Acute airway obstruction can result in life - threatening pulmonary edema. It can develop rapidly, without warning, in otherwise healthy patients. Negative pressure pulmonary edema has been described after acute airway obstruction in situations when a patient is breathing against an obstructed airway such as croup, epiglottitis or laryngospasm. In the following case, we observed a rare occurrence of pulmonary edema in a female following sedation in the prone position. It was then decided to place a laryngeal mask and to supply positive pressure ventilation; however no improvement of the hypercapnia ensued and we could not provide adequate ventilation. The patient was then emergently intubated to secure an airway and provide adequate ventilation. This required interruption of surgery, turning the patient supine, ventilating oxygen by mask and a succinylcholine bolus. During the intubation process, marked edema above the vocal cords was noticed by an experienced anesthesiologist, allowing only for a smaller sized endotracheal tube to be inserted . The patient was given dexamethasone 8 mg and diphenhydramine 25 mg for the airway edema. Following intubation and after securing the tube in place, we started positive pressure ventilation and returned the patient to the prone position, and the end tidal CO2 returned within normal range. However, within a few minutes, the patient developed bibasilar rales, with abundant frothy, blood – tinged secretions appearing in the tube. We maintained the patient prone while the surgical procedure was concluded. The edema fluid was adequately suctioned and abated after 10 minutes of positive pressure ventilation. Postoperatively the patient remained intubated and sedated on a propofol infusion. While in recovery, an arterial blood gas obtained on Synchronized Intermittent Mechanical Ventilation (SIMV) and FiO2 of 30%, showed a PO2 of 92 mmHg, PCO2 of 46 mmHg, pH of 7.34 and HCO3 of 25 mEq/L with a SaO2 of 97%. Chest X-ray performed after surgery indicated clear lung fields with normal size heart and no evidence of pulmonary edema. The patient’s condition steadily improved and she was subsequently extubated two hours later but continued to be closely monitored overnight and was discharged the following day without requiring an ICU stay.A 49-year-old Caucasian female, with a long history of heavy cigarette smoking and a previous diagnosis of supraventricular tachycardia, for which cardiac ablation was performed 2 years ago, was admitted to the hospital for an elective sacral neuromodulator lead change to treat urinary incontinence. In the operating room, the patient received monitored anesthesia care (MAC) in the prone position. During the procedure she required increasing sedation (IV propofol infusion) and after thirty minutes the patient showed progressive hypercapnia from the nasal cannula sample line, while receiving supplemental oxygen (4 L/min). An oral airway was placed, but end-tidal COIn this case report we describe a case of NPPE following supraglottic obstruction with deepening sedation in the prone position. Supraglottic obstruction was possibly induced by intense inspiratory effort in a patient who was sedated and hypoventilating leading to airway obstruction and subsequently pulmonary edema. Another possibility is malpositioning (or biting) of the LMA causing inspiratory efforts against an obstructed airway, which could be the cause for the observed supraglottic swelling. The apparent absence of gastric contents in the pulmonary edema fluid, the absence of left ventricular dysfunction in a previously performed echocardiogram and the sequence of the described symptoms make the diagnosis of acute NPPE likely.2O, but in instances of obstruction, inspiratory pressures can increase to −50 cm of H2O and in extreme circumstances have been known to exceed −100 cm of H2O [NPPE is a relatively uncommon intra-operative and post-operative complication and is still a diagnosis by exclusion of other factors. Generally, NPPE occurs when inspiratory effort against an obstruction creates a large intrapulmonary negative pressure which can increase venous return with subsequent increase in the pulmonary capillary hydrostatic pressure. The transudation of fluid from the pulmonary vasculature causes edema formation into the alveolar space ,2. Normam of H2O . Typicalm of H2O .c-Pi) preventing fluid leak into the interstitium. The sudden relief of the obstruction may lead to loss of the auto-PEEP resulting in a large transcapillary pressure gradient and subsequent pulmonary edema [NPPE has also been described in detail in children following tonsillectomies. In these patients inspiratory efforts against airway obstruction creates a positive end-expiratory pressure (auto-PEEP) which decreases the trans-capillary pressure gradient (Pry edema ,5,6. NPPry edema ,7,8.NPPE is not only observed during or after anesthesia; numerous reports in the literature describe situations of respiratory loading requiring large inspiratory efforts, as in submerged scuba divers or breath-holding divers after resurfacing and even swimmers ,10,11. TTreatment of NPPE is usually supportive and includes maintenance of a patent airway and adequate oxygenation. The uses of dexamethasone and diuretics have not shown to be efficacious in prospective studies and may be deleterious . In seveIn conclusion, negative pressure pulmonary edema is a possible cause of respiratory distress in the operating room, especially in patients breathing spontaneously while in the prone position. Any compromise of the airway in the prone position is difficult to recover. We therefore suggest that these patients receive adequate protection of their airway, comprising aggressive intubation, general anesthesia and continuous ventilatory support.


The cellular mechanism responsible for modulating the activity of mPing remains unknown. Cytosine methylation is a major epigenetic modification in most eukaryotes, and the primary function of which is to serve as a genome defense system including taming activity of transposable elements (TEs). Given that tissue-culture is capable of inducing both methylation alteration and mPing transposition in certain rice genotypes, it provides a tractable system to investigate the possible relationship between the two phenomena.mPing transposition and cytosine methylation alteration were measured in callus and regenerated plants in three rice (ssp. indica) genotypes, V14, V27 and R09. All three genotypes showed transposition of mPing, though at various frequencies. Cytosine methylation alteration occurred both at the mPing-flanks and at random loci sampled globally in callus and regenerated plants of all three genotypes. However, a sharp difference in the changing patterns was noted between the mPing-flanks and random genomic loci, with a particular type of methylation modification, i.e., CNG hypermethylation, occurred predominantly at the mPing-flanks. Pearson's test on pairwise correlations indicated that mPing activity is positively correlated with specific patterns of methylation alteration at random genomic loci, while the element's immobility is positively correlated with methylation levels of the mPing's 5'-flanks. Bisulfite sequencing of two mPing-containing loci showed that whereas for the immobile locus loss of CG methylation in the 5'-flank was accompanied by an increase in CHG methylation, together with an overall increase in methylation of all three types  in the mPing-body region, for the active locus erasure of CG methylation in the 5'-flank was not followed by such a change.mPing activity in rice ssp. indica is correlated with alteration in cytosine methylation patterns at both random genomic loci and the elements' flanks, while the stability of mPing positively correlates with enhanced methylation levels of both the flanks and probably the elements per se. Thus, our results implicate a possible role of cytosine methylation in maintaining mPing stability under normal conditions, and in releasing the element's activity as a consequence of epigenetic perturbation in a locus-specific manner under certain stress conditions.Our results documented that tissue culture-induced  Transposable elements (TEs) are sequences capable of changing their physical locations in their host genomes ,2. TEs aTourist-like and Stowaway-like, based on the similarity of their terminal inverted repeats (TIRs) and target site duplications (TSDs) [vs. absence of a particular MITE being correlated with expression states of the genes in question [MITEs have been classified into two superfamilies, s (TSDs) . The poss (TSDs) ,7, and squestion -9.Oryza sativa L.) revealed that MITEs are major components of interspersed repetitive sequences of the genome [mPing, has been experimentally demonstrated as transpositionally active in the rice genome [nDart [mPing is a 430 bp DNA sequence with terminal inverted repeats or TIRs (15 bp) and target site duplications or TSDs (TAA or TTA) typical of a Tourist-like MITE [mPing can be effectively mobilized by several stressful conditions like tissue culture [mPing has no coding capacity, the transposase (TPase) required to catalyze its transposition is provided in trans by related autonomous element(s) [Arabidopsis thaliana) with mPing, both of the mPing-related, transposase-encoding elements, Ping and Pong, are demonstrated as TPase donors for mPing, though Pong appeared to have a higher mobilizing capacity [Whole genome data mining in rice  ,16. Basecapacity ,14,19.Arabidopsis [Arabidopsis have further strengthened the relationship and even enabled the establishment of causal links between TE activity and its DNA methylation states. For example, it was found in Arabidopsis that silencing of an introduced retrotransposon (Tto1) was caused by hypermethylation of the element, and genome-wide hypomethylation (in the ddm1 mutant background) results in its reactivation and transposition [ddm1 mutation in Arabidopsis, which results in genome-wide methylation reduction by 70% [MET1, CMT3 and DRM2 in Arabidopsis,which have provided unequivocal evidence for the deterministic role of DNA methylation in controlling both transcriptional and transpositional activities of specific families of TEs [Cytosine DNA methylation is an important epigenetic marker that exists in most animal and plant genomes. Whereas in mammalian animals this modification occurs almost exclusively at the CG dinucleotides, cytosines of any sequence context including CG, CHG and CHH (H is any base other than G) can be methylated in plants ,21. Cytobidopsis ,32. Moreposition . The ddmn by 70% , has caun by 70% . Furthers of TEs -37. Thess of TEs .mPing has been studied extensively both in its native host (rice) [Arabidopsis) [mPing activity, we tested whether alteration of status of cytosine methylation of random genomic loci and regions immediately flanking the element copies might be associated with the element's transposition in rice. To address this issue, we employed tissue culture of three rice ssp. indica cultivars in which mPing can be efficiently mobilized and marked alteration in cytosine methylation of various types occurs. We report that statistically meaningful correlations exist between mPing activity and alteration in cytosine methylation at random genomic loci, and between mPing stability and heavy methylation status of mPing per se as well as regions immediately flanking the element. We propose that cytosine methylation likely plays an important role in maintaining mPing stability under normal conditions, and in releasing the element's activity as a consequence of perturbation in the epigenetic modification by certain stress conditions like tissue culture.Although stress-induced mobility of t (rice) ,17,18 anidopsis) , it is uMseI-adaptor-primers with two consecutive mPing-specific primers at the 5' end (named as TAILmp-1 and -2) to assess the transpositional activity of mPing in calli and regenerants of the three rice ssp. indica genotypes. Because amplification by using each of the MseI adaptor-primers alone produced no resolvable bands in the gel-running range (200–1000 bp), all resolvable bands on the TD profiles should have resulted from hetero-amplifications, i.e., a MseI-adaptor