(13) = −.436, p = .670).The discrimination criterion was attained when performances reached rho≥.9 in two out of three consecutive sessions, for both the two hemispheres consecutively. On average, the pigeons needed 10.9±1.8 sessions (ranged from 5 to 28) to reach rho≥.9 with the left hemisphere, and 8.7±1.8 sessions (ranged from 3 to 27) with the right hemisphere. Nine pigeons achieved high performance more quickly with their right hemisphere, four with the left hemisphere, and one pigeon needed equal numbers of sessions with both hemispheres. This difference in acquisition speed was not significant (t(13) = −0.944, During the binocular test session the pigeons were confronted with six stimuli: the four stimuli known from the monocular training as well as the two conflict-producing stimuli that produced a Go-response in one and a NoGo-response in the other hemisphere.p = .600). Interestingly, the pigeons performed the color discrimination task better in the last monocular viewing session compared with the binocular viewing session  = 18.471, p = .001; pairwise comparison monocular vs. binocular performances: .088±.021, p = .001 Bonferroni corrected)  = .196, p = .665).The binocular performances with the monocularly-learned stimuli were rho = .87±.036 (range: from rho = .523 to rho = 1) with the color pair learned by the left hemisphere, and rho = .90±.03 (range: from rho = .625 to rho = 1) with the color pair learned by the right hemisphere . The latrrected) . The perp = .520). Nonetheless, comparing the average bootstrap index of each pigeons to its measured index, as well as looking on the U value from which the rho value was obtained, showed that six pigeons exhibited significant metacontrol. Four pigeons had a significantly negative dominance index, and hence showed RH-metacontrol, whereas two pigeons showed LH-metacontrol as their dominance index was significantly positive  and (LH-NoGo & RH-Go), the pigeons were faced with a conflicting situation. For every test trial, the pigeons had to decide according to which monocularly-learned color pair, i.e. hemisphere, they would react. Hemispheric dominance was determined by the pigeons' relative choices with the two test stimuli. The pigeons showed complete distribution of hemispheric dominance, ranging from .8 to −1. Across the group, the hemispheric dominance was normally distributed  = −.661, positive .p = .004; RH: r(14) = −.438, p = .117). Finally, the dominance index was approaching a significant correlation with number of monocular training sessions needed by the left hemisphere till criterion (LH: r(14) = .522, p = .055; RH: r(14) = .207, p = .472). The dominance index, however, was not correlated with the amount of overtraining sessions that occurred while a hemisphere was waiting for the other to reach criterion (LH: r(14) = .036, p = .904; RH: r(14) = . 389 , p = .170).The degree of metacontrol was correlated with the binocular discrimination of the LH-trained but not the RH-trained stimuli (LH: r (14) = .710, Metacontrol refers to the existence of a preference or choice mechanism that determines which hemisphere will control a task when the two sides of the brain are facing discrepant behavioral options In the initial training the pigeon were taught to discriminate between two color pairs, one with each hemisphere. This is an easy task for pigeons, and as shown previously As previously found in humans The half brain that has a slight advantage, either previously or due to training, seems able to take control over the task, possibly via commissural inhibition. In mammals this could be achieved with the corpus callosum. Since birds do not possess this commissure, other interhemispheric inhibitory pathways at brainstem level are obviously also able to achieve a similar function. Indeed, the intertectal commissures in birds are mostly inhibitory The lines to today's birds and mammals parted about 280 million years ago


Laparoscopic extravesical ureteral reimplantation in children is currently a technically demanding procedure with sparse literature to aid in mastering the learning curve. We present our most recent technique and lessons learned after 20 cases in children 4–15 years of age. The literature is also reviewed to encapsulate the current state-of-the-art. Openextravesical ureteral reimplantation is a successful and well-toleratedprocedure with a proven track record in the surgical management ofvesicoureteric reflux (VUR). Nevertheless, the relentless pursuit of minimallyinvasive ideals has led to the development of alternatives. Most recently, theendoscopic injection techniques have become quite popular, but concerns remainover the success rate and long-term efficacy. Thus, the