Critical Care [2) increased significantly in response to emergency intubation, and the authors suggested that the early normalization of ScvO2 after intubation might not be reliable to reflect successful resuscitation. However, they might have ignored the influence of arterial oxygen tension (PaO2) on ScvO2.We read with great interest the study by Hernandez and colleagues published in cal Care . The stu2. However, a very high PaO2 could significantly influence ScvO2 even if arterial oxygen saturation reaches 100%. Pre-oxygenation may result in a very high PaO2 in the emergency intubation, so PaO2 should be taken as a potentially confounding factor. Very high PaO2 (about 288 mmHg) has a more significant and consistent effect on ScvO2 than a relevant change in cardiac index (>10%) [2 could increase ScvO2 without increasing oxygen delivery [It is well known that when arterial oxygen saturation is approaching 100%, the increase in oxygen delivery would be limited in response to the increase of PaOx (>10%) , and anix (>10%) . Recentldelivery .2 on ScvO2 in the management of critically ill patients.It is worth paying attention to the impact of PaO


DLK1 and MEG3, are located in the same gene cluster at 14q32. Previous studies in bladder cancer have suggested that tumor suppressor genes are located in this region, but these have not been identified.The two oppositely imprinted and expressed genes, DLK1 and MEG3 are frequently silenced in urothelial cancer tissues and cell lines. The concomitant downregulation of the two genes is difficult to explain by known mechanisms for inactivating imprinted genes, namely deletion of active alleles or epitype switching. Indeed, quantitative PCR revealed more frequent copy number gains than losses in the gene cluster that were, moreover, consistent within each sample, excluding gene losses as the cause of downregulation. Instead, we observed distinctive epigenetic alterations at the three regions controlling DLK1 and MEG3 expression, namely the DLK1 promoter; the intergenic (IG) and MEG3 differentially methylated regions (DMRs). Bisulfite sequencing and pyrosequencing revealed novel patterns of DNA methylation in tumor cells, which were distinct from that of either paternal allele. Furthermore, chromatin immunoprecipitation demonstrated loss of active and gain of repressive histone modifications at all regulatory sequences.We observed that both DLK1 and MEG3 in urothelial carcinoma is epigenetic silencing across the 14q32 imprinted gene cluster, resulting in the unusual concomitant inactivation of oppositely expressed and imprinted genes.Our data support the idea that the main cause of the prevalent downregulation of The online version of this article (doi:10.1186/1868-7083-6-29) contains supplementary material, which is available to authorized users. CDKN1C, which is inactivated alternatively by genetic or epigenetic mechanisms in several human cancers, including urothelial carcinoma [The differential expression of alleles inherited from mother or father at genomic imprinted genes is achieved by epigenetic mechanisms, particularly by differential methylation at regulatory regions designated as differentially methylated regions (DMRs). Imprinted genes regulate growth and other physiological functions during embryonic development, but also in adult tissues. Since several maternally imprinted genes limit growth, they possess tumor-suppressive potential and tend to become inactivated in different types of human cancer . Their iarcinoma , 3.DLK1-MEG3 cluster, is affected by allelic losses or epigenetic changes [Delta-like 1 (DLK1), Deiodinase Iodothyronine Type III (DIO3) and Retrotransposon-like Gene 1 (RTL1 or PEG11) [Maternally Expressed Gene 3 (MEG3), Maternally Expressed Gene 8 (MEG8) and RTL1 antisense (RTL1-AS) [MEG3  and 1.3 kb upstream of the MEG3 transcription start site (MEG3 DMR) [DLK1 promoter is also relevant for its expression. The IG DMR, which is methylated on the paternal allele and unmethylated on the maternal allele, serves as the initial imprinting control region (ICR) for the entire cluster during early development [MEG3 DMR usually represents the dominant regulatory region [DLK1 and MEG3 is commonly reciprocal, possibly as a consequence of regulatory effects of the MEG3 RNA [In several cancers, a cluster of imprinted genes at 14q32.2, the  changes . This clr PEG11) . The matRTL1-AS) , 10 encoelopment , whereasy region . The expMEG3 RNA .Figure 1DLK1 expression is associated with changes in DNA methylation at this gene and its control regions [MEG3 has been reported to act as a tumor suppressor in a broader range of cancers [MEG3 exert various functions relevant for cancer development and progression, including regulation of growth factors and Notch signaling by DLK1, and regulation of TP53, pRB1 and NOTCH activity by MEG3 [Loss of imprinting in the 14q32 region due to epimutations at the IG DMR or microdeletions has been implicated in a range of diseases including UPD14mat/pat  and various cancers . In rena regions , 14–16.  cancers –19. Both by MEG3 –22.DLK1 and MEG3 are good tumor-suppressor candidates. Indeed, MEG3 has recently been reported to become downregulated in the majority of urothelial carcinomas and to exert tumor-suppressive functions [Urothelial carcinoma is the most common cancer of the urinary bladder. It can be categorized into two subtypes, namely papillary tumors and the more malignant invasive carcinomas, which are characterized by pronounced chromosomal instability , 24. In unctions . HoweverDLK1 and MEG3 was strongly diminished in urothelial carcinoma tissues and cell lines. This finding raises a conundrum as it is difficult to envision how either allelic loss or epitype switching could lead to the concomitant downregulation of these two imprinted and normally inversely expressed genes, which are located less than 100 kb apart. Indeed, upon closer investigation, we found that inactivation of the two genes is associated with the establishment of a novel epigenetic state in the region, which is distinct from that of either parental allele and is independent of copy number changes in most urothelial carcinoma tissues and cell lines. This epigenetic state involves a characteristic DNA methylation pattern and a strong shift towards repressive histone modifications across three major regulatory regions in this imprinted gene cluster. This mechanism could provide a means to silence both genes despite their normal opposite regulation.Unexpectedly, we found that expression of both DLK1 and MEG3 mRNA by qPCR in urothelial cancer tissues (n = 30) and cell lines compared to benign bladder tissue samples (n = 11) and primary cultured normal urothelial cells. Normal kidney tissue and the hepatoma cell line HepG2 were used as additional positive controls. DLK1 mRNA was significantly reduced in urothelial cancer tissues compared to normal bladder tissue  measured copy numbers varied between 1.7 and 2.2, as normalized to normal diploid leukocytes set at two copies. Of 23 urothelial carcinoma samples (BT), 10 cases displayed increased copy numbers and 5 cases had decreased copy numbers, whereas 8 tumors showed copy numbers in the normal range (1.7 to 2.2). Importantly, gene copy number changes affected all three analyzed loci to the same extent within each sample.Similarly, gene copy number changes affected all analyzed genes to the same extent in urothelial cancer cell lines (UC). Primary urothelial cell cultures (UP) were measured as diploid, as expected. Seven cell lines displayed elevated copy numbers between 2.5 and 3.5 across the analyzed region, for example, the Umuc3 and 639v cell lines. In accord with our results the predicted modal copy numbers are 3 for Umuc3 and 639v . DecreasMEG3 and DLK1 expression was reduced in almost all urothelial cancer tissues and cell lines, irrespective of copy numbers [see Additional file Thus, whereas copy numbers of the 14q region were variously increased or decreased in urothelial carcinoma tissues and cell lines, MEG3 and DLK1 downregulation was associated with changes in DNA methylation, we analyzed the three relevant CpG-rich regulatory regions, the DLK1 promoter, the IG DMR, and the MEG3 DMR, by bisulfite sequencing in selected urothelial cancer tissue and cell line samples  and tumor (n = 23) tissue samples, normal urothelial cells (n = 5) and urothelial carcinoma cell lines (n = 15)  in the majority of urothelial cancer tissues and cell lines , which are retained during bisulfite conversion if methylated, but mutated if unmethylated. Thus, up to five different restriction products are obtained if the sequence is heterogeneously methylated [see Additional file The DNA sequence in the DLK1 and MEG3 expression in urothelial cancer is associated with the acquisition of novel DNA methylation patterns, especially at the DLK1 promoter and the MEG3 DMR that in some cases extend to the IG DMR.Cumulatively, these findings indicate that the concomitant loss of DLK1 and MEG3 mRNA in five urothelial cancer cell lines. Treatment with 5-aza-dC or SAHA individually did not significantly induce MEG3 or DLK1 expression except for 5-aza-dC in the BFTC905 cell line. Combined SAHA/Aza-dC treatment consistently restored DLK1 and MEG3 gene expression to detectable levels [see Additional file To determine to which extent DNA methylation contributes to the silencing of the two genes, we tested the effects of the DNA methyltransferase inhibitor 5-aza-2-deoxycytidine (Aza-dC) alone or in combination with the pan-HDAC inhibitor suberoylanilide hydroxamic acid (SAHA) on the expression of DLK1 promoter, the IG DMR and the MEG3 DMR in normal urothelial cells, HepG2 cells and seven urothelial carcinoma cell lines , we quantified the H3K4me3 histone modification associated with active genes, the H3K9me3 and H3K27me3 modifications associated with repression, and H4K16ac, a marker of transcriptional competence but also of fixed nucleosomes, at the DLK1 promoter. The IG DMR likewise displayed enrichment of both active and repressive histone modifications, H3K4me3 and H3K9me3, and more strongly H3K27me3. Interestingly, H4K16ac was strongly enriched at both DMRs. At the MEG3 DMR, in agreement with the high expression in normal bladder, the active histone modification H3K4me3 was enriched, whereas repressive histone modifications (H3K9me3 and H3K27me3) were low.Because the variable results of DNA methylation analyses had suggested that the epigenetic state of the 14q32 region changes during culture of normal urothelial cells Figure . As expeDLK1 expressing control cell line. In HepG2 cells, active and repressive histone modifications were enriched to comparable extents at the DLK1 promoter and the MEG3 DMR. The active mark H3K4me3 was highly enriched at the IG