primer plus the mPing-specific primer  relative to their donor seed-plants in a given genotype  that is further internal to the two primers (TAILmp1 and 2), mentioned above, authenticity was validated in each case as judged by the expected band size differences in agarose gels (data not shown).Transposon display (TD) analysis was performed using combinations of mPing, both excisions and insertions varied markedly among them  and target site duplications (TSD) of TAA or TTA, suggesting they were de novo mPing insertions induced by tissue culture. Again, locus-specific primers flanking each of the "mPing insertion-loci were designed based on the 93–11 draft genome sequence, and used to amplify the "complete" loci . Further sequencing of the complete loci confirmed that they all were bona fide mPing de novo insertions in the calli and/or regenerants  but have differential sensitivity to certain methylation states of the two cytosines: HpaII will not cut if either of the cytosines is fully (double-strand) methylated, whereas MspI will not cut if the external cytosine is fully- or hemi- (single-strand) methylated [thylated . Thus, fthylated -45. For EcoRI + HpaII/MspI primer combinations  to a Ty1-copia retrotransposon, while the rest 10 showed no significant similarity to the available database sequences -methylation display (TMD) assay. TMD is a modified version of transposon-display (TD) by substituting the original MseI digestion with methylation-sensitive HpaII/MspI-digestions (see Methods). Another modification we made here was that mPing-specific primers targeting at both the 5' and 3' ends were included (Methods). To rule out confounding polymorphic bands due to mPing transpositions (excisions or insertions), only those changing TMD patterns that appeared in one but not both of the digestions (HpaII and MspI) were scored for a given genotype , CG hypermethylation (C2), CHG hypomethylation (D1) and CHG hypermethylation (D2)  and mPing activity was found irrespective of whether the three genotypes were considered separately or together (data not shown). However, when excisions and insertions were considered separately on a per-genotype basis, and methylation alteration being dissected into specific types, i.e., CG or CHG, the correlation coefficients were statistically significant in four cases. These are: (1) between mPing insertions and CHG hypomethylation in genotype V14 ; (2) between mPing insertions and CG hypomethylation in genotype V27 ; (3) between mPing insertions and CHG hypomethylation in genotype V27 , and; (4) between mPing excisions and CHG hypomethylation in genotype R09  . If all three genotypes were considered together, the separation of excisions and insertions produced even more meaningful correlations. These are (1) between mPing excisions and three of the four types of methylation alteration , with CHG hypomethylation being the only exception, and; (2) between mPing insertions and each of the four types of methylation alteration   mPing activity was separated into excisions and insertions, and; (2) the methylation alteration were dissected into specific patterns.To test if there exists any intrinsic correlation between tissue culture-induced ese are:  between ese are:  between mPing activity and alteration in cytosine methylation patterns at random genomic loci from a global perspective (based on the MSAP data), then an intuitive question to ask is whether the mPing activity should be equally or even more correlated with cytosine methylation of the genomic regions immediately flanking the element copies. To investigate this possibility, we calculated correlation coefficients between levels of the two major types of methylation, CG and CHG, of each of the 5'- and 3'-mPing flanking regions detected by mPing-TMD and mPing immobility. It should be pointed out that, in contrast to the situation of random loci sampled genome-widely (described above), with TMD only mPing immobility (or stability) can be considered because the genomic regions flanking active mPing copies can not be amplified from the calli and/or regenerants (due to excision) by the TMD assay (see Methods). Nonetheless, we reasoned that if methylation status of the flanking regions plays a role in the mPing activity, then we would expect to find a meaningful correlation between high levels of methylation and mPing stability, i.e., a positive correlation should exist. Indeed, the correlation analysis ; (2) in genotype V27, mPing stability correlates with three of the four types of methylation levels, i.e., CG of the 5'-flank, CG of the 3'-flank and CHG of the 5'-flank ; (3) in genotype R09, mPing stability correlates with two of the four types of methylation levels, i.e., CG of the 5'-flank and CG of the 3'-flank ; (4) when all three genotypes being considered together, mPing stability correlates with three of the four types of methylation levels, i.e., CG of the 5'-flank, CG of the 3'-flank and CHG of the 5'-flank . A conclusion emerged from the correlation data is that whereas CG methylation of both the 5'- and 3'-flanking regions likely plays important roles in maintaining mPing stability, CHG methylation of only the 5'-flanking regions appeared important for the purpose (Table If meaningful correlations exist between tissue culture-induced is Table  establismPing copies and active ones (showing excision), we determined the cytosine methylation status of portion of the mPing body-regions and their immediate 5' flanks by bisulfite sequencing for one locus of each kinds, ITDTG8 (inactive) and ITDTA6 (active), which were arbitrarily chosen from the TD profiles , the 5'-flank was slightly methylated (< 5%) in the seed-plant for all three types of methylation, CG, CHG and CHH; the residual CG methylation (3%) was completely lost in the callus, and which was accompanied by a increase in CNG methylation (from 6% to 10%), while the residual CHH methylaiton (2%) remained unchanged; the methylation status of all three types were restored to those of the seed-plant in the regenerated plant  and moderately methylated in CHG (52%) and CHH (35%) in seed-plant, and the degree of all three types of methylation in the mPing body-region was further increased in callus, particularly in CHG and CHH, followed by a decrease to roughly the original levels of seed-plant in the regenerated plant , in the seed-plant the 5'-flank was partly methylated in CG (24%), residually methylated in CHG (4%) and non-methylated in CHH  was also heavily methylated in CG (96%) which was even higher than that of the inactive copy (77%), but the methylation levels of CHG (41%) and CHH (6%) of this active mPing copy were markedly lower than those of the inactive copy . It is not possible to analyze possible methylation changes at the mPing body-region of this locus during the callus stage, as it was excised. Collectively, the bisulfite genomic sequencing data suggest that methylation status of both the 5'-flanks and the body-regions of mPing may be important for its activity or inactivity, depending on the loci. Thus, under the tissue culture stress conditions, whereas CG hypomethylation in the element's 5'-flanks might have played a part in facilitating the excision of active mPing copies  s Figure . We fount Figure . In contt Figure . 2) ForH Figure ; notablys Figure , this CGs Figure . The mPis Figure , furthers Figure  might ha ForH FigmPing is the most active TE so far documented in any organism, and hence, provides an ideal system for studying the cellular mechanism controlling a TE's activity, as well as a tool for elucidating impact of its activity on adjacent genes. Induced transposition of mPing has been firstly discovered independently in three laboratories working with different rice materials, long-term somatic cell cultures of indica rice [japonica rice [japonica rice lines [mPing between the two cultivated rice subspecies, indica and japonica, as well as between the two groups of japonica cultivars  has led to the suggestion that its transpositional activity has also been induced by other sources of factors. Indeed, it was found that mPing can be induced to transpose by interspecific hybridization [japonica rice mPing has undergone dramatic amplifications associated with domestication and breeding [It has been demonstrated that among all kinds of TEs, MITEs are most closely associated with plant genes -9. This,ica rice , newly iica rice , and gamce lines . The obsdization  and hydrdization . More rebreeding , implicajaponica rice, mPing activity is less studied in indica rice. In this study, somatic cell-derived calli and their regenerants of three rice ssp. indica genotypes which are currently under cultivation in large acreages in Burundi and several other African countries showed high frequencies of transpositional activation of mPing, though genotypic difference in both excision and insertion frequencies are evident.Compared with the situation of mPing in any of the hitherto reported cases. Nonetheless, given the inducible nature of mPing transposition by various stressful conditions and under which epigentic modifications are known to alter, it is likely that epigenetic mechanisms like cytosine methylation are involved. To address this issue, it is important to have a system wherein both mPing activity and alteration in cytosine can be concomitantly induced.Accumulated evidence in various organisms has pointed to the importance of epigenetic modification in the form of cytosine methylation as an important mechanism for repressive control of TEs activity (see Introduction). It is unknown whether alteration in this epigenetic modification has contributed to the activation of indica genotypes provides a system whereby the possible relationship between mPing activity and cytosine methylation can be addressed. Indeed, the often-observed phenomenon of somaclonal variation in plant tissue cultures is the results of concerted action of both genetic and epigenetic instabilities induced by the tissue culture process [mPing activation and perturbed patterns or levels in cytosine methylation in the rice genome.We have shown in this study that various types of cytosine methylation alteration occurred in calli and their regenerants in all three studied rice genotypes, which included both hypo- and hyper-methylation that occurred at CG or CHG sites. Therefore, the tissue culture system  of these rice ssp.  process , and act process ,48. Furt process . TherefomPing activity is indeed correlated with alteration in cytosine methylation at randomly sampled loci across the genome. However, this correlation is cryptic, and which becomes evident only when both the mPing activity and methylation alteration are further dissected into more specific aspects. Under such conditions, significant correlations were detected between mPing activity and specific types of cytosine methylation alteration both within each genotype and when all three genotypes were considered as a whole.As a first step to address this issue, we have demonstrated in this study that tissue culture-induced mPing) appeared to reside in genomic regions with different degrees of cytosine methylation modifications [mPing activity and alteration in cytosine methylation at globally sampled random genomic loci, discussed above, it is pertinent to ask two questions: (1) Are the genomic regions immediately flanking the mPing copies also underwent methylation alteration? (2) Are the methylation states of the mPing immediate flanking regions important for the element's stability? To answer these two questions, we performed TMD analysis targeting at the various immobile mPing copies in the calli and regenerants from each of the three rice genotypes. We found that both the 5'- and 3'-mPing flanks underwent extensive methylation alteration that could also be classified into four types as in the case of random genomic loci detected by MSAP. However, a striking feature characterizing the methylation alteration of the mPing flanks is that a specific type of alteration, i.e., CNG hypermethylation, occurred at markedly higher frequencies relative to that of the random genomic loci, in all three genotypes  was accompanied by rapid hypermethylation of CNG. Given that only the genomic regions flanking