laparoscopic approach offers another option which improves onthe open procedure with better cosmesis and convalescence, while providing adurable and successful procedure compared to injection therapy.Despitemultiple reports in the early 1990s of experimental surgery in animal models and few A total of 20children aged 4–15 years (mean 7.3 years) have undergone laparoscopic extravesical ureteral reimplantation overa 5-year period. The subjects were mostly female (15 of 20) with 11 (55%) casesbeing bilateral. All cases were diagnosed with VUR after urinary infection andthe indication for surgery included breakthrough infection in 18 of 20 andpersistent high-grade VUR in 2 of 20.The highest gradeof reflux per patient ranged from 2 to 4, with only 1 case of unilateral grade2, that being a case of failed injection therapy. The distribution of VUR perpatient by highest grade was grade 4 in 7 patients (35%), grade 3 in 10 (50%),and grade 2 in 3 (15%). Megaureters, duplicated ureters, and neurogenicbladders were excluded initially. Previous open ureteral surgery remains anexclusion criterion. Bilateral cases areselected such that one side is not high-grade VUR, so as to minimize the riskof urinary retention. This hypothesis is based on the postulates that bladderdysfunction should not occur with unilateral extravesical dissection, and thathigh-grade VUR is a risk factor for postoperative bladder dysfunction [The postoperativefollow-up regimen includes a routine abdomino-pelvic ultrasound 1 month aftersurgery and a voiding cystourethrogram 3 months after surgery, with maintenanceof antibiotic prophylaxis until the VCUG is done. In the absence of newfindings on the first post-op ultrasound, another routine abdomino-pelvicultrasound is planned 1 year after surgery.Wefind it useful for learning purposes to divide the case into four specifictasks: 1-access, 2-uretero-vesical junction exposure, 3-detrusor tunneldissection, and 4-tunnel suturing.A 4-port approachis utilized with the patient in Trendelenburg position, legs spread apart, andthe arms tucked in at the side. A sterile Foley catheter is placed in theoperative field and controlled with a Toomey syringe. A mechanical bowelpreparation can be helpful in patients with constipation. The first port issupraumbilical, and the 3 others form an arc along the level of theanterior-superior iliac spine . The levblunt dissection adjacent to thebladder, often with the superior vesical artery coursing parallel. The assistantthen controls the ureter with a vessel loop  havehad resolution of reflux, with 2 cases refusing the post-op VCUG and 1 beinglost to follow-up. One case developed denovo contralateral grade 2 VUR. Three cases were converted to opensurgery, the first 2 cases, both bilateral, because surgical time had surpassed4 hours. Case 7 had a nonneurogenic neurogenic bladder with a severelyhypertrophied detrusor which made tunnel dissection difficult.The first 5 cases,4 of which were bilateral, can be considered the learning curve with operativetimes falling consistently below 3 hours for a unilateral case, and 5 hours fora bilateral case thereafter. Mucosal perforation remains the main determinantof operative time, in its absence the operative time averages 2 hours perureter. Mucosal perforation also remains the main determinant of hospital stay.The usual case is discharged the following day after having voided, whereasthose with suction drains remain for an extra day of observation. Three caseshave had a mucosal perforation including cases 4, 6, and 20, none of whichleaked postoperatively. There has been 1 complication, that of a distalureteral necrosis in case 5 which necessitated open revision with a Boari flap.In this case, the ureter was held on prolonged traction with a Babcock clamp,which is no longer used. None of thecases have experienced postoperative voiding dysfunction.Afew technical aspects merit greater commentary, especially where there may bedifferences with other authors. To begin, though exposure of the ureter isfastest from the bladder up to the pelvic brim, it may be helpful for the firstfew cases to mobilize the ureter from the pelvic brim caudally until one isfamiliar with the anatomical orientation of the juxtavesical ureter.Cystoscopically placed ureteral catheters are not necessary though they wereused in the first few cases to document that the ureter was not obstructed byan errant detrusor suture. The direction of the detrusorotomy should bestraight up; a medial orientation will lead to kinking of the ureter whereas alateral orientation makes for tedious dissection of the submucosal tunnel. Theinverted Y-type detrusorotomy is used sparingly so as to limit the chances ofmucosal injury. Instead, the