DMR in HepG2 cells, with higher levels of H3K9me3 compared to H3K27me3, which is the inverse pattern compared to that in normal urothelial cells.HepG2 cells were used as a DLK1 promoter and the MEG3 DMR. As a consequence, these latter two regions assumed similar patterns of histone modifications across the urothelial carcinoma cell lines. Interestingly, in contrast to the H3K4me3 mark, the H4K14ac modification was less severely depleted or even retained in some cell lines, with lowered levels especially at the IG DMR.The most striking difference in the urothelial carcinoma cell lines towards the controls was the severe depletion of H3K4me3 at all three regulatory regions analyzed. In comparison, repressive histone modifications (H3K9me3 and H3K27me3) were generally increased, in particular H3K9me3 at both DMRs, whereas increases in H3K27me3 were more evident at the In summary, the ChIP analyses revealed the predominance of repressive histone modifications and a nearly complete loss of H3K4me3, with partial retention of H4K16ac, a modification characteristic of fixed nucleosomes, across the entire analyzed region in all urothelial carcinoma cells. This finding supports the contention that the region acquires a repressive chromatin state in urothelial carcinoma.DLK1 and MEG3 within this cluster are tumor suppressor candidates in urothelial carcinoma due to their known functions in the regulation of cell growth and development [DLK1 is downregulated by epigenetic mechanisms in renal cell carcinoma [MEG3 was reported to act as a tumor suppressor, too, but in a more consistent manner [DLK1 argue strongly that this cell line retains only a paternal allele.Previous molecular and cytogenetic analyses of urothelial cancers have suggested at least one tumor suppressor gene residing at chromosome 14q32.2 , 27, 32.elopment , 33. Indarcinoma . Howeverarcinoma , 34. Thet manner , 19, 22.t manner . Of notet manner , 16, 30.t manner , 30. WitDLK1 or MEG3 was reported to become deregulated, but not both genes, as we observed in urothelial carcinoma. Unfortunately, many papers do not comment on whether they have investigated the other gene at all. In benign bladder tissues, DLK1 and MEG3 were well detectable with MEG3 being expressed more strongly than DLK1, like in normal kidney, liver and pituitary gland [MEG3 and is required for silencing of the paternally expressed genes such as DLK1. Despite their reciprocal relationship in benign bladder tissue, MEG3 and DLK1 expression were found to be both significantly diminished in urothelial cancer tissues and cell lines. With respect to MEG3, our findings are fully consistent with those of Ying et al. [DLK1 in urothelial carcinoma.In previous reports on other cancer types, either ry gland , 30, 35.ry gland . Most mog et al. . UnfortuDLK1 is expressed from the paternal allele and MEG3 from the maternal allele. Their concomitant downregulation is therefore difficult to explain by allelic loss. Accordingly, we found a range of copy numbers between one and four in urothelial carcinoma tissues and cell lines indicating that both losses and gains of this region are frequent, accounting for the high frequency of apparent ‘loss of heterozygosity’ in previous reports [DLK1 and MEG3 can indeed not be explained by chromosomal deletions.Importantly,  reports , 26. HowMEG3 expression should be retained or even increased, and conversely, if the gene cluster assumed the paternal state, DLK1 expression should be retained or increased. Likewise, DNA methylation at the regulatory regions should become homogeneous and resemble either the maternal or the paternal pattern. This type of change is exemplified by the HepG2 cell line, which shows strong DLK1 expression associated with a paternal epigenetic state.By a similar argument, we can exclude conventional loss of imprinting or epitype switching as a plausible cause of the concomitant downregulation. If the gene cluster assumed the maternal state, DLK1-MEG3 cluster  and between RTL1 and DIO3  have also been described to be significantly reduced or silenced by DNA hypermethylation in bladder tumor tissues and the cell lines RT4, RT112 and T24 [A clue to the actual mechanism is provided by the observation that the DNA methylation patterns at the three regulatory regions in urothelial carcinoma cells are indeed homogeneous, but are different from both the maternal and paternal patterns in normal bladder and renal tissues. These methylation patterns therefore suggest that a novel repressed epigenetic state is established during urothelial carcinogenesis at the 14q32 gene cluster. Of note, several microRNAs encoded in the  and T24 , 29 sugg and T24 .DLK1-MEG3 cluster in urothelial carcinoma cell lines is also reflected in the predominance of repressive histone modifications such as H3K9 and H3K27 trimethylation  with our experimentally obtained methylation patterns suggest strongly positioned and potentially newly phased nucleosomes in cancer compared to benign urothelial cells  became significantly hypomethylated in cancer cells, while methylation of flanking sites rather increased. It could therefore be interesting to map the nucleosomal positioning in the 14q imprinted gene cluster in normal and cancer cells in future work.The repressed state of the n Figure . InteresDLK1-MEG3 cluster in urothelial carcinoma was hampered by the lack of an epigenetically stable normal urothelial cell line. Upon culturing, normal urothelial cells acquire a considerable degree of plasticity, including the ability to differentiate into epidermis-like as well as urothelial-like structures [DLK1-MEG3 cluster appears particularly susceptible to such changes, as reduced expression or silencing of MEG3 has also been observed in normal cell lines originating from other tissues [Meg3, have also been reported during establishment of cell cultures of mouse embryonic fibroblasts [DLK1 and MEG3 that varied between individual urothelial cell cultures. These changes did not extend to the IG DMR and were not as pronounced as in the cancer cell lines and tissues. For that reason, we used freshly isolated, noncultured urothelial cells, which are unfortunately only available in limited amounts, for the chromatin immunoprecipitation experiments.Our study of the epigenetic changes at the ructures . Upon imructures . The DLK tissues , 44. Charoblasts . In our DLK1 and MEG3 expression, overcoming the normally antagonistic regulation of these two imprinted genes. One target of these changes is evidently MEG3, which emerges as a tumor suppressor in many different tissues [et al. [MEG3 alone could be achieved by conventional mechanisms such as allelic loss or by epitype switching. The findings reported here and the observation of others that several smaller RNA species encoded in the cluster are downregulated by DNA hypermethylation [DLK1. The question of which of these changes support tumor progression will therefore have to be addressed by future research.In conclusion, our data suggest that the 14q32 imprinted gene cluster acquires a novel epigenetic state in urothelial cancer that allows the concomitant inactivation of  tissues . In urot [et al.  have demhylation , 29, colThe bladder cancer and benign tissue samples were a subset of those described in previous studies , 47 compAll urothelial cancer cell lines  and the hepatocellular carcinoma cell line HepG2 were cultured in DMEM  supplemented with 10% fetal calf serum . They weDLK1 and TBP in a reaction volume of 25 μl. MEG3 primers were described by Kawakami et al. 2006 [TBP was used as a reference gene. Primers and QuantiTect assays are listed in Additional file Total RNA was isolated from subconfluent cell cultures and cell lines or from powdered tissues using the RNeasy Mini or Micro Kit . Two μg RNA was reverse transcribed using 200 U SuperScriptII reverse transcriptase , with 300 ng oligo-dT and 25 ng random hexamer primers in a reaction volume of 20 μl. Real-time PCR assays were performed with the ABI7900HT System using the QuantiTect SYBR Green PCR Kit  and QuantiTect primer assays for DLK1 promoter and the MEG3 DMR using primers described in [Total genomic DNA was isolated from subconfluent cell cultures using the Blood and Cell Culture DNA Midi Kit . For DNA methylation analyses, 1 μg DNA was bisulfite converted using the EZ DNA Methylation-Gold Kit  following the supplier’s protocol with incubations at 95°C for 10 min, followed by 64°C for 2.5 h. After purification, the bisulfite converted DNA was analyzed for methylation of the ribed in . PCRs weMEG3 DMR bisulfite-treated DNA samples were used with newly designed primers [see Additional file MEG3 DMR region analyzed by standard bisulfite sequencing.For pyrosequencing analysis of the DLK1, IG DMR and MEG3 DMR as in ChIP analysis [see Additional file GAPDH was used as a reference gene. Gene copy numbers were calculated as the ratio of the measured sequence to the reference gene GAPDH and normalized to the value from leukocyte DNA set as two copies.For gene copy number analysis by qPCR 25 ng total genomic DNA was used with the QuantiTect SYBR Green PCR Kit  and primers for 5 primary urothelial cells freshly prepared from a ureter after nephrectomy using the True MicroChIP kit . The precipitated DNA fractions were quantified by real-time PCR with the QuantiTect SYBR Green PCR Kit  and primers for the DLK1 promoter, IG DMR and MEG3 DMR. ChIP assays [see Additional file CTCFL was used as a control for a silenced gene with inactive histone modifications (H3K9me3 and H3K27me3), whereas GAPDH was used as control for a highly expressed gene with active histone modifications . The input DNA purified from the sheared unprecipitated chromatin was used to make a standard curve for each gene investigated and each sample was measured in duplicate with less than 12% variation. The 100% enrichment refers to the levels at the respective control genes. The results remained in principle unchanged, if the values were normalized to input DNA directly  was performed on triplicate samples with the ChIP-IT Express Kit  according to the manufacturer’s instructions as previously described  or 105 pTable S2. Copy number changes and relative to gene expression of DLK1 in urothelial cancer cell lines. Figure S1. Additional bisulfite sequencing results in urothelial cultures. Method S1. Treatment with epigenetic inhibitors. Figure S2. COBRA analysis of the DLK1 promoter sequence. Method S2. DNA methylation analysis by COBRA. Figure S3. Effects of treatment with epigenetic inhibitors on DLK1 and MEG3 expression. Figure S4. Comparison between two normalization methods for the ChIP experiment. Figure S5. Bioinformatic prediction of nucleosome positioning at the DLK1-MEG3 locus. (DOCX 1 MB)Additional file 1: Table S1: Primer assays.