the immobile mPing copies were analyzed by the TMD method, this observation may implicate that one factor for ensuring stability of the immobile mPing copies is due to the rapid CHG hypermethylation which might have been capable of compensating for the almost inevitable loss of CG methylation during callus culture  showed that in the rice genome various TEs -RNA-based RNA-directed DNA methylation (RDM) mechanism is most likely responsible [mPing copies (they were excised), it is probably safe to speculate that it would not have undergo similar enhancement in methylation. Although in theory the siRNA-based RDM mechanism should act in trans, it is easy to imagine that the different mPing copies being residing at different epigenetic chromatin environments [mPing activity and alteration in localized and/or more global cytosine methylation modifications in the rice genome.The bisulfite sequencing data further suggested that cytosine methylation alteration in the hylation . Therefoponsible -53, and ronments  may causronments -53. FurtmPing, we analyzed the relationship between TD-based mPing transpositions and MSAP- or TMD-based alteration in cytosine methylation in callus and regenerants of three indica rice genotypes. We found that, on one hand, mPing transpositional activity is correlated with alteration in cytosine methylation patterns at randomly sampled genomic loci , and on the other hand,mPing stability is positively correlated with methylation levels of genomic regions immediately flanking the immobile mPing copies . In addition, high frequency of CNG hypermethylation occurred specifically at the genomic regions flanking immobile mPing copies, suggesting that this particular type of methylation modification probably plays an immediate role in fortifying local epigenetic control and ensuring mPing stability at these loci, while the failure in this fortification at other mPing-flanking regions might be associated with the element's transposition. Bisulfite sequencing of a locus containing an immobile mPing copy and one containing an active one further bolstered this possibility.To investigate a possible role of cytosine methylation in the transpositional activity of Oryza sativa L., ssp. indica cultivars , widely cultivated in Burundi and some other Africa countries, were used as source of calli. Sterilization and culture/subculture conditions were essentially as reported [Embryos from mature seeds of three rice, reported  with thereported , micronureported  and vitareported  were useGenomic DNA was extracted from fully expanded leaves of five randomly selected regenerated plants for each genotype , two pools of calli for each genotype , and the donor plants (pools plants germinated from five seeds) for each genotype . A modified CTAB (hexadecyltrimethylammonium bromide) protocol  was usedmPing-specific primers together with a primer designed according to the MseI adapter sequences . The MSAP (methylation-sensitive amplified polymorphism) analysis was essentially as reported [HpaII and MspI digestions  in combination with the MseI primer  mPing copies, and loci showing alteration in cytosine methylation. BlastX was used for homology analysis of the isolated loci.The calli and regenerants together with their corresponding donor plants were subjected to transposon-display (TD) analysis , using nreported  using vamPing-TMD was performed essentially as TD, with modifications only in the restriction enzymes used, namely, MseI in TD being replaced by a pair of isoschizomers, HpaII and MspI. Two sets of consecutive mPing – specific primers respectively targeting the 5'- and 3'-mPing ends were designed and combined with HpaII/MspI adaptor-primers , only altering patterns in one of the two enzyme digests (HpaII or MspI) but not in both, which can be unequivocally assigned as due to alteration in cytosine methylation were scored  is a combination of transposon-display (TD) and methylation-sensitive amplified polymorphism (MSAP) . Thus, tmPing excisions and insertions, respectively . For MSAP, the changing patterns were divided into four major types, CG hypomethylation, CG hypermethylation, CNG hypomethylation and CNG hypermethylation for each genotype  relative to the donor plants were scored as mPing activity  and alteration in cytosine methylation at random genomic loci (based on the MSAP data) were tested by using the Pearson correlation analysis. By the same method, Possible correlations between mPing stability and two types of methylation levels respectively at the 5' and 3' immobile mPing-flanking regions (based on the TMD data) were tested. In both cases, the software SPSS 11.5 for Windows, Bivariate Correlation, Two-tailed, Correlation coefficients, Pearson"  was used, and the statistical significance was determined.Possible correlations between ) according to the manufacturer's instructions. Briefly, 900 μl ddH2O, 50 μl M-dissolving buffer and 300 μl M-dilution buffer were added per tube of CT conversion reagent (Zymo Research) prior to use. Then, 130 μl of bisulfite-containing CT conversion reagent was added to 1 μg of DNA in a volume of 20 μl  and mixed, and the samples were then incubated at 98°C for 10 min, and 64°C for 2.5 h. Modified DNA was purified using a Zymo-Spin IC column (Zymo Research) and stored at -20°C until use. The bisulfite sequencing amplifications primer pairs respectively for a locus containing an immobile mPing copy (ITDTG8) and a locus containing an active mPing  (ITDTA6), both being arbitrarily chosen from the sequenced bands isolated from the TD profiles  and are given in Additional file Genomic DNA from a pool of calli (V27Ca2), one regenerant (V27Reg5), and their corresponding seed-plant of cv. V27 was modified using the EZ DNA Methylation-Gold kit  and validated by cloning, sequencing and locus-specific PCR amplification.Click here for filemPing insertions in callus and regenerated plants of the three rice ssp. indica cultivars, V14, V27 and R09Characteristics of isolated target sites flanking de novo . A total of 30 de novo mPing insertion events which occurred in calli and/or some of the regenerated plants in one or more of the three cultivars were identified by mPing-specific transposons-display (TD) and validated by cloning, sequencing and locus-specific PCR amplification.Click here for filessp. indica cultivars  detected by MSAP and TMDCategory of patterns of cytosine methylation alteration induced by tissue culture in the three rice . Types of alteration in methylation patterns occurred at random genomic loci (detected by MSAP) and regions flanking the immobile mPing copies (detected by TMD) summarized.Click here for filessp. indica cultivars, V14, V27 and R09Characterization of the isolated variable MSAP fragments from the calli and regenerants of the three rice . Chromosomal location, predicted homology and restriction map of the isolated variable MSAP fragments from the calli and/or regenerants of the three rice ssp. indica cultivars, V14, V27 and R09.Click here for fileSequences of primer pairs used for PCR amplification from bisulfite-treated genomic DNA of a seed-plant, a pool of calli and a regenerated plant of cv. V27. Pairs of bisulfite sequencing primers were designed to amplify a locus containing an inactive mPing copy and a locus containing an active mPing copy, respectively.Click here for file


The present studies were initiated to quantitate the oxygen dependence of bioreductive metabolism-induced binding of EF5, a pentafluorinated derivative of the 2-nitroimidazole, etanidazole. Two different assays were compared: first, radioactive drug incorporation into cell lysates, which provides a direct measure of drug metabolism or uptake; second, monoclonal antibody detection of cellular macromolecular adducts of EF5 after whole cell permeabilisation and fixing. The antibodies (a single clone designated ELK3-51) were conjugated with the fluorescent dye Cy3, with fluorescence determined by fluorescence microscopy and flow cytometry. For the two cell lines tested (V79 Chinese hamster fibroblasts and 9L rat glioma), the oxygen dependence of binding was found to be the same for the two techniques. Using the antibody binding technique, the fluorescence signal was highly reproducible between experiments, resistant to light or chemical bleaching and stable over time following cell or tissue staining. Flow cytometric analysis of cells from rat 9L tumours treated with EF5 in vivo or in vitro showed a distribution of fluorescent signal which was very compatible, on both a relative and absolute basis, with the in vitro results. Our results indicate that immunofluorescent techniques provide a quantitative assay for bioreductive drug adducts, and therefore may be able to measure the absolute oxygen concentration distribution in cell populations and tissues of interest.


We report a rare adverse effect of ondansetron in a 14-year-old girl, presenting as ventricular tachycardia.Ondansetron is a serotonin receptor antagonist used widely in the prophylaxis and treatment of postoperative nausea and vomiting (PONV) and vomiting associated with cancer chemotherapy. The common side effects of ondansetron are fever, malaise, diarrhoea, constipation, and allergic reactions. Extra-pyramidal reactions are rare and cardiovascular side effects are even rarer. Even though its clinical safety has been established in a large number of studies, its adverse effects have been reported and these include cardiovascular events like acute myocardial ischemia and arrhythmias in adults. A 14 year olds girl with unremarkable past medical history presented to a local hospital with vomiting and pain in abdomen of eight hours durations.[Abdominal examination showed no distension and normal bowel sounds. Bilateral crepiataions were observed on auscultation of chest. An ECG showed ventricular tachycardia  and she 3  receptor antagonists used in the management of PONV and cancer chemotherapy induced vomiting.[3Ondansetron belongs to a group of 5HTvomiting. It also omiting.3 Its safeomiting.3 Two case+  and K+  channels are determinants of cardiac muscle action potential and the electrocardiogram (ECG). Cardiac Na+  channels are responsible for depolarisation and propagation of the cardiac action potential. In ECG, the QRS complex represents the ventricular depolarisation. Two voltage dependent K+  channels represented as rapid repolarising current (IKr) and slow repolarising current (IKs) are responsible for rapid and slow repolarisation of cardiac muscle and QT interval in the ECG. Of the two channels, rapid repolarising current is the main repolarising channel in the heart, which is encoded by human ether-a-go-go-related gene (HERG). Ondansetron can block this channel, resulting in lengthening of the repolarisation.[+  and K+  channels to prolong QRS or QT interval, resulting in ventricular arrhythmias.[+  channels is possibly responsible for the prolongation of cardiac repolarisation, thus resulting in conduction disturbances.[1 , 5HT2 , 5HT3 , 5HT4  receptors, which are distributed throughout the cardio vascular system. 5HT3  receptors mediate Bezold-Zarish reflex, which is an autonomic reflex consisting of bradycardia, hypotension and apnea. Suppression of this reflex by ondansetron leads to tachyarrhythmias.[3  receptor blockade could possibly lead to unopposed action of 5HT2  and 5HT4  receptors, resulting in tachyarrhythmias and hypertension.[Ondansetron is known to cause cardiac arrhythmias by several mechanisms.6 FirstlyThough the cause of the death is inconclusive in our case, we presume it was possibly due to ondansetron, because the patient was previously healthy and we could not establish any other cause for ventricular tachycardia. Although she presented with vomiting and pain in the abdomen, she had normal abdomen clinically. She died within fours hours of administration of ondansetron presenting in shock. This cardiogenic shock is secondary to ventricular tachycardia, resulting in ventricular fibrillation. However, the possibility of idiopathic ventricular arrhythmia also cannot be ruled out. Further studies are required to confirm ondansetron induced ventricular arrhythmia and its clinical safety. Till such time, this drug should be used judiciously in patients.