detrusor tunnel edges are reapproximated withsutures further away from the ureterovesical junction, so as to limit ureteralobstruction by compression. If there is tension with the closure, a limited invertedY-type dissection is performed.Though all otherauthors describe the use of a single traction suture, this author believes thatthe use of 2 suspension stitches provides superior exposure of the mucosa asdissection progresses. In addition, the method of traction suture placementdeserves greater attention. Most authors describe percutaneous passage of aKeith needle into the abdomen, whereas this author passes an intracorporealsuture extracorporeally with the use of a fascial closure device. This approachpermits one to better judge the exit site of the stitch based on optimalexposure and orientation. The opposite and more commonly described approachcommits the surgeon to an exit site before one has a chance to test the effecton bladder exposure. The direction of tunnel dissection is ergonomically bestfrom the neohiatus downwards towards the ureterovesical junction.Unfortunately, this can lead to nuisance bleeding obscuring the exposure of theremaining mucosa. Ideally, one would want to dissect from the ureterovesicaljunction upwards towards the neohiatus that way the bleeding does not obscurevision, which is impossible with rigid instruments. Perhaps this is an area where the superiordexterity of the robot may be of benefit.Consideringthe multiple options for thesurgical management of VUR currently available, the indications for alaparoscopic extravesical approach are debated. The families electing to choosethis option are concerned with the success rate of injectables and the mountingevidence that the product is not durable over the long term. These familieswant a successful procedure so as to avoid multiple postoperative VCUG’s or tominimize the risk of another pyelonephritis in those who have experiencedrecurrent pyelonephritis. The advantages of reduced pain and convalescence areless in the infant population such that the procedure is offered mainly toschool age children. Cosmetic considerations become more important in thepostpubertal population. As a result of this selection process, the case loadis smaller relative to the overall cohort of surgically managed VUR, which doesimpact on operative time.Havingchosen a laparoscopic approach, other considerations include whether to use atransvesical approach or an extraperitoneal approach. Though the extravesicalapproach is ideally suited to an extraperitoneal exposure, this author feelsthat the extra surgical time involved in creating the space is not warranted.When one considers that the bowel is not mobilized and that the peritonealwindow used for transperitoneal exposure is so small, it is difficult toimagine significant adhesions occurring in such a context. I have been impressed in thecases converted open at how small the peritoneal window was; in fact the boweldid not enter the wound. Likely forthese reasons, there are no published reports on extraperitoneal ureteralreimplantation, though extraperitoneal pelvic laparoscopy has been reported forvarious procedures .Thetransvesical approach with pneumobladder was first described by Okamura et al.with the technique of endoscopic trigonoplasty . This prLaparoscopicextravesical ureteral reimplantation was popularized by Lakshmanan and Fung with excNevertheless, inmost series, the occasional problem of mucosal perforation and its attendantprolonged catheter drainage persists in comparison to open extravesicalsurgery. It remains to be seen if the ergonomic advantage of robotic assistancewill be helpful in this regard. Improvements in instrumentation such as a hookelectrocautery which is shielded posteriorly and thus does not cause mucosalperforation by thermal injury would be of tremendous benefit. Furthermore,prospective experimental study of the facility of mucosal exposure at differentinsufflation pressures and different bladder filling volumes deserves greaterattention.Laparoscopicextravesical ureteral reimplantation is another option in the surgicalmanagement of vesicoureteric reflux. It offers a greater success rate anddurability compared to injection therapy, while offering cosmetic andconvalescence advantages over open surgery in the older child. The learningcurve of the procedure is reasonable and facilitated by an analysis based on 4components, namely, access, ureter exposure, tunnel dissection, and tunnelclosure. The component of tunnel dissection is the only one which could benefitfrom further improvement, likely accomplished with refinements in instrumentsor greater study of the variables which contribute to mucosal perforation.