Myocyte pO2 values in normal dogs were calculated from the myocardial deoxymyoglobin (Mb- δ) levels using 1H-spectroscopy (MRS) and were normalized to Mb-δ obtained after complete LAD occlusion. During Protocol 1 (n = 6), Mb-δ was measured during sequential reductions of the oxygen fraction of inspired gas (FIO2) from 40, 21, 15, 10, and 5%, while in protocol 2 (n = 10) Mb-δ was measured at FIO2 of 3%. Protocol 3 (n = 9) evaluated time course of Mb-δ during prolonged exposure to FIO2 of 5%. Myocardial blood flow (MBF) was measured with microspheres and high energy phosphate (HEP) levels were determined with 31P-MRS. MVO2 progressively increased in response to the progressive reduction of FIO2 that is accompanied by increased LV pressure, heart rate, and MBF. Mb-δ was undetectable during FIO2 values of 21, 15, 10, and 5%. However, FIO2 of 3% or prolonged exposure to FIO2 of 5% caused progressive increases of Mb-δ which were associated with decreases of PCr, ATP and the PCr/ATP ratio, as well as increases of inorganic phosphate. The intracellular PO2 values for 20% reductions of PCr and ATP were approximately 7.4 and 1.9 mmHg, respectively. These data demonstrate that in the in vivo system over a wide range of FIO2 and arterial pO2 levels, the myocyte pO2 values remain well above the Km value with respect to cytochrome oxidase, and oxygen availability does not limit mitochondrial oxidative phosphorylation at 5% FIO2.Decrease of ambient oxygen level has been used in myocytes culture experiments in examining the responsiveness to stress secondary to hypoxia. However, none of these studies measure the myocytes oxygenation levels resulting in ambiguity as to whether there is insufficient oxygen delivery. This study examined the hypothesis that at a basal myocardial work state, adequate myocyte oxygenation would be maintained until extremely low arterial pO The unpaired electron spin in the heme-Fe(II) complex of deoxymyoglobin (Mb-δ) extends over the proximal histidyl Nδ proton to cause a chemical shift that produces a characteristic resonance on 1H NMR spectroscopy 2 of approximately 2 mmHg and fell to 80% of the basal value at an intracellular pO2 of 1.8 mmHg 2 during basal conditions and 5% saturated during total coronary occlusion, the intracellular pO2 values for 20% reductions of PCr and ATP were calculated to be approximately 4.4 and 0.9 mmHg respectively 2 where significant decreases in high energy phosphates occurred could have been influenced by a decrease in myocardial oxygen consumption caused by a decrease in coronary flow and perfusion pressure 2 levels. Thus, the true critical PO2 level that leads to a reduction of HEP can be better estimated with graded hypoxia through decrease in FIO2 such as in the present experimental setting.in vivo. Graded reductions of FIO2 were produced to document the threshold level of myocyte oxygenation at which perceptible reductions of the PCr/ATP ratio  occurred, and to examine the effect of progressive reductions of myocyte oxygenation on myocardial high energy phosphate content.Consequently, the present study was carried out to examine the effect of decreasing myocyte oxygenation by graded hypoxia on myocardial high energy phosphate content in the intact heart Studies were performed in 25 adult mongrel dogs of either sex weighing 20–27 kg. All experimental procedures were approved by the University of Minnesota Animal Resources Committee. The investigation conformed to the “Guide for the Care and Use of Laboratory Animals” published by the US National Institutes of Health .The dogs were anesthetized with sodium pentobarbital , intubated and ventilated with a respirator with supplemental oxygen to maintain arterial blood gases within the physiologic range. A heparin-filled polyvinyl chloride catheter, 3.0 mm o.d., was introduced into the right femoral artery and advanced into the ascending aorta. A left thoracotomy was performed through the fourth intercostal space and the heart suspended in a pericardial cradle. A heparin-filled catheter (3.0 mm o.d.) was introduced into the left ventricle through the apical dimple and secured with a purse string suture. A similar catheter was inserted into the left atrium through the atrial appendage. A homemade intra-cardiac vein catheter (0.3 mm o.d) was inserted directly into the great cardiac vein for coronary vein blood sampling. A 1.5–2.0 cm segment of the proximal left anterior descending coronary artery (LAD) was dissected free and a hydraulic occluder constructed of polyvinyl chloride tubing (2.7 mm o.d.) was placed around the artery. A silicone elastomer catheter (0.75 mm i.d.) was placed into the LAD distal to occluder by the method of Gwirtz 31P and 1H-MRS frequencies were 81 MHz and 200.1 MHz, respectively.Measurements were performed in a 40 cm bore 4.7 Tesla magnet interfaced with a SISCO  console. The left ventricular pressure signal was used to gate MRS data acquisition to the cardiac cycle, while respiratory gating was achieved by triggering the ventilator to the cardiac cycle between data acquisitions 1H-MRS detection of the proximal histidyl N-δ proton resonance of Mb-δ has been described in detail 1 value of Mb-δ. Each spectrum is acquired within 6 min . Although the short T1 of Mb-δ and the fast acquisition prevent gating of data acquisition to the cardiac cycle, signal loss as a result of heart motion was negligible because of the inherently broad line width of the Mb-δ peak. Although the Mb-δ resonance is temperature sensitive, the chemical shift of this resonance which appeared at 71–72 ppm (relative to H2O), remained virtually constant during the study protocol. No other resonances were detected within a 10 ppm region. In phantom studies we have established that the detection sensitivity for Mb-δ is essentially flat across the wall of the left ventricle. Therefore, the Mb-δ resonance reflects average whole wall Mb-δ without the need for correction for differences in sensitivity in the deeper myocardial layers as is the case for HEP measurements (see below).The method for 31P MR spectra were acquired in late diastole with a pulse repetition time of 6–7 seconds. This repetition time allowed full relaxation for ATP and Pi resonances, and approximately 90% relaxation for the PCr resonance. PCr resonance intensities were corrected for this minor saturation. RF transmission and signal detection were performed with a 28 mm diameter surface coil. A capillary containing 15 µl of 3M phosphonoacetic acid was placed at the coil center to serve as a reference. The proton signal of the water resonance was used to homogenize the magnetic field and to adjust the position of the animal in the magnet so that the coil was at or near the magnet and gradient isocenter. This was accomplished using a spin-echo experiment with a readout profile. The information gathered in this step was also utilized to determine the spatial coordinates for spectroscopic localization. Chemical shifts were measured relative to PCr which was assigned a chemical shift of −2.55 ppm relative to 85% creatine phosphate at 0 ppm. Spatial localization across the left ventricular wall was performed with the RAPP-ISIS/FSW method. The technical details of this method including voxel profiles, voxel volume, and the accuracy of spatial localization obtained in phantom studies and in vivo have been published elsewhere 0 gradients and adiabatic inversion pulses to an 18 mm×18 mm column coaxial with the surface coil and perpendicular to the left ventricular wall. Within this volume, the signal was further localized using the B1 gradient to 5 voxels spanning the left ventricular wall from epicardium to endocardium. Each set of spatially localized transmural spectra consisted of a total of 96 scans accumulated in a 10 minute block. Resonance intensities were quantified using integration routines provided by SISCO software. The values for PCr and ATP in each voxel were normalized to those present in the basal state and the PCr/ATP ratio was determined for each voxel. Pi resonances were also measured and ratio of Pi/PCr was calculated.51Cr, 85Sr, 95Nb and 46Sc). Microsphere suspension containing 2×106 microspheres was injected through the left atrial catheter while a reference sample of arterial blood was withdrawn from the aortic catheter at a rate 15 ml/min beginning 5 seconds before the microsphere injection and continuing for 120 seconds. Radioactivity in the myocardial and blood reference specimens was determined using a gamma spectrometer  at window settings chosen for the combination of radioisotopes used during the study. Activity corrected for overlap between isotopes and for background was used to compute blood flow as ml/g of