A hallmark of cancer is the deregulation of the dna methylation machinery and aberrant dna methylation patterns dna methylation play a critical role in the changes in gene expression involved in cancer progression and metastasis.Methylation of dna play a role very similar to that of genetic mutations in cancer; however, unlike a genetic alteration, dna methylation is potentially reversible with pharmacologic intervention. The dna methylation machinery was therefore proposed— almost a decade and a half ago—to be an attractive anticancer target Methylation changes in dna methylation machinery occur in cancer:Three principal kinds of aberration in the Hypermethylation of tumour suppressor genes 7dna (cytosine-5-)-methyl-transferase 1 (dnmt1) and other dnmts that methylate genomic dna involved in processes of gene inactivation, chromatin organization, X chromosome inactivation, and genomic imprintingAberrant expression of Hypomethylation of unique genes and repetitive sequencesdna methylation have very important diagnostic significance, and using current whole-genome techniques, the methylation signatures of specific cancer types are being delineated. These signatures will play an increasingly important role in diagnosis and prognosis of all cancers These aberrations in dnmt1 is tightly regulated with the state of cell growth by transcriptional and posttranscriptional mechanisms DNMT1 DNMT1 in non-transformed cells causes cellular transformation, which supports the idea that inhibition of dnmt1 would block tumour growth dnmt1 inhibition were demonstrated both pharmacologically, using antisense oligonucleotide inhibitors dnmt1–/– mice The expression of dnmt1 inhibition, it is essential to understand why dnmt1 transforms cells and why dnmt1 inhibition blocks tumour growth. The commonly accepted and attractively straightforward model is that dnmt1 inhibition causes loss of methylation during dna synthesis and therefore activation of tumour-suppressor genes that are silenced by dna methylation. Activation of tumour-suppressor genes by demethylation would be expected to block tumour growth. Another possible utility of demethylation agents is demethylation of tumour antigen promoters, which can lead to reactivation of the expression of those promoters, thus increasing the sensitivity of tumour cells to immunosurveillance.To properly design and apply therapeutic strategies that involve dnmt1 inhibitors, then the goal should be to develop catalytic inhibitors of the dna methylation reaction that would cause global demethylation of dna. However, there are several reasons to question whether dnmt1 inhibition blocks tumour growth exclusively through inhibition of dna methylation:If demethylation is the only mechanism of action of dnmt1 by small interfering rna (sirna) or antisense oligonucleotides blocks the growth of cancer cells by mechanisms independent of dna methylation through induction of tumour-suppressor genes dna damage response and inhibition of dna replication Knockdown of dnmt1 has multiple domains. To be able to target the clinically relevant actions of dnmt1, delineating the functional domains of dnmt1 that are critical for cancer growth will be important  The multifunctional protein dnmt inhibitor, 5-azacytidine (5-azaC), and its deoxy analogue, 5-deoxycytidine (5-azaCdR), a potent and non-selective dna methylation inhibitor, have been launched Several clinical trials with a nucleoside analogue pan-dna methylation, mediated through 5-azaC binding of dnmt1 and other dnmts; or whether the main activity of this agent relates to its non-specific toxicity as a nucleoside analogue. Similarly, the specific dnmt1 isoforms that are responsible for the anticancer activity of 5-azaC remain undefined.Although 5-azaC has been tested in clinical and preclinical settings for decades, basic questions remain regarding its mechanism of action. It is unclear whether the clinical activity of 5-azaC requires demethylation of candidate tumour-suppressor genes; whether the anticancer effect is a result of activities independent of dnmt inhibitors. A major issue is the possibility that non-selective demethylation would induce pro-metastatic genes.These unresolved issues have implications for the dosing and scheduling of 5-azaC and for the development of new, more potent p15 before treatment Recent trials have focused on low-dose 5-azaC (well below the maximum tolerated dose), under the supposition that 5-azaC causes demethylation at low doses but is mainly toxic at high doses dnmt inhibitors is zebularine, a nucleoside analogue, which, unlike 5-azaC, is chemically stable and orally bioavailable. Zebularine was originally identified as a cytidine deaminase inhibitor dna demethylation activity with reduced potency and toxicity as compared with 5-azaC. Nevertheless zebularine belongs to the same class of nucleoside analogues, raising similar problems to those seen with 5-azaC.Another nucleoside analogue that has recently been introduced to the arsenal of dnmt1 in the clinic is an “old” nucleoside analogue that must be incorporated into dna to perform its action. Thus, although the goal of dna methylation therapy is to target the cell’s machinery in a way that is fundamentally different from classical chemotherapy and thus anticipated to exhibit limited toxicity, the use of a classical nucleoside analogue seems to defeat that purpose.It is unfortunate that the only drug targeting dnmts. The only non-nucleoside, isotypic, specific dnmt1 inhibitor that has undergone clinical trial is MG98, a second-generation antisense oligonucleotide that specifically targets dnmt1 messenger rna dnmt1. With MG98, the expression of the dnmt1 protein is entirely eliminated, and thus all functional activities of dnmt1 are targeted, including methylation-independent activities. Knockdown of dnmt1 results in inhibition of dna replication dnmt1 knockdown dramatically limits the demethylation induced by dnmt1 inhibition, thus avoiding the potential deleterious impact of global demethylation The many toxicities of 5-azaC result from its properties as a nucleoside analogue and might perhaps mask its activity on rnas— carries great promise. Searching for agents that knock down dnmt1 rather than inhibit its catalytic activity is a priority that should be pursued.The chief remaining issue with antisense oligonucleotides is their delivery to solid tumours. The clinical trials of this promising class of drugs were recently stopped because of a lack of objective response. Nevertheless, the overall strategy—and therapeutic siAlthough the principal attention in the field of cancer has been directed at the phenomenon of hypermethylation, a hallmark of the methylation pattern in many tumours is hypomethylation dnmts (such as 5-azaC) that cause global hypomethylation and that are now used in anticancer therapy, might increase the propensity of cancer cells to metastasize.First, catalytic inhibitors of dnmt1 inhibitors that do not cause demethylation of metastatic genes. A new goal in dna methylation therapy should be the development of dna demethylation inhibitors  Second, might demethylation inhibitors be a new approach to cancer therapy? It is therefore critical to develop S-adenosylmethionine (sam). S-Adenosylmethionine contributes the methyl moieties to the methylation reactions and has also been shown to inhibit demethylase activity in vitro and in cells sam was shown to be chemoprotectant in a liver cancer model in rodents in vitro with sam resulted in inhibition of invasion in vitro, and metastasis and tumour growth when the cells were transplanted into nude mice in vivo. The sam molecule is notoriously unstable sam analogues with improved pharmacokinetics.Two different approaches were recently used to block demethylation in cancer. The first approach involved treatment with the methyl donor dna binding 2 (Mbd2)—was previously suggested to be involved in silencing methylated genes and demethylation alike in vivo MBD2 antisense oligonucleotides, MBD2 sirna inhibitors, and the Mbd2 antagonists are therefore potentially promising antimetastasis candidates.Another important line of investigation involves identifying the proteins responsible for demethylation of metastatic genes in cancer and targeting them for inhibition. One protein—methylated domain dna methylation and demethy-lation represents an attractive therapeutic target; however, certain questions need to be answered before the full potential of this approach is realized. The dna methylation inhibitor currently in use is crude and, by demethylation, could unleash pro-metastasis genes that might increase metastasis. The specific functions of dnmt1 that are involved in tumorigenesis must be isolated from the functions involved in metastasis. Not only dnmts, but also the dna demethylation machinery, are emerging as new targets for inhibition of metastasis—one of the most intractable facets of cancer. The challenge is to design and target the various compartments of the dna methylation machinery to achieve both growth-control induction of tumour antigens and inhibition of metastasis in the absence of adverse effects on methylation.The machinery of


Hantaviruses, similar to several emerging zoonotic viruses, persistently infect their natural reservoir hosts, without causing overt signs of disease. Spillover to incidental human hosts results in morbidity and mortality mediated by excessive proinflammatory and cellular immune responses. The mechanisms mediating the persistence of hantaviruses and the absence of clinical symptoms in rodent reservoirs are only starting to be uncovered. Recent studies indicate that during hantavirus infection, proinflammatory and antiviral responses are reduced and regulatory responses are elevated at sites of increased virus replication in rodents. The recent discovery of structural and non-structural proteins that suppress type I interferon responses in humans suggests that immune responses in rodent hosts could be mediated directly by the virus. Alternatively, several host factors, including sex steroids, glucocorticoids, and genetic factors, are reported to alter host susceptibility and may contribute to persistence of hantaviruses in rodents. Humans and reservoir hosts differ in infection outcomes and in immune responses to hantavirus infection; thus, understanding the mechanisms mediating viral persistence and the absence of disease in rodents may provide insight into the prevention and treatment of disease in humans. Consideration of the coevolutionary mechanisms mediating hantaviral persistence and rodent host survival is providing insight into the mechanisms by which zoonotic viruses have remained in the environment for millions of years and continue to be transmitted to humans. N and GC), and an RNA polymerase (Pol), respectively. More than 50 hantaviruses have been found worldwide Hantaviruses are negative sense, enveloped RNA viruses (family: Bunyaviridae) that are comprised of three RNA segments, designated small (S), medium (M), and large (L), which encode the viral nucleocapsid (N), envelope glycoproteins  may support replicating virus in the absence of overt disease Peromyscus maniculatus) and European common voles  infected with SNV and Puumala virus (PUUV), respectively, with nucleotide and amino acid mutations identified in immunodominant regions of the GN protein Mechanisms of immune evasion, including viral mutation and segment reassortment, may contribute to viral persistence. Cyclical rises of Seoul virus (SEOV) L segment deletions and S segment insertions in regions necessary for initiation of transcription are observed in vitro N protein of hantaviruses Hantaviruses can infect macrophages and endothelial cells in rodents and humans Infection of HMVEC-Ls and HUVECs indicate that at least some of the hantaviruses that cause disease in humans  delay induction of factors in the type I IFN pathway  as compared with hantaviruses that cause no known disease in humans  Rig-I and Tlr7) is reduced or remains unchanged throughout SEOV