Sir,Biotinidase deficiency is a rare metabolic disorder with an estimated incidence of 1:61,067 population, although severe or profound disease is much rarer .[Clinical manifestations include neurological, dermatological, immunological and ophthalmological abnormalities. BiochemiA 6-month-old male child born of nonconsanguineous marriage presented with a history of seizures from 3 months of age and was being treated with sodium valproate, but seizure control was not observed. Antenatal and natal history was uneventful. There was no family history of seizure disorders. According to the mother, the child developed normally for the first 3 months and after that was not growing normally. On examination, the child had normal neurological findings, except for hypotonia, irritability and alopecia . The chiBiotin is a cofactor required by acetyl CoA carboxylase, pyruvate carboxylase, propionyl CoA carboxylase and 3 methylcrotonyl CoA carboxylase. It is covalently attached to the apocarboxylases by the epsilon amino group of a lysine residue, where it functions at the active site as a carbon dioxide carrier in the carboxylation reactions. Biotinylation of the apocarboxylases is catalyzed by holocarboxylase synthetase in an adenosine triphosphate-dependent reaction, with the intermediate formation of biotinyl adenosine monophosphate. The turnover of carboxylases yields biotinyllysine (biocytin) from which biotin is regenerated by the action of a specific amidolyase, biotinidase. This enzyme is also required for the release of dietary protein-bound biotin.[Individual inherited disorders of all four biotin-dependent carboxylases have been reported in addition to reports of patients with simultaneous defects of all four enzymes (combined carboxylase deficiency). In a majority of patients with late-onset combined carboxylase deficiency, the underlying defect is biotinidase deficiency. The earlBiotinidase deficiency can be profound (<10% enzyme level) or partial (10-30% enzyme level). Clinical presentation depends on the severity of enzymatic defect. Profound defects usually manifest between 3 and 6 months of age, with neurological manifestations , skin manifestations  and (c) respiratory problems . Older children and adolescents may exhibit limb weakness, neurosensory hearing loss and eye problems, e.g. optic atrophy and scotomas. The present case had only seizures and alopecia. Many symptomatic children with biotinidase deficiency exhibit various neuroimaging abnormalities, e.g. cerebral edema, attenuated white matter signal, cerebral atrophy and compensatory ventricular enlargement. Neuroimaging features may improve or become normal after biotin treatment.[Laboratory findings include metabolic acidosis and abnormal organic acids in the urine, and diagnosis can be established by estimating biotidinase in the serum. Biotinidase deficiency may be detected on screening of the newborn.Biotinidase deficiency may be confused with holocarboxylase deficiency, previously called early-onset or infantile multiple or combined carboxylase deficiency, which presents early and the biotidinase level is normal.Because biotinidase deficiency can be treated readily with biotin, this disorder should be considered in children with infantile seizures, especially in the presence of other characteristic neurological or cutaneous features.


Our data show how mitochondrial function may be adapted in response to external stimuli, and support the concept that such adaptation is critically involved in cellular survival and in lifespan extension by calorie restriction.There is increasing evidence that longevity and stress resistance are connected, but the mechanism is unclear. We report that mitochondria are regulated in response to oxidative stress and calorie restriction through a shared mechanism involving peroxisome proliferator-activated receptor-γ co-activator 1α (PGC-1α). We demonstrate that PGC-1α subcellular distribution is regulated, and its transcriptional activity is promoted through SIRT1-dependent nuclear accumulation. In addition, the duration of PGC-1α activity is regulated by glycogen synthase kinase beta (GSK3β), which targets PGC-1α for intranuclear proteasomal degradation. This mechanism of regulation permits the rapidity and persistence of PGC-1α activation to be independently controlled. We provide evidence that this pathway of PGC-1α regulation occurs  Cellular response to stress generally reflects a balance between cell survival and death. Stress resistance is a measure of the cell's ability to survive under conditions that are detrimental. Manipulations that extend lifespan often increase stress resistance at the cellular level, and a number of factors