myocardium/minute.Myocardial blood flow was measured using radionuclide labeled microspheres, 15 µm in diameter labeled with 4 different radioisotopes  2 during each experimental period can be calculated from the relationship Mb-O2  = 1 - Mb-δ. The fractional contents of Mb-O2 and Mb-δ and the temperature corrected [PO2]50 value can then be employed to calculate intracellular PO2 using equation 1. Because of continuing collateral blood flow we assumed that the Mb-δ resonance measured during total coronary occlusion represented 95% of the total myoglobin, and normalized the other Mb-δ resonances in relation to that value. We assumed that the fractional Mb-O2 content in the basal state  was ∼90%. This also follows from calculations using equation 1. At 37°C, calculated intramyocyte PO2 would be 21 and 46 mm Hg for Mb-O2 saturations of 90% and 95%, respectively. Hence, we assumed that a fractional Mb-O2 content of ∼90% was present because this value is most compatible with the presence of an oxygen gradient between the capillary and intracellular myoglobin and reported values for coronary venous PO2In equation 1, Mb-O31P and 1H MRS spectra were first obtained under basal conditions.Aortic and left ventricular pressures were measured with fluid filled pressure transducers positioned at mid-chest level and recorded on an 8-channel direct writing recorder . Left ventricular pressure was recorded at normal and high gain for measurement of end-diastolic pressure. Hemodynamic measurements and In protocol one, of 6 dogs, baseline measurements were acquired at a FIO2 of 40% which leads to an arterial oxygen saturation of approximately 100%. This was followed by a two minute LAD occlusion. In the meantime the deoxymyoglobin MRS data were obtained for pO2 calculation. After the total occlusion Mb-δ data were acquired, FIO2 was progressively decreased to 21, 15, 10 and 5% respectively and interleaved P-31 and H-1 spectra obtained during each experimental condition every 4 minutes.in protocol 2, 10 dogs (after baseline data were obtained) were exposed to 3% FIO2 and LAD occlusion, respectively. The P-31 and H1 MRS data were obtained similarly.To examine if more severe hypoxia would produce an association between myoglobin desaturation and reduction of myocardial energetic state and alterations in OXPHOS, 2 would continue to decrease during prolonged hypoxia and whether this would be accompanied by a decrease in myocardial oxygenation level and PCr/ATP, in protocol 3, 9 dogs (after baseline data were obtained) were exposed to prolonged 18 minutes 5% FIO2, with P-31 and H1- MRS, myocardial blood flow and venous O2 measured every 4 minutes. Finally, the LAD was completely occluded  and all measurements were repeated.To examine whether the venous O31P spectra were analyzed as described above. Transmural blood flow distribution was determined from the microsphere measurements. Data were analyzed with one-way analysis of variance for repeated measures. A value of p<0.05 was considered significant. When a significant result was found, individual comparisons were made using the method of Sheffé.Hemodynamic data were measured from the chart recordings. The hemodynamic, myocardial blood flow, arterial and coronary vein blood gas and myocardial oxygen consumption, myocardial HEP and oxygenation data are summarized in 2 when mean aortic pressure and LV systolic pressure increased with subsequent increase in RPP [2, MVO2 actually increased [2 . However, myocardial HEP remained relatively stable [Hemodynamic measurements during each experimental condition are shown in e in RPP . Even thncreased  and thisy stable . Mb-δ rey stable .2. The mean aortic pressure and LV systolic pressure increased substantially resulting in a significant increase in rate pressure product [2 increased [2 decreased to 12±0.9, which is significantly lower than the myocardial ischemia induced increase of oxygen extraction. The deoxymyoglobin reached approximately 50% of maximum deoxymyoglobin level [To examine whether more severe hypoxia would produce an association between myoglobin de-saturation and reduction of myocardial HEP, 10 dogs were exposed to 3% FIO product . MVO2 inncreased  again asncreased . Coronarin level .2 would result in continuous reduction of coronary venous pO2 and therefore detectable Mb-δ. Prolonged 5% FIO2 caused a significant increase in LV systolic pressure and subsequent RPP. MVO2 increased as a result of increase in MBF but there was a significant decrease in HEP which was associated with an increase in deoxymyoglobin after 8 minutes exposure. It was evident that when the 5% FIO2 was maintained the venous pO2 continued to decrease. Once it was below 14 mm Hg, the Mb-δ became detectable.Protocol 3 was performed to examine whether prolonged 5% FIO2 beyond 5% or prolonged exposure to low FIO2 was associated with a decrease in PCr/ATP and increase in Pi/PCr which was associated with a decrease in intracellular PO2. The relationships of PCr and ATP with intracellular PO2 are shown in 2 content in the basal state was 90%. Values for PCr decreased when intracellular PO2 approached 10 mmHg. PO2 values that are associated with significant reductions of HEP have been termed “critical” with the implication that they reflect the presence of O2 limitation of oxidative phosphorylation. Based on 2 value for 20% and 50% reduction of PCr were approximately 7.4 and 0.6 mmHg respectively. Similarly, the (PO2)80 for ATP was approximately 1.9 mmHg. A (PO2)50 value for ATP could not be determined because this degree of ATP reduction was not achieved. The lower critical PO2 for ATP than for PCr is not surprising because the fall of ATP is buffered by PCr through the creatine kinase reaction.Decreasing FIO1H- Mb-d and 31P- MR spectroscopic measurements can detect the signals of reduction of oxygen delivery to the mitochondria. Prior studies did not measure Mb-d, therefore, no evidence of decrease of oxygen delivery to the mitochondria under these conditions. The findings from the present study demonstrate that the in vivo compensate mechanisms can maintain the normal mtOXPHOS even during extreme low FIO2. The present study has further elucidated the response of the myocardial oxidative phorsphorylation (OXPHOS) regulation to hypoxia. Firstly, it has reiterated the hemodynamic response of the in vivo heart to graded hypoxia as a result of a decrease in the fractional inspired oxygen concentration. Secondly, it has depicted the increase in transmural blood flow during hypoxia, which helps in maintaining a normal mitochondrial OXPHOS. Thirdly, it has defined the relationships between myocyte PO2 and HEP levels in vivo myocardium under conditions where myocardial flow is not limited.The present study examined whether mitochondria lack oxygen under the extreme low FIO2 conditions, and if so, how long it takes the compensation systems to fail. We examined whether the During myocardial hypoxia, the lactate production and glycolysis pathway of ATP production are increased 2 up to 10% [2 caused no changes in systemic hemodynamics up to 6% Our study shows that the mean arterial pressure, heart rate, left ventricular systolic and end diastolic pressure and rate pressure product are maintained with graded reductions in FIOp to 10% . With fup to 10% . These c2 [2 caused a decrease in myocardial blood flow but it was still higher than baseline. This increased myocardial blood flow caused myocardial oxygen consumption to be maintained throughout hypoxia, as opposed to LAD occlusion [The graded hypoxia caused a proportional increase in myocardial blood flow up to 5% FIO2 . This wa2 . Howevercclusion .2). The effect of hypoxia on myocardial HEP has been studied in both of the above mentioned conditions in isolated working rat hearts perfused with Krebs-Henseleit buffer at 25°C 2 below 1.5 mmHg, with (PO2)80 of 1.1 mmHg and (PO2)50 of 0.5 mmHg 2 while coronary flow rate was maintained constant, and demonstrated that PCr began to decrease at an intracellular PO2 of 2 mmHg, with a (PO2)80 of 1.8 mmHg 2)80 of 4.3–4.5 and (PO2)50 of 1.0–1.2 mmHg 2 for PCr in the perfused heart. As the critical PO2 in the perfused rat hearts is higher in the graded perfused PO2 setting as compared to graded coronary flow setting, we hypothesized that we may find a similar trend in the in vivo setting. In the present study, we examined the critical PO2 in the vivo setting with graded hypoxia and found that the (PO2)80 is 7.4 mmHg, which is significantly higher than the hypoperfusion setting.Under normal baseline conditions, the myocardium has adequate supply of oxygen for oxidative phosphorylation and oxygen does not seem to play a regulatory role. Even at high cardiac work states, no