infection, suggesting that inhibition of viral recognition may contribute to the establishment of persistent infection Il6 and Tnfα expression in the lungs within physiological ranges, does not affect SEOV persistence in male rats nor does it cause observable disease In the lungs of rats with high amounts of virus , the expression of pattern recognition receptors  in cultured CD4+ T cells, which are not evident in CD4+ T cells isolated from persistently infected deer mice Mus musculus), the transfer of functional CD8+ T cells is necessary for clearance of HTNV Cellular immune responses, in particular CD8+ T cells, contribute to clearance of hantaviruses in humans at the expense of causing disease Foxp3 mRNA and proportions of CD4+CD25+FoxP3+ regulatory T cells are elevated locally at a site of elevated SEOV replication  in male rats during persistent SEOV infection Tgfβ than do CD4+ T cells isolated from deer mice during the acute phase of infection Regulatory T cell responses suppress proinflammatory and effector T cell responses locally at the site of infection to allow pathogen persistence, as well as to mitigate proinflammatory-mediated pathogenesis Hantaviruses persist in their rodent hosts despite the presence of neutralizing antibody. Antibody against hantaviruses is usually detectable after the first 2 weeks of infection, rapidly increases for the next 4–6 weeks, and declines, but remains detectable presumably for the lifetime of the rodent  [9], [13Tlr7, Myd88, Rig-I, Visa, Ifnβ, and Mx2) and proinflammatory  factors is higher in the lungs of female than male rats Foxp3 and TGF-β, is elevated in the lungs of males as compared with those of females In natural populations of rodent reservoirs, males are more frequently infected with hantaviruses and are more likely to engage in aggressive encounters than are females, which may result in elevated exposure and transmission of hantaviruses among males Sex differences in response to hantavirus infection in humans are beginning to receive attention. During acute PUUV infection in humans, circulating concentrations of CXCL8 and CXCL10 are higher, whereas concentrations of IL-9 and GM-CSF are lower in men than in women Foxp3 and production of TGF-β) and expression of the glycogenase, matrix metalloprotease (Mmp)-9, in the lungs of male, but not female, rats during SEOV infection Glucocorticoids are potent immunosuppressive steroid hormones that can suppress proinflammatory and cellular responses and have been administered to patients with HFRS or HCPS to reduce immunopathology DQA-09, which is positively, and Cgl-DQA-05 and Cgl-DQA-12, which are negatively, associated with the likelihood of bank voles being infected with PUUV Host genetic factors may contribute to susceptibility to and outcome of hantavirus infection in both humans and rodent hosts. Patients with TNF polymorphisms associated with elevated or reduced TNF-α transcription are predisposed to exhibit more severe disease during PUUV infection Hantaviruses and their rodent reservoirs represent highly coevolved systems in which virulence and host responses have been adapted to ensure survival of both the virus and the host. The current literature suggests that hantavirus persistence is mediated by both the virus and the host. Although several potential mechanisms mediating the persistence of hantaviruses in their rodent hosts have been discovered in recent years, there are a number of intriguing questions that remain to be addressed:Do hantaviral NSs inhibit type I IFN responses in rodents and therefore contribute to viral persistence?Is DC and macrophage activity  suppressed by hantavirus infection in rodent reservoirs?Does suppression of excessive proinflammatory cytokine responses  prevent disease in rodents at the cost of causing viral persistence?Is the activity of hantavirus-specific CD8+ T cells suppressed during infection to mediate viral persistence in rodents?How is regulatory T cell activity induced by hantaviruses in reservoir hosts?What is the mechanism of regulatory T cell–mediated hantaviral persistence ?Do host genetic factors, in addition to MHC alleles, contribute to the susceptibility of rodents to hantaviruses?What is the role of non-immune mediators, including MMP-9 We propose that comparing immune responses in rodents to those in humans may provide insight into ways to prevent pathology in humans. Although advances are being made in the development of a hantavirus vaccine, there currently is no FDA-approved vaccine or drug for prevention or treatment of hantaviral disease


We analyzed serial concentrations of multiple inflammatory mediators from serum and induced sputum obtained from patients with stable COPD and controls. The objective was to determine which proteins could be used as reliable biomarkers to assess COPD disease state and severity.Forty-two subjects; 21 with stable COPD and 21 controls, were studied every 2 weeks over a 6-week period. Serum and induced sputum were obtained at each of 3 visits and concentrations of 19 serum and 22 sputum proteins were serially assessed using multiplex immunoassays. We used linear mixed effects models to test the distribution of proteins for an association with COPD and disease severity. Measures of within- and between-subject coefficients of variation were calculated for each of the proteins to assess reliability of measurement.There was significant variability in concentrations of all inflammatory proteins over time, and variability was greater for sputum proteins (median intra-subject coefficient of variation 0.58) compared to proteins measured in serum . Of 19 serum proteins and 22 sputum proteins tested, only serum CRP, myeloperoxidase and VEGF and sputum IL-6, IL-8, TIMP-1, and VEGF showed acceptable intra and inter-patient reliability and were significantly associated with COPD, the severity of lung function impairment, and dyspnea.Levels of many serum and sputum biomarkers cannot be reliably ascertained based on single measurements. Multiple measurements over time can give a more reliable and precise estimate of the inflammatory burden in clinically stable COPD patients. There is increasing evidence that chronic obstructive pulmonary disease (COPD) is associated with chronic systemic inflammation as well as inflammation in the airways and lung parenchyma ,2. PreviA recent meta-analysis of 652 studies showed that only sputum neutrophils, IL-8, TNFα, and CRP were able to distinguish between differing stages of COPD disease severity [Our study had two major objectives. The first objective was to use multi-analyte profiling of numerous inflammatory proteins in induced sputum and serum to determine which proteins could be used as reliable biomarkers to assess COPD disease state and disease severity. The second objective was to describe the intra- and inter-patient variability in inflammatory proteins in induced sputum and serum in patients with stable COPD compared to controls.We recruited 4 categories of subjects aged 35 years or older into this study; 1) Current smokers with COPD, 2) Former smokers with COPD, 3) Current smokers with no evidence of COPD or airflow obstruction, and 4) Lifetime non-smokers with no evidence of COPD or airflow obstruction. Groups 3 and 4 were control groups. This design allowed us to assess for current smoking as an independent risk for altered inflammatory cytokine profiles, and allowed us to assess potential interactions of smoking and disease state. All patients with COPD had to have at least GOLD stage II-IV COPD and a 10 pack-year history of smoking. We excluded patients with a history of COPD exacerbation within one month prior to study entry, asthma or atopy, patients using chronic oral prednisone, those not able to perform spirometry, and those subjects with other conditions which may cause inflammation, including; current cancer, infections, rheumatoid arthritis, lupus, scleroderma, or a recent myocardial infarction within 3 months. The study was approved by The Research Ethics Board of The Ottawa Hospital and all subjects signed written, informed consent.This was a prospective cohort study. Subjects were seen on 3 occasions at 2-week intervals over a 6 week period. On each visit, patients underwent assessments of measurements of dyspnea intensity using the Baseline Dyspnea Index  and measg to sediment cellular constituents. Cell supernatants and centrifuged serum were collected and stored frozen at -70°C. Samples were batched and shipped on dry ice at the halfway point of the study and at the study termination. Samples were kept frozen from 1 day up to 3 months. Frozen samples were immediately thawed on receipt at the laboratory and processed for assessment of protein levels.Induced sputum and blood samples were collected at each visit. Sputum induction was performed according to previously described protocols . SubjectSerum and sputum levels of inflammatory mediators were measured using two multiplex platforms: Luminex multi-analyte profiling performed at Rules Based Medicine  ,10 and SWe tested the distribution of biomarkers for an association with COPD using a linear mixed effects model including disease (COPD or control), smoking, and the interaction between disease and smoking as fixed effects, and visit number within each subject as a random effect for each protein. A log transformation was used for those analytes whose concentration distributions were highly skewed. A second linear mixed effects model was also constructed adding age and gender into the original model as covariates to adjust for potential effects of these variables. We assessed associations between individual protein levels and lung function and dyspnea using a linear mixed effect model adjusting for age and gender. In these models lung function variables and dyspnea scores were fixed effects and patient visit number  was modeled as a random effect.The analytical variation of the measurements was obtained from the assay manufacturers. Within-subject coefficient of variation (WCV) and the between-subject coefficient of variation (BCV) were calculated for each of the proteins to assess for the intra- and inter-patient variability respectively in the levels of the inflammatory proteins. The reference change value for each analyte measurements was calculated from the analytic variation and the within subject coefficient of variation using the Harris formula . BootstrWe studied 42 subjects prospectively over a 6-week period. Twenty-one subjects had COPD; 8 were active smokers, and 13 were former smokers. Twenty-one controls without COPD were studied; 9 were active smokers and 12 had never smoked. Patient characteristics of the 4 groups of patients are listed in Table Table Table 1 .1 1, dyspnea, and diffusion capacity, suggesting that higher serum levels of these proteins are found in patients with more advanced lung disease.Four of 19 serum proteins tested  were elevated in patients with COPD compared to control subjects. However of these proteins only CRP, myeloperoxidase and VEGF showed acceptable coefficients of variation to suggest they were sufficiently reliable over repeated measurements, and hence clinically useful as reliable predictors to assess disease state. These were also the only three proteins that showed significant correlations with FEVOur study also found that seven proteins from induced sputum were significantly elevated in COPD subjects compared to controls, however of these seven sputum proteins only four  showed acceptable intra and inter-patient reliability. We again found significant correlations of these four proteins with measures of lung function, air-trapping and dyspnea.It should be noted that sputum biomarker levels were reported normalized for the sputum total protein concentration. We corrected for total sputum protein concentration in order to overcome variability associated with sample dilution of induced sputum. All of our sputum analytes except four showed a significant correlation with total protein. The failure of these four to correlate with total