that play a role in the stress response have also been implicated in longevity. It is unclear if regulation of metabolism is a feature of cellular survival or how the metabolic state of the cell influences stress resistance. Mitochondria are the key organelle in substrate utilization and energy production. Transcriptional profiling studies demonstrate that genes involved in mitochondrial energy metabolism are coordinately up-regulated in multiple tissues with calorie restriction (CR), suggesting a change in dynamic of the electron transport system and a role for this alteration in mitochondrial metabolism in the mechanisms of CR . BiochemThe transcriptional co-activator peroxisome proliferator-activated receptor-γ (PPAR-γ) co-activator 1α (PGC-1α) plays a multifaceted role in the regulation of metabolism . PGC-1α We first examined PGC-1α function and regulation using a cell culture model. As shown, extra copies of PGC-1α conferred increased resistance to oxidative stress in mouse fibroblasts , in agreTo determine the cellular localization of PGC-1α, we used a strain over-expressing GFP-tagged PGC-1α. Antibodies against GFP or the N-terminal region of PGC-1α reveal that PGC-1α is localized both in the cytoplasm and in the nucleus . These dSIRT1 NAD-dependent deacetylase is a member of the sirtuin family that has previously been shown to play a role in the oxidative stress response . SIRT1 fPrompted by these findings, we set out to explore the immediate response of PGC-1α to a low-intensity stress in cells that are not genetically manipulated. Under normal conditions, PGC-1α was detected both in the nucleus and the cytoplasm. Following oxidative stress, SIRT1 and PGC-1α accumulated in the nucleus and were colocalized . This diThe transient nature of PGC-1α accumulation in the nucleus led us to ask how stress subsequently resets PGC-1α distribution to the cytoplasm. There are two most likely explanations: (i) PGC-1α is sequestered in the nucleus and, following transcriptional activation of target genes, is released back to the cytoplasm; and (ii) PGC-1α is sequestered in the nucleus but now, following transcriptional activation, the nuclear pool is degraded, and the cytoplasmic pool is replenished by transcriptional activation of the PGC-1α gene. To test this, we looked for evidence of nuclear degradation of PGC-1α. Subcellular fractionation revealed lower molecular weight forms and reduced levels of PGC-1α in the nuclear pool 1 h following stress . One of In the presence of proteasomal inhibitor with subsequent exposure to hydrogen peroxide (45 min), immunofluorescent staining for PGC-1α was further elevated, and bright intranuclear foci containing PGC-1α were detected . The proPrior to ubiquitination and degradation, the target protein is usually ‘tagged’ by phosphorylation allowing it to be recognized by the ubiquitination apparatus. GSK3β has been previously associated with the stress response where it regulates levels of β-catenin, and as a result, activity of the forkhead transcription factor Foxo4 . FurtherTo confirm that GSK3β plays a role in PGC-1α processing, we generated cells with reduced levels of GSK3β using siRNA. In the absence of any other treatment, PGC-1α staining is elevated 24 h after knockdown of GSK3β, and the increase is most evident in the nuclei . To testTo investigate this further, we conducted immunoprecipitaion experiments using extracts from cells grown in the absence or presence of GSK3β inhibitor and treated with hydrogen peroxide for 15 min. Immunoprecipitation of GSK3β coprecipitated PGC-1α, and this association was impaired in the presence of GSK3β inhibitor . These dBased on these data, we propose a model for PGC-1α regulation in the early response to stress . A key cin vivo in mice. Mice were exposed to the oxidative stressor paraquat, and tissue was harvested and processed for subcellular fractionation at the indicated time points  a response to increased energy demand as cellular survival pathways are induced; (ii) a change in flux of endogenous mitochondrial-derived reactive oxygen species (ROS) implemented as a protective mechanism to minimize mitochondrial damage; or (iii) the alteration in mitochondrial function is itself a secondary signal in the response to stress. While studies in isolated mitochondria indicate that increased ROS causes a decrease in membrane potential, the experiments described here differ significantly in that the cells are intact upon exposure to hydrogen peroxide retaining the possibility for extra-mitochondrial signalling and nuclear response.in vivo stress response and in CR. In exploring the relationship between SIRT1 and PGC-1α, we utilized the inhibitors nicotinamide and