deoxymyoglobin signal is detected despite a decrease in high energy phosphates 2 required for decrease in HEP in the present study suggests that there were other factors which are affecting the intracellular PO2 in the graded coronary artery stenoses setting. Indeed, the response of myocardial HEP levels to decreased coronary perfusion can be affected by changes in myocardial oxygen demand. Gregg initially described that myocardial oxygen consumption can be influenced by coronary flow and perfusion pressures. Several mechanisms have been proposed to modulate oxygen demand during hypoperfusion. The pressure in the coronary system may distend the ventricles, leading to increased contractility and oxygen consumption by an increase in sarcomere length or ventricular stiffness et al showed that in the range of coronary pressure of 60–160 mmHg, decreases in coronary perfusion pressure in isolated ferret hearts resulted in preload independent decrease of the calcium transient which they hypothesized represented a protective mechanism that minimizes energy demands during low flow ischemia 2 levels. Thus, the true critical PO2 level that leads to a reduction of HEP can be better estimated with graded hypoxia through decrease in FIO2 such as in the present experimental setting. This reveals a (PO2)80 of 7.4 mmHg for PCr and a (PO2)80 of 1.9 mmHg for ATP.The higher PO2 that far exceeds the apparent Michaelis-Menten constant of cytochrome oxidase with respect to O2. It was shown that the critical PO2 at which oxidative phosphorylation began to decrease in isolated mitochondria was approximately 0.01 mmHg, which is more than 100 fold lower than the PO2 at which HEP levels started to decrease in the present study. This could be attributed to inhomogeneities of O2 availability at the cellular level, if the PO2 gradient between the cytosol and the inner mitochondrial membrane was significant. However, mathematical models of O2 diffusion suggest very low perimitochondrial O2 gradient 2 gradient near the sarcolemma, while PO2 values toward the center of the cell are nearly flat et al postulated that since the mitochondrial membrane is impermeable to myoglobin, a significant PO2 gradient can occur because oxygen must diffuse through a myoglobin free volume before it can react with cytochrome oxidase et al have recently shown that in the myocardium, Mb-facilitated O2 diffusion contributes increasingly more than free O2 diffusion when the PO2 falls below 1.77 mmHg 2 is around 10 mmHg, myoglobin facilitated oxygen diffusion does not play a major role but under hypoxic conditions such as in the present study it plays a significant role in oxygen diffusion.Another important question to address is why any oxygen limitation occurs at an intramyocyte PO2, significant loss of PCr occurred at an intracellular PO2 of 7.4 mmHg. Thus, in normal in vivo heart, oxygen availability plays an important role in regulation of oxidative phosphorylation only when mean intracellular PO2 falls below 7.4 mmHg.In conclusion, during graded hypoxia caused by decreased FIO


Blastocystis, is one of the most common human intestinal protozoan, which has many conflicting reports on its pathogenic role. Gut conditions which obviously varies in asymptomatic individuals, symptomatic and irritable bowel syndrome (IBS) patients in terms of gut flora, pH, osmotic pressure and water potentials could play an important role in its pathogenicity. The present study is the first study to investigate phenotypic characteristics of Blastocystis sp. ST3 isolated from asymptomatic, symptomatic and IBS isolates.Blastocystis isolates were obtained from four IBS patients (IBS1-4) and four symptomatic patients (S1-4) at a local gastroenterology clinic. Asymptomatic isolates (A1-4) were obtained from a field survey at a local village.A total of 8 Blastocystis showed a very coarse and intensely folded surface. The IBS isolates also exhibited a dense material and a thicker layer of surface coat can be seen compared to asymptomatic and symptomatic isolates.All 12 isolates were determined as subtype 3 (ST3). A1-4 isolates showed the highest peak growth followed by IBS1-4 isolates and S1-4 isolates for the growth profile. Parasites from IBS isolates (IBS1-4) showed the largest diameter with a mean 18.43 ± 2.22 μm compared to parasites of symptomatic isolates (isolates S1-4) 15.54 ± 3.02 μm and asymptomatic isolates (isolates A1-4) 11.76 ± 0.82 μm. The symptomatic isolates (average generation time: 9.87 ± 2.97 h) grew faster than the IBS isolates (average generation time: 7.56 ± 1.06 h) and asymptomatic isolates (average generation time: 5.97 ± 1.52 h). The parasites isolated from IBS isolates showed strong aggregation and clumping, which had seen reduced in parasites of isolates S1-4. No clumping was seen in parasites from A1-4. The outer surface of parasites in IBS isolates showed greater binding affinities towards FITC-labelled Concanavalin A (Con A) than symptomatic isolates and asymptomatic isolates. Scanning electron microscopy showed that in IBS isolates, the surface of .There have been no studies thus far providing evidence for phenotypic variation within a particular subtype. The present study is the first to demonstrate the phenomenon of gut environment facilitating adaptation of parasites possibly for survival leading to phenotypic differences for Blastocystis Blastocystis, the most common human intestinal protozoan 32] and tBlastocystis to be the influencing factors for the parasite’s pathogenicity especially subtype 3 where in Malaysia [Blastocystis ST3 was also found to be the only subtype in 100% of patients suffering from urticaria in Egypt [Blastocystis.Studies have implicated that genotypes of Malaysia  SingaporMalaysia  and USA Malaysia  evidencein Egypt  Blasto-Ain Egypt . Tan et in Egypt  also demThe present study however is the first to demonstrate phenotypic variation within a subtype 3 population from three environments namely asymptomatic, IBS and symptomatic. Hence environment adaptability for survival purposes could be a possible reason for the phenotypic variation to exist.Blastocystis towards the intestinal lining of the gut. Blastocystis lysate and live parasite have been shown to trigger cytopathic effect on Chinese Hamster Ovary cells [The thicker surface coat shown by the ultrastructural study in IBS isolates could influence cytopathic effect of ry cells , 42. Thery cells  as well Blastocystis infected IBS patients [In IBS condition inflammation of intestinal layer could cause leaky gut syndrome which facilitates undigested food substances to pass between the cells of the intestinal epithelial layer to the bloodstream causing damage on the intestinal wall  which inpatients .The present finding has a very important implication. Previously all evidences have been pointing to subtype 3 to be the pathogenic one. However the present study cautions on forming such a conclusion and suggest that ascribing subtype to pathogenicity could be an over generalization. It is evident that gut environment can influence phenotypic expression of even the same subtype. A similar study had been done on assessing the phenotypic characterization of entamoeba hystolytica whereby the interaction between the parasites and host component such as bacterial flora and mucins can trigger the parasites to be more pathogenic by exhibiting their virulences factor .It is beyond the scope of the present study to postulate the details of the gut environment although others have suggested that an unhealthy gut has a complex open ended ecosystem which can be a host for various microorganisms –51. ThesBlastocystis.There have been no studies thus far providing evidence for phenotypic variation within a particular subtype. The present study is the first to demonstrate the phenomenon of gut environment facilitating adaptation of parasites possibly for survival leading to phenotypic differences for


Please see the corrected Figure S2 here.There is an error in the title for Figure S2. The complete, correct Figure S2 title is: “Relationship between the diet diversity (H’) and the prey consumption specificity  and the prey consumption specificity (PSi) at the territory level (n  =  71).(DOC)Click here for additional data file.Table S2Explanatory variables used in the GLMMs to assess their potential effect on Bonelli’s eagle productivity, classified either as spatiotemporal parameters, breeding pair parameters or diet parameters.(DOC)Click here for additional data file.Table S3Summary of model parameter estimates and standard error of parameter estimates for each model included in the GLMMs. Models are showed following the same order as in Table 3.(DOC)Click here for additional data file.