protein concentration suggests dilution alone does not account for the differences seen. However there is also a potential drawback to this method since airway inflammation may increase local production of cytokines as well as protein leakage into the airway from the interstitium. If total protein in the airway is increased due to inflammation, correction of individual biomarkers for total protein concentration will dampen the inflammatory biomarker signal. However we felt that it was important to be conservative and to correct for any variability in dilution of sputum which is likely to be large.Our study findings are somewhat complementary to those of a recently published study by Sapey and colleagues . These iResults from our study suggest that variability in serum inflammatory biomarkers is somewhat less than that seen in biomarkers from induced sputum. This finding makes intuitive sense, since the amount of sputum yielded from the lower respiratory tract, and its purulence and relative dilution, can vary in clinically stable subjects from day to day. Correction of results by normalizing for the sputum total protein concentration, such as we did, can partially, but not completely account for variability in sputum yields. In contrast, peripheral blood analysis, which yields a standard amount of serum, is less subject to variability in sampling, and this may explain why inflammatory protein measurements are more reproducible from serum than from induced sputum.Previous studies have associated serum values of CRP with COPD ,14., andA previous study by Gompertz and colleagues suggested that the sputum inflammatory markers neutrophil elastase and IL-8 were significantly elevated in COPD patients with alpha-1 antitryspin deficiency and chronic sputum expectoration compared to levels in similar patients who did not chronically expectorate . Our stuThere are some limitations to our study. Other sputum inflammatory markers aside from IL-8 may potentially also be higher in the subgroup of COPD patients with chronic bronchitis, however our study did not have adequate patient numbers or power to explore the association of sputum analytes in the chronic bronchitis subgroup in greater detail. We did not perform high-resolution CT scans in our patients to quantitatively describe their degree of emphysema. This would have been useful, in order to try to better phenotype our patients, and in order to relate relative levels of serum biomarkers to CT scan findings of emphysema. Also we did not test for all of the possible proteins that might participate in the complex inflammatory mechanisms that underlie COPD. Other potential limitations relate to potential sources of variability in measurements. Serum and sputum samples were frozen and batched, and a variable duration of freezing, and the freeze-thaw cycle, may introduce variability in measurement values. Two complementary multiplex assays were used, and each platform has its own analytical variation of measurement for each protein. Although we performed spiking experiments for serum proteins, spiking experiments were not performed to measure the same proteins in sputum supernatants. It is possible that the coefficient of variation for analytes measured in serum may not be applicable to that measured in sputum if the biologic fluid affects the assay. Finally, our sample size of 42 subjects was relatively small, and an increase in sample size would be expected to improve the precision around the estimates of mean analyte values in patients with COPD.The acceptability of single biomarker measurements can only be interpreted in the context of total variability of the measurements over time. Based on relatively high intra-subject variability and large reference change values for individual biomarkers measurements seen in this study, we would conclude that levels of many serum and sputum biomarkers cannot be reliably ascertained based on single measurements, and that multiple measurements over time can give a more reliable and precise estimate of the inflammatory burden in clinically stable patients.Our study did identify 3 biomarkers from serum and 4 biomarkers from sputum that were significantly associated with COPD, and with COPD disease severity. Importantly, all of these biomarkers also showed acceptable inter- and intra-subject reliability over repeated measurements. Increasing use of multiplexed immunoassay technology may help to identify new clusters of important biomarkers that may be associated with ongoing lung inflammation of COPD.COPD: chronic obstructive pulmonary disease; TNF alpha: tumor necrosis factor alpha; IP-10: interferon-inducible protein 10; TNF-R1: tumor necrosis factor receptor I; TNF-RII: tumor necrosis factor receptor 2; TIMP-2: tissue inhibitors of metalloproteinases 2; HGF: hepatocyte growth factor; KGF: keratinocyte growth factor; MMP-2: matrix metalloproteinase 2; MMP-13: matrix metalloproteinase 13; GROα: growth-related oncogene alpha; CRP: C-reactive protein; ENA-78: epithelial neutrophil-activating peptide 78; IL: interleukin; MCP-1: monocyte chemotactic protein 1; TIMP-1: tissue inhibitors of metalloproteinases 1; VEGF: vascular endothelial growth factor; IP-10: interferon-inducible protein 10; KGF: keratinocyte growth factorSDA has no competing interests.KLV has no competing interests.TR has no competing interests.CZ, ZA, TN and AQ are employees of Roche Pharmaceutical Inc.SA and KV designed the study and collected the patient data. SA wrote the first draft of the paper.TR and CZ performed the statistical analysis of the data and edited the paper. ZA, TN, and AQ helped design the study, coordinated assays of the blood and sputum samples, and edited the paper. All authors have read and approved the final manuscript.


Drosophila adult external structures. GIPCs  were first identified in mammals and bind to the Gαi GTPase activating protein RGS-GAIP. They have been proposed to act in a G-protein coupled complex controlling vesicular trafficking. Although GIPCs have been found to bind to numerous proteins including Frizzled receptors, which participate in PCP establishment, there is little in vivo evidence for the functional role(s) of GIPCs. We show here that overexpressed Drosophila dGIPC alters PCP generation in the wing. We were however unable to find any binding between dGIPC and the Drosophila receptors Fz1 and Fz2. The effect of overexpressed dGIPC is likely due to an effect on the actin cytoskeleton via myosins, since it is almost entirely suppressed by removing a genomic copy of the Myosin VI/jaguar gene. Surprisingly, although dGIPC can interfere with PCP generation and myosin based processes, the complete loss-of-function of dGIPC gives viable adults with no PCP or other detectable defects arguing for a non-essential role of dGIPC in viability and normal Drosophila development.Epithelia often show, in addition to the ubiquitous apico-basal (A/B) axis, a polarization within the plane of the epithelium, perpendicular to the A/B axis. Such planar cell polarity (PCP) is for example evident in the regular arrangement of the stereocilia in the cochlea of the mammalian inner ear or in  all  Drosophila adult external structures, including the body wall, where sensory bristles point posteriorly , the perfect alignment of the ommatidial units in the eye, or in the wing, where cellular hairs all point distally Drosophila, each adult wing cell forms one hair, an actin based process. Mutations affecting core PCP or actin cytoskeleton components result in missing or extra wing hairs Epithelial cells are polarized along their apico-basal axis through the asymmetric segregation of protein complexes. Many epithelia also show a second axis of polarization, within the plane of the epithelium and perpendicular to the apico-basal axis. For instance, this planar cell polarity (PCP) is evident in the regular arrangement of the stereocilia in the cochlear epithelia of the mammalian inner ear. Strikingly, it is apparent in  all per se that mediates the actin nucleation and polymerization. The proximo-distal polarity in the Drosophila wing is established between 12–30 hours after pupation formation (APF). It is controlled by a very well conserved PCP gene cassette used to polarize all adult structures in Drosophila , and whose homologues also control PCP in vertebrates. The core PCP factors include the seven-pass transmembrane receptor Frizzled (Fz1), the scaffold protein Dishevelled (Dsh), the Ankyrin repeat protein Diego (Dgo), the 4-pass transmembrane protein Strabismus , the scaffold protein Prickle (Pk), and the 7-pass transmembrane Cadherin Flamingo  multiple wing hairs (mwh), tricornered, and furry, are also involved in the formation and orientation of wing hairs, but their role remains less well understood This process is under the control of both the polarity determinants that define where the wing hair grows, and the machinery Drosophila wing, and identified the GIPC homologue (dGIPC). Given the observation that the Xenopus GIPC (Kermit) binds to the C-terminal cytoplasmic tail of several Frizzled receptors including Xfz7, involved in the control of the PCP regulated process of convergent extension movements during gastrulation Drosophila.To gain further insight in the control of PCP establishment we performed a gain–of-function screen in the GIPCs  were first identified in mammals and bind to the Gαi GTPase activating protein RGS-GAIP and they have been proposed to be involved in a G-protein coupled complex controlling vesicular trafficking in vivo evidence for their functional role(s). We show here that overexpressed dGIPC alters PCP generation in the Drosophila wing. This effect of overexpressed dGIPC is likely due to an effect on the actin cytoskeleton via the myosins, since it is almost entirely suppressed by removing one copy of the Myosin VI/jaguar gene. We were however unable to find any binding between dGIPC and the Drosophila receptors Fz1 and Fz2. The complete loss-of-function of dGIPC gives viable adults with no PCP defects arguing for a dispensable role of dGIPC in viability and normal development of the fly, including PCP generation.Even though GIPCs have been found to bind to numerous proteins including Frizzled receptors (see above), there is little apterous-Gal4  driver. A similar approach has proven fruitful and led to the description of the core PCP gene diegoap-Gal4, the EP line drives the expression of the gene(s) it is inserted next to , which could be interpreted by the fact that having a uniform or mislocalized polarity signal is as bad as having no polarity signal at all, resulting in the same loss of polarization for a given cell. We have taken advantage of this property and conducted an overexpression screen for genes potentially involved in PCP by screening EP libraries driven on the dorsal thorax by the  next to  [19], [2engrailed-Gal4  with wing hairs showing an impaired orientation creating swirls and waves instead of pointing distally. Furthermore, a high occurrence of multiple wing hairs was observed  arguing that the gene associated affects only a subset of PCP events.Two such EP lines, EP(2)493 and GS2053 gave strong PCP defects on the notum . These Eobserved . No PCP Drosophila GIPC homologue 02045, dGIPC encodes a 336 aa protein with a single central PDZ domain, closely related to mammalian GIPCs dGIPC is responsible for the phenotype observed, we first raised a monoclonal antibody. Upon overexpression of GS2053 driven by en-Gal4, a strong signal is observed in the posterior compartment of third instar wing discs proving that GS2053 drives the overexpression of dGIPC 2431, located just downstream of the dGIPC ORF, between dGIPC and the predicted gene CG8709 ED1725 and Df(2R)ED1735). There were no wing hair defects (orientation or number) or any other noticeable defect associated with the loss of dGIPC either in homozygous animals , or in homozygous somatic clones. In particular there were no detectable defects in epithelial cells with respect to the cortical actin  phenotype was a