sirtinol. These inhibitors are not specific to SIRT1, but also influence other members of the sirtuin family , which is about 90% of the average ad libitum intake for these mice. The restricted mice were fed 58 kcal week−1 (a 41% reduction) from 8 weeks of age. The restricted diet was nearly isocaloric to the control diet, but was enriched in proteins, vitamins and minerals to avoid malnutrition. Under this regimen of controlled intake, animals ingest all the allocated food, and the calorie intake is precisely known. For the oxidative stress experiment, the mice were treated with paraquat  and sacrificed at 1, 3, 5 and 7 h after exposure. Tissues were collected, snap frozen in liquid nitrogen and stored at –80 °C until further processing by microarray or immunoblotting.Wild-type male C57B16 mice were housed under controlled, specific pathogen-free conditions at the Shared Aging Rodent Facility at Madison VA Geriatric, Research, Education, and Clinical Center. The mice were cared for in accordance with the Institutional Animal Care and Use Committee at UW Madison. To control calorie intake, the mice were housed singly and fed less than m) in serum-free media for up to 1 h. For inhibitor/viability experiments, cells were grown 1 h pre-incubation in the presence of nicotinamide , PSI proteasomal inhibitor , or 2 h pre-incubation GSK3β inhibitor VII , Sirtinol  and JNK inhibitor II  prior to and during stress. Cell viability was determined by measuring fluorescence from cells in phosphate-buffered saline (PBS) with carboxyfluorescein diacetate  on a LS50B Perkin Elmer luminescence spectrometer  . Experiments were performed in triplicate, and values from four wells each were corrected for background fluorescence and normalized against identically treated cells without peroxide exposure. For localization and live imaging studies, cells were grown on cell-culture-treated cover slips . For protein stability experiments, cells were incubated in either PSI (10 µm) or lactacystin  for the indicated times. For mitochondrial membrane potential measurement by JC-1 staining, ∼1 × 106 cells in triplicate were treated in the absence or presence of hydrogen peroxide 1 h and stained with presonicated JC-1  for 15 min. Cells were trypsinized, resuspended in PBS and fluorescence detected at λex527 nm and λex590 nm using Perkin Elmer LS50B.NIH3T3 cells  were cultured in Dulbecco's modified Eagle's medium with 10% foetal bovine serum and antibiotics. Transfections were performed with Lipofectamine  according to the manufacturer's instructions using pcDNA3.1 and pcDNA–PGC-1α . Clones were isolated, and expression of PGC-1α was confirmed by Western blot. The GFP-tagged PGC-1α was generated by polymerase chain reaction cloning the PGC-1α cDNA from pcDNA–PGC1α into pcDNA3.1/NT–GFP–TOPO fusion vector (Invitrogen). Knock down of PGC-1α or GSK3β was performed by RNA interference using Silencer pre-designed siRNAs , and introduced into cells by electroporation using siPORT electroporation kit (Ambion). To determine sensitivity to oxidative stress, subconfluent cells were exposed to hydrogen peroxide  containing protease inhibitors (Sigma) and where indicated phosphatase inhibitors (Sigma). Proteins were detected by immunoblotting using standard techniques. Antibodies used were PGC-1α, SIRT1, lamin A, ubiquitin , GSK3β , COX IV , GFP, MnSOD, catalase , GSK3β, phospho-GSK3β, JNK, phospho-JNK, Ac-lys , actin and tubulin (Sigma). Subcellular fractionation was performed using nuclear/cytoplasmic fractionation kit . For immunoprecipitation, 500 µg of extract was incubated overnight with control IgG or specific primary antibody; antibodies were precipitated with Protein A or Protein G Agarose beads (Santa Cruz Biotechnology). Immunoprecipitates were analysed by Western blot. Densitometric analysis was performed using NIH ImageJ software (http://rsb.info.nih.gov/ij/); cytoplasmic and nuclear band densities normalized against actin loading control and were analysed separately.Proteins from mouse tissues and cultured cells were extracted in modified RIPA buffer , with a Spot Insight Color camera  and Spot 3.3.1 software.Cellular localization was analysed by immunofluorescence using standard techniques. Following exposure to stress, cells were fixed  at the indicated times. Cells were incubated overnight in primary antibody, and cellular distribution of proteins was visualized using fluorescent-tagged secondary antibodies . For live imaging, cells were exposed to stress in media lacking phenol red, incubated in Mitotracker Red [, 300 mt-test assuming equal variance. Differences were considered statistically significant at P < 0.05.Effects of treatments were analysed by two-tailed