SLD5 is a member of the GINS complex composed of PSF1, PSF2, PSF3 and SLD5, playing a critical role in the formation of the DNA replication fork with CDC45 in yeast. Previously, we had isolated a PSF1 orthologue from a murine hematopoietic stem cell DNA library and were then able to identify orthologues of all the other GINS members by the yeast two hybrid approach using PSF1 as the bait. These GINS orthologues may also function in DNA replication in mammalian cells because they form tetrameric complexes as observed in yeast, and gene deletion mutants of both PSF1 and SLD5 result in a lack of epiblast proliferation and early embryonic lethality. However, we found that PSF1 is also involved in chromosomal segregation in M phase, consistent with recent suggestions that homologues of genes associated with DNA replication in lower organisms also regulate cellular events other than DNA replication in mammalian cells. Here we analyzed the function of SLD5 other than DNA replication and found that it is active in DNA damage and repair. Attenuation of SLD5 expression results in marked DNA damage in both normal cells and cancer cells, suggesting that it protects against DNA damage. Attenuation of SLD5 delays the DNA repair response and cell cycle restoration in normal cells but not in cancer cells. These findings suggest that SLD5 might represent a therapeutic target molecule acting at the level of tumor stromal cells rather than the cancerous cells themselves, because development of the tumor microenvironment could be delayed or disrupted by the suppression of its expression in the normal cell types within the tumor. Cells are constantly exposed to genomic DNA damage caused by internal and external agents such as oxidative stress and UV, respectively. Errors in DNA damage repair can result in cancer cell development SLD5 is a member of the GINS complex composed of PSF1, PSF2, and PSF3. This complex regulates the DNA replication fork in budding yeast We identified a mouse orthologue of PSF1 in a DNA library derived from hematopoietic stem cells during embryogenesis in which this cell population actively proliferates +/−mice, which were healthy and fertile, were born at Mendelian frequency and exhibited normal growth. Moreover, there is no large difference of bone marrow cellularity between wild and SLD5+/− mice +/− mouse embryonic fibroblasts (MEFs) to analyze DNA damage repair and cell growth after DNA damage. Moreover, we compared the function of SLD5 in DNA damage repair using siRNA knock-down experiments in cancer cells.High expression of GINS genes has been observed in cancers and a correlation of their level of expression with malignancy has been suggested 2. MEFs were prepared from wild-type (WT) or SLD5+/− mice at embryonic day (E) 15.5 according to the usual method MEFs, B16 cells (mouse melanoma cells), and colon26 cells (mouse colon cancer cells) were grown in Dulbecco's modified Eagle's medium (DMEM) (Sigma) with 10% fetal bovine serum , and penicillin/streptomycin (Sigma) at 37°C under an atmosphere of 5% COSLD5 expression in B16 and Colon26 cells was transiently knocked down with small interfering RNA (siRNA). Lipofectamine RNAiMAX (Invitrogen) was used for the transfection of plasmid and siRNA into cells, following the manufactureŕs protocols, and experiments were done 48 h after transfection. We used two different siRNA oligonucleotides specific for SLD5; target siRNA sequences were: 5′-GGA CCA CAC GGA GAC CCA CUU UAA A-3′ (#1); 5′-GAU GAG CAG AGA GAC UAC GUG AUU G-3′ (#2).3 cells were seeded into each well of a 24-well culture plate  in 500 µl of medium. After 24 h, the wells were exposed to 1 µM etoposide for 12 h. After the drug was removed, cells were harvested immediately (0 h). Cell viability was evaluated by trypan blue exclusion. The same number of living cells was resuspended in fresh medium, and cells were cultured for 24, 48 or 72 h. They were then detached by adding 100 µl Trypsin-EDTA to each well; cells were then washed and re-suspended in 400 µl of medium. 20 µl of re-suspended cells were mixed with 20 µl of 0.4% solution of trypan blue dye (Life Technologies) for 1 min. Cells were immediately counted using a Neubauer microchamber with a light microscope. All counts were done using four technical duplicates of each sample. Means and standard deviations were calculated for each subculture.5×10s (Amersham). The immunoreactive proteins were visualized using the ECL Prime Western Blotting Detection system . The blots were scanned using the imaging densitometer Las-300 mini .Cells were collected and lysed in SDS sample buffer  containing a cocktail of protease inhibitors. Proteins were separated on 10% or 15% SDS-PAGE and transferred onto PVDF membranes. After blocking for 1 h in TBST  containing 2% non-fat dry milk, membranes were incubated with anti-SLD5 , anti-γ-H2AX , anti-Rad51 , or mouse anti-β-actin antibody in blocking buffer overnight. Membranes were then washed with TBST and incubated with HRP-conjugated anti-rat, rabbit or goat antibodies (1∶10000) for 1 h. Bound antibodies were detected with ECL kitt test was used for statistical analysis. Differences were considered statistically significant when p<0.05.Results are expressed as the mean ± SD. Student's +/− mice +/− MEFs was approximately half of that in WT MEFs  directed against SLD5 (siRNA B16 and siRNA Colon26). We confirmed that SLD5 expression could be efficiently silenced by specific siRNA in B16 and Colon26 cells but not by scrambled control siRNA (SCR B16 and SCR Colon26) (We predicted that attenuation of SLD5 expression would delay DNA repair and cell cycle restoration in cancer cells as observed in normal cells. However, although the level of Rad51 expression was the same in SCR B16 and SLD5 siRNA B16 cells, and in SCR Colon26 and SLD5 siRNA Colon26 cells before treatment with etoposide, thereafter, a rapid increase of Rad51 was similarly observed in all four cell lines (To assess how the rapid response to DNA damage in SLD5 knocked-down cancer cells affects cell cycle restoration, we enumerated the cells after treatment with etoposide. Cell proliferation itself was not affected by knocking down SLD5 in either siRNA B16 or siRNA Colon26 cells in the presence of control DMSO treatment (It has been reported that SLD5 forms a GINS complex with other moieties such as PSF1, PSF2, and PSF3 and is involved in DNA replication in yeast As described above, the GINS complex is involved in DNA replication; however, GINS components PSF1, PSF2, PSF3, and SLD5 do not always form complexes and may have other functions. For instance, PSF1 regulates microtubule organization in M phase and is involved in chromosome segregation +/− MEFs compared with WT MEFs for repairing heavily damaged DNA. However, SLD5 attenuation was found to delay Rad51 expression in MEFs, resulting in severe retardation of cell cycle restoration. These findings suggested that SLD5 not only protects against DNA damage but regulates the rapidity of DNA repair. Recently, it has been reported that PSF2, also a member of the GINS complex, is phosphorylated by ATM upon DNA strand breakage DrosophilaIt was to be expected that a longer period of time would be required for DNA repair in cells with worse DNA damage as a result of lack of SLD5. We hypothesized that rapid Rad51 expression would be induced in SLD5Recent studies have shown that tumor growth and metastasis are not determined by cancer cells alone but also by various stromal cells. The stroma constitutes a large part of most solid tumors, and the cancer-stromal cell interaction contributes functionally to tumor growth and metastasis


Bacillus thuringiensis is a potent microbial control agent against insect pests. Here, we present the draft genome of the Egyptian strain Btm27 that shows high toxicity toward the cotton leafworm. The genome contains three insecticidal genes cry1Ac9, cry2Ab1, and vip3V that have been implicated in conferring toxicity toward lepidoptera. Bacillus thuringiensis has been successfully used as a biopesticide to control many agricultural pests and insect vectors of human disease  collection as 4AC2.This genome sequence has been deposited in GenBank under the accession no.