strong multiple wing hair defect (see above). Multiple wing hairs can also be generated by overexpressing core PCP proteins such as Fz. The removal of one copy of It has been reported that Xenopus Kermit1, the Xenopus homolog of dGIPC binds to a specific subset of Fz receptors (namely Xfz7 and Xfz3) and controls the Wnt1/Xfz3 mediated neural crest induction dGIPC could affect the canonical Wg pathway. However, we did not notice any Wg pathway associated phenotypes in the dGIPC loss of function alleles. Furthermore, we did not detect any genetic interaction between overexpressed Dfz2 and dGIPC either by reducing the dose of, or by co-overexpressing dGIPC with dFz2 (data not shown). We were also not able to detect a molecular association between dGIPC and the Fz1 or Fz2 Cterm cytoplasmic tails in any of the three assays employed . Finally, dGIPC does no get relocalized to the plasma membrane upon co-transfection of Drosophila S2 cells with Fz1 or with Fz2 (data not shown).Therefore, it was possible that dGIPC plays a role at all in either the canonical or the non-canonical Fz signaling pathways.In light of these results, it is very unlikely that Drosophila transmembrane proteins . We did not detect any strong interaction with any of the following: InR (insulin) - Semaphorins 1A, 1B, 5C - EGFR (mapk pathway) - ECadherin - Notch - Dome (jak/stat pathway) - Tkv (BMP pathway) - Stbm (PCP pathway).Apart from the Frizzleds, vertebrate GIPCs have been shown to interact with a wide variety of transmembrane proteins with a carboxy end PDZ binding motif see  to promoIn order to identify the genes affected downstream of overexpressed dGIPC, we have assayed for modification of the strong multiple wing hair phenotype associated with GS2053 by removing one copy of candidate genes. The results are summarized in fz, dsh, dgo, stbm, pk-sple, and fmi, confirming further that dGIPC is not involved in PCP establishment per se.We first tested for interactions with core PCP genes. No interaction was observed with the PCP mutants for rac1, rac2, mtl, or of rhoA, and of the RhoA associated kinase drok did not modify the multiple wing hair phenotype of overexpressed dGIPC, indicating that the cdc42 interaction is specific. Interestingly, a dominant enhancement was also observed with fy and mwh, two genes whose mutant phenotype are multiple wing hairs. Finally an enhancement was also noted with zipper (zip), the Myosin II light chain (spaghetti squash (sqh), the Myosin II regulatory subunit, previously implicated downstream of RhoA/dRok in the control of wing prehair numbers. The most striking results was an almost complete suppression of the overexpressed dGIPC phenotype by the removal of one copy of myosin VI/jaguar  generation in Drosophila, we have identified the fly homologue for the PDZ domain containing gene GIPC. Overexpression of dGIPC affects PCP in the body wall and wing of the adult fly, but not in the eye. In particular, the orientation and number of wing hairs is affected. However, dGIPC null mutants are homozygous viable without any PCP defects arguing that dGIPC is not normally involved during PCP generation in the fly. Furthermore, we did not observe any obvious phenotype in homozygous null dGIPC individuals, even when the maternal contribution was removed, and even in tissues of high dGIPC expression such as the embryonic midline glia. This suggests that although GIPCs have been implicated in several important aspects of vertebrate development such as neural crest formation and head induction in the frog Xenopus laevis (kermit1 and 2 respectively) This not essential role of dGIPC, could be due to redundancy, but it would be a functional redundancy, since there is only one identified GIPC homologue in the fly genome. The existence of a clear relationship between Drosophila and Vertebrates GIPCs argues for a selection to keep these genes, so that wilt type flies are fitter than dGIPC mutant flies. Thus, it could be that we were unable to uncover more subtle phenotype such as glial cell morphology or biology, or that the dGIPC function is revealed only after a challenge, absent in standard lab conditions of Drosophila culture. There are other examples of genes conserved between flies and vertebrates, that despite having well characterized roles in vertebrates, appear to have elusive functions in flies. For instance, the only p120ctn homologue in fly is not essential and homozygous null individuals are viable with no phenotype myoII/zipper inhibits dGIPC function on the actin cytoskeleton, while myoVI/jaguar is required to mediate dGIPC function. This antagonism between MyoII and MyoVI has previously been suggested in Drosophila, in particular during the asymmetric segregation of cell fate determinants in the developing neuroblasts, where MyoII is required to exclude determinants from the apical cortex, while MyoVI is required to translocate determinants basally highlighting different mode of action of these two Myosins ex2dGIPC and ex2/Df(2R)ED1725dGIPC null animals, suggesting that this role of Myosin VI is independent of dGIPC.Finally, we have provided evidence that, like its vertebrate homologues, dGIPC functions with the non-muscular Myosins II and VI. Genetic interactions suggest that apterous-Gal4 driver. Interesting lines were rescreened using the engrailed-Gal4 (posterior compartment of the wing) and sevenless-Gal4  drivers. Flies carrying a UASt transgene coding for full length dGIPC were generated by amplifying by PCR from genomic fly DNA the ORF of dGIPC and cloning in the UASt vector.An EP screen for potential new PCP components was performed by driving the expression of several EP collections on the notum of the adult fly using the dGIPC, dGIPCex2 and dGIPCex31, were generated by imprecise excision of the P element EP2431. Other dGIPC alleles used were the EP lines GS2053dGIPC and EP493dGIPC, and the deletions Df(2R)ED1725 and Df(2R)ED1735. dGIPC mutant clones were generated by crossing FRT42D ex31dGIPC and eyFLP; FRT42D arm-LacZ flies.Null alleles for GS2053 using the different alleles stated in the text or in the figures .Genetic interactions were performed at 25°C assaying for modification of the multiple wing hair phenotype of engrailed-Gal4 driven overexpression of dGIPCrd instar larval heads, following standard protocols, and using mouse anti-dGIPC , mouse anti-Arm , mouse anti-GFP .Yeast two-hybrid, GST-Pull down, and co-immunoprecipitation experiments between full-length dGIPC and the C-terminal cytoplasmic tails of frizzled1 and frizzled2 were performed as described previously Total RNA from 5 adult females egg chambers was isolated by Trizol (Ambion) and reverse transcribed using the M-MLV RT kit (Promega) and random hexamers (Promega). The levels of the cDNA for dGIPC and for CG8709 were quantified by real-time PCR using QuantiTec Sybr Green PCR mix (Qiagen), after normalization to the levels of the ubiquitous gene rp49. The calibration curves for each primer pair were constructed from serial dilutions of genomic DNA.The primers used were:S: GACTGACGATCACCGACAAC AS: ACCATATTCTGGCCGTTGAGdGIPC S:AGCGCAAGAACTCTTCAAGC AS: GTTGTTGTTTGTGGCACTGGCG8709 S:GTCGCCTGCGTTCTCAAGAG AS:GACAATTGAACTCGGCACTCrp49 Embryo collection, and dissection of larval brain and imaginal discs and antibody stainings were performed using standard protocols. Primary antibodies used were: mouse anti-dGIPC , rat anti-LacZ , rat anti-E-Cad , rhodhamine-phalloidin .


Myostatin is an endogenous, negative regulator of muscle growth determining both muscle fiber number and size. The myostatin pathway is conserved across diverse species ranging from zebrafish to humans. Experimental models of muscle growth and regeneration have implicated myostatin as an important mediator of catabolic pathways in muscle cells. Inhibition of this pathway has emerged as a promising therapy for muscle wasting. Here we discuss the recent developments and the controversies in myostatin research, focusing on the molecular and cellular mechanisms underlying the actions of myostatin on skeletal muscle and the potential therapeutic role of myostatin on muscle-related disorders. In commactivity -6. Zimme) family . However1 [1) [Although the mechanism of activation of the latent myostatin complex remains to be determined, the bioavalaibility and the function of myostatin appear to be dependent of a network of protein interactions. Indeed, in addition to the propeptide, several other proteins have been identified as inhibitory binding proteins of myostatin. Through a number of yeast two-hybrid system experiments, four binding partners of myostatin  have been defined in muscle -10 where1 , 7-17. I1 [1) -15; a ph1 [1) . FurtherAlthough not fully characterized, the signalling pathway and mechanism of myostatin action appear similar to those exhibited by members of the TGF-β superfamily. The mature myostatin peptide binds to one of the two activin type II receptors (ActRIIB to a greater degree than ActRIIA), which recruits, phosphorylates and thereby activates the activin type I receptor propagating signals along the Smad pathway , 18. MyoLike TGF-β family members, myostatin also induces the activation of various MAPK cascades. Philip and coworkers  establisTaken together, these results demonstrate that myostatin signalling can recruit various intracellular signalling cascades and suggest that myostatin signalling might be regulated by cross-talks with tyrosine kinases-linked growth receptor signalling pathways and/or other pathways that remain to be elucidated. Muscle size depends on fiber number and fiber size. The number of fibers is usually set during development while their size continously adapts during the entire lifetime depending on activity, nutrition, diseases and aging. In the last decade myostatin has emerged as a key maintenance regulator of muscle mass. The role of myostatin in muscle comes from the phenotype of myostatin-deficient animals. Myostatin was first found to regulate muscle mass in mice from which the gene encoding myostatin has been knocked-out. The resulting “mighty mice” displayed muscle overgrowth due to both hyperplasia (increased number of muscle fibers) and hypertrophy  . These emdx mouse model of Duchenne muscular dystrophy, in the caveolin 3 deficient model of limb-girdle muscular dystrophy 1C (LGMD1C) and in two rodent models of amyotrophic lateral sclerosis [w/dywdy mouse model of laminin-deficient congenital muscular dystrophy, did not ameliorate the muscle pathology but increased postnatal lethality [et al. [-/-scgd) (a model of human limb-girdle muscular dystrophy referred as LGMD2F). However, this effect is obtained only in young dystrophic mice but not in older ones. In addition, their results also showed that the effectiveness of myostatin blockade could be dependent on specific muscle groups studied. Taken together these studies indicate that myostatin inhibition may not be beneficial in all dystrophic contexts. This is supported by data showing that AAV-mediated delivery of a mutated myostatin propeptide in the muscles of animal models of two limb-girdle muscular dystrophies  leads to different outcomes: it ameliorates calpain 3 but not α -sarcoglycan deficiency [The function of myostatin appears to have been conserved accross diverse species as indicated by the doubling of muscle mass in cattle, sheep, dog and human carrying natural mutations in the myostatin gene , 32-36. clerosis , 37-40. ethality . With re [et al.  reportedficiency . Therefoet al. [There is also controversy about the potential improvement of muscle strength in response to myostatin inhibition. Using front paw grip strength, several studies have reported an improvement of force generation in myostatin knock-out mice as well as in wild type mice treated with antibody anti-myostatin or immunized with a DNA vaccine against myostatin , 44, 45.et al.  showed aet al.  reported1). Interestingly, myostatin has been proposed to be a key signalling molecule that signals the quiescence of satellite cells and their progeny, the myoblasts. Direct analysis of satellite cells in myostatin mutant mice has shown an increased number of satellite cells per single myofiber and a higher proportion of activated satellite cells compared to wild-type mice [mdx mice that are known to have cycles of degeneration also reveals evidence for improved muscle regeneration in lack of myostatin [mdx mice [et al. [Growth and repair of skeletal muscle are carried out by a group of quiescent muscle stem cells known as satellite cells. In response to tissue damage or hypertrophic stimuli, these cells are activated and will proliferate and differentiate -50. Howeype mice . In addiype mice , 45, 52. [et al.  showing  [et al. . Therefo1). Although the mechanism underlying the anti-proliferative action of myostatin has yet to be elucidated, but one possibility presented by Yang et al. [via promotion of degradation of cyclin D1, an important component of the cell cycle machinery. Their results suggest that myostatin is capable of stimulating Akt inhibition, GSK-3β activation and cyclin D1 destabilization, and that this process is dependent upon the activin receptor IIB, but not Smad3. How does the myostatin signalling act on satellite cell activation, proliferation and differentiation? Recombinant myostatin is able to arrest the cell cycle at the G1 phase of cultured myoblasts including C2C12 myoblasts, primary bovine myoblats and mouse satellite cells, and thereby induces inhibition of proliferation and differentiation , 51, 55.g et al.  indicateThe inhibitory effect of myostatin on differentiation is associated with down regulation of the myogenic differentiation factors MyoD, Myf5 and myogenin . Therefoet al. [via a Pax7-dependent mechanism to regulate satellite self-renewal . Myostatin also inhibits satellite cell activation and self renewal . Althouget al.  suggestsThe control of muscle mass is determined by a dynamic balance of anabolic and catabolic processes. Muscle hypertrophy is characterized by an increased in diameter and in total protein content of fibres and occurs as a result of an enhanced rate of protein synthesis while muscle atrophy induced by a decrease in activity and load or by catabolic loss of muscle mass is a decrease in the size of pre-existing muscle fibers, resulting from a dramatic increase in protein degradation and turn-over. A wealth of data supports that myostatin functions by inducing muscle atrophy. For example, elevated myostatin expression correlates with several settings of muscle loss including prolonged bed rest in young men , chronicet al. [in vivo to adult mice produces the signs and symptoms characteristic of the muscle wasting syndrome, cachexia. In addition, the muscle wasting observed in these mice can be partially reversed by systemic delivery of the myostatin propeptide or follistatin in the mice indicating that the observed muscle wasting was caused by excess myostatin. Similarly, ectopic expression of myostatin through gene electrotransfer of a myostatin expression vector induces atrophy of skeletal adult muscle associated with decreased muscle gene expression [et al. [et al. [Relevant to muscle wasting diseases, it has been shown that inhibition of myostatin rescued the muscular atrophy of caveolin-3-deficient mice . The inhet al.  demonstrpression . The eff [et al.  showed t [et al.  demonstr [et al. , 66. TheIn contrast to the above findings, several independant studies have reported that myostatin might not be required in some types of muscle atrophy. Indeed, myostatin levels are not increased in muscle atrophy due to denervation or hindlimb suspension , 68. Unevia myostatin. However, future researches should be focused on exploring how myostatin is connected with the cellular signalling pathways regulating the anabolic and catabolic responses in muscle. Another question that remains to be investigated is whether myostatin also plays a role in inactivating hypertrophic pathways. There is substantial evidence to suggest that myostatin could be connected with pathways regulating hypertrophy. The recent work of McFarlane  has showIncreasingly, research into the mechanisms by which myostatin controls muscle mass has revealed both new components of this signalling pathway and the participation of parallel pathways in the mediation of myostatin signal. It has been established that myostatin signalling can recruit various intracellular signalling cascades, however it remains unclear how these pathways are intercalated. With respect to new components of myostatin signalling pathway, decorin, a component of the extra-cellular matrix (ECM) has been discovered recently that binds myostatin and modulates its activity. This provides another means by which myostatin signalling can be controlled and raises the question of a potential link between myostatin activity and ECM integrity. Also, the demonstration that other TGF-β related ligands cooperate with myostatin to suppress muscle growth makes the search for manipulating TGF-β signalling pathways attractive.It is well documented that myostatin is important for proper development and homeostasis of adult musculature. In this line, the link between myostatin and the Akt/Foxo pathway may account, at least in part, for observations that support a role for myostatin in breaking or promoting muscle wasting. It will be challenging and informative to determine whether myostatin can also interfere with anabolic pathways.et al. [In the last decade, the blockade of myostatin has been represented an ideal tool to attenuate muscle wasting related to diseases, and there is a multi center clinical trial on adult patients with muscular dystrophy underway. The beneficial effects of loss of myostatin activity reside in enhancing muscle mass, however, there is also evidence that this increased muscle mass may in fact not be beneficial to skeletal muscle function. Indeed, Amthor et al. , recentl


Plasma levels of phenylalanine in homozygous (HMZ) PAHenu2 mice were >12-fold those of heterozygous (HTZ) littermates while tyrosine levels were reduced. Phenylketones, including PA, were also markedly elevated to the range seen in the human disease. Mice were subjected to 7,12 dimethylbenz[a]anthracene (DMBA) carcinogenesis, a model which is sensitive to the anticancer effects of the PA derivative 4-chlorophenylacetate (4-CPA). Tumor induction by DMBA was not significantly different between the HTZ and HMZ mice, either in total tumor development or in the type of cancers that arose. HMZ mice were then treated with 4-CPA as positive controls for the anticancer effects of PA and to evaluate its possible effects on phenylalanine metabolism in PKU mice. 4-CPA had no effect on the plasma concentrations of phenylalanine, phenylketones, or tyrosine. Surprisingly, the HMZ mice treated with 4-CPA developed an unexplained neuromuscular syndrome which precluded its use in these animals as an anticancer agent. Together, these studies support the use of PAHenu2 mice as a model for studying human PKU. Chronically elevated levels of PA in the PAHenu2 mice were not protective against cancer.Phenylketonuria (PKU) is a metabolic disorder caused by impaired phenylalanine hydroxylase (PAH). This condition results in hyperphenylalaninemia and elevated levels of abnormal phenylalanine metabolites, among which is phenylacetic acid/phenylacetate (PA). In recent years, PA and its analogs were found to have anticancer activity against a variety of malignancies suggesting the possibility that PKU may offer protection against cancer through chronically elevated levels of PA. We tested this hypothesis in a genetic mouse model of PKU (PAH About the time of the discovery of phenylketonuria (PKU) in 1934 This information inexorably leads one to ask the following question. Since PA is one of the metabolites of phenylalanine that is elevated in individuals with PKU, is the cancer incidence/types/cancer mortality different in this population of people? If so, PKU may be “nature's model” for assessing the anticancer properties of this compound. To our knowledge, no such study has yet been conducted. One reason for the dearth of information on this subject may be that the oldest early-detected adults with PKU are only now in their 40′s, since national newborn screening did not start until 1963 eun2/eun2 (HMZ) mice and in the range reported in the human disease enu2/wild mice (HTZ) were similar to that seen in the wild-type BTBR strain and in normal humans enu2 animal model and human PKU. Together with reported genetic, neurological, and biochemical comparisons with human PKU DMBA is a “pro-carcinogen” and must be metabolized to produce the proximate carcinogenic metabolite 3,4-dihydrodiol To determine whether the HMZ mice show a reduced incidence of tumor induction, we employed a protocol similar to that previously used in our aromatase transgenic mouse study These results do not support the hypothesis that chronically elevated PA at serum levels seen in the HMZ mice (∼13 μM) can influence DMBA-mediated tumor induction. As such, these results did not provide evidence that PKU can protect against cancer. A possible reason for the lack of PA effects in this study versus the potent suppression of 4-CPA on tumor induction in aromatase-transgenic mice could be that tumors induced in the latter are almost exclusively estrogen-driven When interpreting these results, we emphasize that effects of certain carcinogens have been shown to be dependent on age, sex, and animal strain in different mouse models. For example, Horlings and Demant In PKU, there is defective hydroxylation of phenylalanine to produce tyrosine. This condition results in reduced levels of tyrosine metabolites such as thyroxin, melanin, and the dopamine family of neurotransmitters enu2 mouse has shown to be a remarkably good model for human PKU. These animals show no residual activity of the liver PAH along with very high plasma Phe levels. Heterozygous PAHenu2/wild (HTZ) and homozygous PAHenu2/enu2 (HMZ) mice were produced by crossing HMZ fathers and HTZ mothers. In our experiments, only female mice were utilized with the HMZ experimental group and were compared with female HTZ animals as the control group. Due to the well known carcinogenic effects of ENU eun2/wild, like humans who are heterozygous for the PKU genotype, have plasma Phe levels that are similar to wild-type who carry no PAH mutation All procedures were carried out in accordance with the National Institutes of Health Guidelines on the Care and Use of Animals and an animal study protocol approved by Emory University's Institutional Animal Care and Use Committee. Mice used in this study were created in the BTBR strain by ENU germline mutagenesis in the gene for phenylalanine hydroxylase (PAH) which results in a loss of enzyme activity such that, in liver, phenylalanine hydroxylase activity is minimal To get reproducible tumor induction in the mice, we used a combination of oral administration of 7,12-dimethylbenz[a]anthracene (DMBA) along with subcutaneous implantation of a slow releasing (90-day) medroxyprogesterone acetate (MPA) pellet Treatment of mice with 4-CPA was performed by continuous intake of the compound in the animal's drinking water at a concentraion of 6 mg/ml as previously reported by our group Phenylalanine and metabolites in the plasma of HTZ and HMZ mice were analyzed by an amino acid analyzer and GC/MS as described To measure CYP1B1 activity, the ethoxyresorufin-O-deethylase (EROD) assay was used After the mice were sacrificed, necropsy and tumor evaluation was performed by the Diagnostic Laboratory, Division of Animal Resources, Emory University. The following tissue were collected and examined for gross lesions or abnormalities: heart, lungs, mammary glands, reproductive organs, liver, spleen, kidneys, stomach, cecum, large and small intestine, brain, salivary glands, thymus. Any suspicious tissue, tumors, and routinely mammary glands (as being the main target of DMBA-induced tumors) were dissected free from skin and processed for histology as previously described