There is growing recognition that epigenetic regulation contributes significantly to the fidelity of biological control. Compromised epigenetic pathways have now been shown to provide indications of transformation and tumor progression; they suggest potential targets for cancer prevention and intervention . A formiPost translational modifications of histone3 lysine4 (H3K4) may constitute epigenetically mediated regulatory signatures that can be informative for the molecular characterization of breast cancer pathology. Both methylation and acetylation of H3K4 are associated with gene activation. Tri-methylation of histone3 lysine4 has been extensively studied and is a well-documented marker for active or poised gene transcription. The enzyme complexes that regulate H3K4me3 have been implicated in tumorigenesis, pointing to the relevance of this epigenetic modification in cancer. Histone acetylation as well as histone acetyltransferases and deacetylating enzymes are recognized as important in tumor progression. However, the acetylation of H3K4 in particular has been less extensively investigated to date.Messier, et al., addressed the coordinated contributions of H3K4me3 and H3K4ac to breast cancer tumorigenesis in three well-established human mammary cell lines that represent a normal-like subtype  and two cancer subtypes, luminal  and basal-like metastatic  [In contrast to methylation and acetylation of H3K9 and H3K27, which activate or repress transcription respectively, methylation and acetylation of H3K4 are both associated with gene activation. Observed increases in H3K4 acetylation marks at specific gene promoters in both the MCF7 and MDA-MB-231 cell lines, compared to normal-like MCF10A cells, suggests that H3K4 acetylation may be an early step in cancer initiation or progression. The global increase in H3K4me3 at gene promoters in MDA-MB-231 cells suggests alterations in the epigenetic landscape that may be functionally related to late stage, metastatic breast cancer.From a mechanistic perspective, the presence of H3K4ac and H3K4me3 at specific gene promoters provides a blueprint for regulatory pathways that are functionally linked to epigenetic control of breast cancer onset and progression. The dynamic acetylation of H3K4 is associated with estrogen-receptor responsiveness in the MCF7 cells and the initial loss of mammary tissue–specific gene expression in MCF7 cells that represent early stage breast cancer. Enhancing the pivotal role of H3K4ac in initial stages of breast cancer tumorigenesis, this epigenetic mark appears to poise genes for expression that leverages continued acquisition and persistence of metastatic properties. In contrast, H3K4me3, with persistence of H3K4ac reflects the epithelial to mesenchymal transition in MDA-MB-231 cells that represent late stage breast cancer. Important mechanistic linkages between histone H3K4ac and H3K4me3 modifications and breast cancer tumorigenesis remain to be established.While H3K4ac is emerging as an informative predictor of pathways that are deregulated during the onset and progression of breast cancer, the full potential for exploitation of this epigenetic modification as a biomarker and therapeutic target remains to be explored. Future studies must further define the enzymes that support acetylation and deacetylation of H3K4. Furthermore, the dynamic balance between H3K4 acetylation and methylation must be elucidated within the context of the full spectrum of histone modifications that contribute to combinatorial control of the epigenetic landscape. This will provide a drastically improved understanding of how epigenetic modifications and chromatin organization regulate mammary epithelial phenotype and gene expression related to transformation and tumor progression—the complexity of chromatin organization that supports breast cancer initiation and progression is increasingly evident. Consequently, the clinical value of epigenetic biomarkers and related therapeutic targets can be expanded by detailed characterization of the regulatory signals responsible for the deposition of specific “histone marks”. Adding to the biological challenge inherent in these analyses, the plasticity observed in post translational histone modifications must also be considered. It will be important to establish the relationships between histone modifications and other parameters of epigenetic control and chromatin organization in breast cancer that include perturbations in inter- and intra-chromosomal interactions -8. Altho


BRCA1 and BRCA2 mutation carriers. We conducted fine-scale mapping at 9p22.2 to identify potential causal variants in BRCA1 and BRCA2 mutation carriers. Genotype data were available for 15,252  BRCA1 and 8,211 (631 ovarian cancer cases) BRCA2 mutation carriers. Following genotype imputation, ovarian cancer associations were assessed for 4,873 and 5,020 SNPs in BRCA1 and BRCA 2 mutation carriers respectively, within a retrospective cohort analytical framework. In BRCA1 mutation carriers one set of eight correlated candidate causal variants for ovarian cancer risk modification was identified . These variants were located up to 20 kb upstream of BNC2. In BRCA2 mutation carriers one region, up to 45 kb upstream of BNC2, and containing 100 correlated SNPs was identified as candidate causal . The candidate causal in BRCA1 mutation carriers did not include the strongest associated variant at this locus in the general population. In sum, we identified a set of candidate causal variants in a region that encompasses the BNC2 transcription start site. The ovarian cancer association at 9p22.2 may be mediated by different variants in BRCA1 mutation carriers and in the general population. Thus, potentially different mechanisms may underlie ovarian cancer risk for mutation carriers and the general population.Population-based genome wide association studies have identified a locus at 9p22.2 associated with ovarian cancer risk, which also modifies ovarian cancer risk in  BRCA1 and BRCA2, which account for approximately 24% of the familial risk among first-degree relatives [BRCA1 mutation have a lifetime risk of 35–60% with an average age of diagnosis of 50 years [BRCA2, with a lifetime risk of 12–25% and an average age of diagnosis of 60 years [BRCA1/2 associated ovarian cancers present as high-grade serous histology in advanced stage [Once age is taken into account, family history is the strongest risk factor for ovarian cancer. Women with a first-degree relative with ovarian cancer are at a 3-fold increased risk of developing the disease, indicating the importance of genetic factors in ovarian cancer predisposition. The most important genes in the context of genetic counseling for ovarian cancer susceptibility are BRCA1 and BRCA2 mutation carriers [BRCA1 and BRCA2 mutation carriers [Basonuclin 2(BNC2) and Centlein (CNTLN). BNC2 is a zinc-finger protein spanning nucleotides 16409503 to 16870706. It is expressed in ovary, testis and the male germ line where it regulates cell cycle progression [CNTLN spans nucleotides 17134982 to 17503923, it is ubiquitously expressed and localises at centrosomes to ensure centrosome function during cell division [Genome wide association studies have identified several common germline variants associated with ovarian cancer risk. The 9p22 locus was first found to be associated with ovarian cancer risk in the general population, and subsequently to be an ovarian cancer risk modifier in carriers ,5. The Scarriers . rs38141BRCA1 and 8211 BRCA2 mutation carriers of European ancestry. We comprehensively characterized the associations of genetic variants in the region with ovarian cancer risk for BRCA1 and BRCA2 mutation carriers.Here, we report the fine-scale mapping of the 9p22.2 locus using data from 15252 BRCA1 and BRCA2 mutation carriers participating in the Consortium of Investigators of Modifiers of BRCA1/2 . Eligib2>0.1 with the SNP that had been identified through the GWAS (rs3814113), and the set of SNPs that tagged all remaining SNPs in the region with r2>0.9. A total of 407 and 401 SNPs that were included on iCOGS in the 9p22.2 region passed QC and were available for the analyses for BRCA1 and BRCA2 mutation carriers, respectively. Imputation of genotypes was based on the phase 3 release of the 1000 Genome Project spanning nucleotides 16407967 to 17407967 (build 37) at chromosome 9 with a buffer region of 500bp, using IMPUTE2 v2 [Genotyping was performed using the iCOGS Illumina array . The quaS rs38141, and theBRCA1 and BRCA2 mutation carriers and all analyses were stratified by country of residence and year of birth. The USA and Canada strata were further subdivided by reported Ashkenazi Jewish ancestry.The primary analysis evaluated the association between each variant and ovarian cancer risk. To account for the non-random sampling of mutation carriers with respect to disease status, the analysis was conducted within a retrospective cohort framework by modeling the likelihood of the observed genotypes conditional on the disease phenotypes as previously described . Each muBRCA1 and BRCA2 mutation carriers. A fixed effect meta-analysis weighted by the inverse variance was conducted for imputed and genotyped SNPs when risk estimates were available in both datasets. For BRCA1 and BRCA2 mutation carriers, logarithms of per-allele HR estimates were used. The Cochran Q test was carried out to assess heterogeneity.Ovarian cancer associations were combined in a meta-analysis between BRCA1 and BRCA2 mutation carriers, each SNP was included in a Cox-regression model conditioned on the most strongly associated variant for each dataset and further adjusting by year of birth, and stratifying by country of residence. This approach has been shown to yield valid tests of association [-8. The most parsimonious model in the conditional analyses was identified using a threshold of p<10−4 for retaining SNPs in the model.To assess the number of variants independently associated with ovarian cancer risk in ociation . All SNPr2) with the top SNP higher than 0.1 [The set of potential causal SNPs was defined by those SNPs for which their likelihood ratio relative to the most significant variant was equal or less than 100 and having a pair-wise correlation .BEDTools was used to intersect positions of ovarian cancer risk-associated variants with functional genomic features generated by Coetzee et al  includinEach of the host institutions recruited under ethically approved protocols. A list of the local Institutional Review Boards that provided ethical approval for this study is given in BRCA1 mutation carriers of whom 2,462 were censored at ovarian cancer diagnosis . The correlation between the top SNP and the rs3814113 was 0.56 , given the strong prior evidence of association between SNPs in the region and risk for BRCA1 carriers and in the general population we selected the most significant SNPs as associated with ovarian cancer risk. Results for all SNPs with p<0.01 are presented in A total of 8,211 iagnosis . The assiagnosis . SNP rs3BRCA1 and BRCA2 ovarian cancer associated cancer tumors are high-grade serous . In the meta-analysis, the strongest associated variant was the genotyped SNP rs7046326 with a MAF of 0.25 and 0.24 in BRCA1 and BRCA2 mutation carriers, respectively. It displayed an HR = 0.74 .Since the majority of both  serous , referred hereafter as the "BRCA1 peak". These variants clustered in a 20kb region around the transcription start site of BNC2 . The SNPs in this set displayed MAFs of 0.24–0.28 and imputation accuracy higher than 0.95 and two out of the eight were genotyped  in BRCA2 mutation carriers r2 = 0.76, All except one (imputed SNP rs139555631) of the likely causal variants in BRCA2 mutation carriers, but was rejected from being potentially causal in BRCA1 mutation carriers.The original GWAS hit, rs3814113, was within the set of the strongest associated SNPs in BRCA1/2 meta-analysis, eleven SNPs were the set of potentially causal variants, which included the eight identified in BRCA1 plus three only present in the BRCA2 set. These eleven variants were highly correlated with the lead SNP of the meta-analysis rs7046326 (r2>0.8). Of note, the set excluded the original GWAS hit rs3814113  for the BNC2 gene in whole blood samples .Intersection of variants exhibiting the strongest associations with genomic features derived from cultured ovarian and fallopian tube cells revealed several SNPs that may be functionally relevant in influencing risk. variants . Althouganalysis , four ofBRCA1 and BRCA2 mutation carriers of European ancestry. We identified a set of variants that provided stronger evidence of association than the original GWAS hit.In this study, we performed fine-scale mapping of the 9p22.2 locus using dense genotype data from the iCOGS array in BRCA1 mutation carriers, one independent set of eight highly correlated (r2>0.8) SNPs could not be excluded as being potentially causal for the reported association with ovarian cancer, designated the "BRCA1 peak". The BRCA1 peak covers positions 16847520 to 16891647, which lie within or up to 20 kb upstream BNC2. Of note, the original GWAS hit rs3814113 was excluded from the candidate causal variants in this peak.In BRCA2 mutation carriers, 100 correlated variants (r2>0.4) could not be excluded as potentially causal ("BRCA2 peak"). The BRCA2 peak spanned positions 16847520 to 16915021, which are up to 44 kb upstream of BNC2 and more than 200kb upstream of CNTLN. The increased number of variants in this case is most likely due to reduced statistical power, as the number of BRCA2 mutation carriers diagnosed with ovarian cancer was only one quarter of the number of affected BRCA1 carriers. The candidate causal SNPs in the BRCA1 peak were mostly contained within the BRCA2 peak but the strongest associated SNP in BRCA2 was excluded from the BRCA1 peak. The current analysis was underpowered to investigate whether the association in BRCA2 mutation carriers is driven by a different set of genetic variants.For BRCA1 and BRCA2 mutation carriers, the meta-analysis would be expected to increase power for refining the set of potential causal variants. However, the combined analysis of BRCA1 and BRCA2 mutation carriers defined a set of eleven variants as potentially causal, which corresponded to the eleven strongest associated variants in BRCA1. This set excluded rs3814113 that was reported in the ovarian cancer GWAS [BRCA1 carriers only.Under the model of one shared causal variant explaining the association in both cer GWAS . The setBRCA1 mutation carriers and results for the most strongly associated SNPs in samples from the general population.Important differences emerged when we compared the patterns of association in the fine-scale mapping of 9p22.2 between BRCA1 mutation carriers, suggesting that the associations in BRCA1 mutation carriers and in the general population may be driven by different causal variants at the 9p22.2 locus. These results may indicate differences in the underlying causal mechanisms explaining the ovarian cancer associations between BRCA1 mutation carriers and the general population. In support of this possibility, differences in the association patterns with ovarian cancer between BRCA1 and the general population have been reported before. The 4q32.3 locus is associated with ovarian cancer risk in BRCA1 but not in BRCA2 mutation carriers or the general population [Fine-mapping results based on iCOGS data from the Ovarian Cancer Association Consortium indicate that SNP rs3814113 remains the most strongly associated SNP at the 9p22.2 region with serous ovarian cancer, the original GWAS hit . Based on our results, this SNP can be confidently rejected from the set of possible causal variants in pulation . HoweverBNC2 gene, and some candidate causal SNPs are eSNPs for BNC2 in whole blood, they may modulate the expression of BNC2 through similar, or different, mechanisms. The possibility that the BRCA1 association signal may differ from that in the general population adds extra complexity and reinforces the value of fine-scale mapping in different populations. These subtle differences in the patterns of associations depending on the underlying genetic landscape may be difficult to uncover by means other than fine-scale mapping, and thus strengthens the value of this approach for generating hypotheses about the functional basis of different sets of variants.As both signals lie in close proximity to the BRCA1 or BRCA2 mutations have shown that BRCA1 and BRCA2 carriers predominantly develop serous disease [BRCA1and BRCA2 mutation carriers.This study cannot exclude the possibility that the actual causal variants were not included in the set of genotyped or well-imputed variants. However, the iCOGs array included variants specifically for fine-scale mapping of the 9p22.2 locus based on data from the 1000 Genomes Project and therefore the region coverage is expected to be high. The relatively low number of ovarian cancer cases with tumor morphology information did not allow performing stratified analyses by ovarian cancer histological subtype. Studies of ovarian tumours in women with  disease ,22. Of tBRCA1 mutation carriers will assist functional studies to identify the gene/s targeted by these variants. BNC2 is an obvious candidate gene, given that the putative causal variants are located in/around its transcription start site. Identifying more strongly associated variants with ovarian cancer in the 9p22.2 region relative to the initial GWAS hit in BRCA1 and BRCA2 mutation carriers will refine the cancer risks associated with this locus further. These novel variants can be included in polygenic risk scores for ovarian cancer and hence inform the identification of patients at greater risk of disease. The results may also help to deepen our understanding of the biology of ovarian cancer development in BRCA1 and BRCA2 mutation carriers, potentially leading to the development of more effective and personalized treatments.Having narrowed down the potential set of causal variants to only eight SNPs in S1 FigThe colour code indicates the linkage disequilibrium with respect to the variant used for adjustment.(TIFF)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.S2 Table(PDF)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.S4 TableBRCA1 and BRCA2 mutation carriers, respectively.'T' corresponds to genotyped; 'Info' measures the accuracy of the imputation; 'Ref' and 'Eff' correspond to reference and effector allele, respectively; 'MAF' to minor allele frequency, 'HR' hazard ratio and 'CI' confidence interval. Bold cells correspond to the strongest associated SNP in the indicated dataset. Green and violet text indicates the set of potentially causal variant/s in (PDF)Click here for additional data file.S5 TableBRCA1 and BRCA2 mutation carriers and their meta-analysis, respectively.'T' corresponds to genotyped; 'Ref' and 'Eff' correspond to reference and effector allele, respectively; 'MAF' to minimum allele frequency, 'HR' hazard ratio and 'CI' confidence interval. Bold cells correspond to the strongest associated SNP in the indicated dataset. Green, violet and orange text indicate those SNPs within 100 times likely of being the causal variant/s in (PDF)Click here for additional data file.S6 Table(XLSX)Click here for additional data file.S1 Text(DOCX)Click here for additional data file.


The unconscious trauma patient with a possible unstable spinal injury constitutes a clinical challenge. To protect the unintubated airway, some guidelines ,2 recommWe surgically created a global ligamentous instability between C5 and C6 in five fresh cadavers . Four diCompared to RC, LTP created significantly less movement during lateral bending (p=.037), while H1and H2 had significantly less movement than RC in axial translation (p=.009 and .033). There was a tendency towards LTP and H1 and H2 performing better than RC also for other movements.Our results indicate that in unconscious trauma patients, LTP or one of the two HAINES techniques is preferable to the classic recovery position in the setting of an unstable cervical spine injury.


G93A mice. One and five CCO terms related to cytoskeleton were described from the spinal cord deregulated genes of 40 days (actin cytoskeleton) and 80 days  old transgene mice, respectively. Also, four terms were depicted from the deregulated genes of sciatic nerve of 60 days old transgenes . Kif1b was the unique deregulated gene in more than one studied region or presymptomatic age. The expression of Kif1b [quantitative polymerase chain reaction (qPCR)] elevated in the lumbar spinal cord (40 days old) and decreased in the sciatic nerve (60 days old) of presymptomatic ALS mice, results that were in line to microarray findings. Upregulation (24.8 fold) of Kif1b was seen in laser microdissected enriched immunolabeled motor neurons from the spinal cord of 40 days old presymptomatic SOD1G93A mice. Furthermore, Kif1b was dowregulated in the sciatic nerve Schwann cells of presymptomatic ALS mice (60 days old) that were enriched by means of cell microdissection (6.35 fold), cell sorting (3.53 fold), and primary culture (2.70 fold) technologies. The gene regulation of cytoskeleton molecules is an important occurrence in motor neurons and Schwann cells in presymptomatic stages of ALS and may be relevant in the dying back mechanisms of neuronal death. Furthermore, a differential regulation of Kif1b in the spinal cord and sciatic nerve cells emerged as key event in ALS.Early molecular events related to cytoskeleton are poorly described in Amyotrophic Lateral Sclerosis (ALS), especially in the Schwann cell (SC), which offers strong trophic support to motor neurons. Database for Annotation, Visualization and Integrated Discovery (DAVID) tool identified cytoskeleton-related genes by employing the Cellular Component Ontology (CCO) in a large gene profiling of lumbar spinal cord and sciatic nerve of presymptomatic SOD1 Amyotrophic Lateral Sclerosis (ALS) is a progressive, rapid and fatal neurodegenerative disease that affects motor neurons of the spinal cord, brainstem, and cerebral cortex  develop clinical and pathological features similar to those seen in human ALS and are considered an excellent model to study the pathogenic mechanisms of the disease  were crossbred and the colony was maintained in a specific pathogen-free environment of the animal facility of University of São Paulo Medical School  as described previously  were used in the experiments. No motor neuron death was seen in those animal ages   and reference (100 ng) were reverse transcribed by the Low-input RNA Linear Amplification kit and then transcribed to Cy3-labelled (samples) or Cy5-labelled (reference) according to the manufacturer's instructions  and to previous descriptions  and scanned (Agilent Microarray Scanner). All steps were performed according to the manufacturer's instructions .The raw data from hybridizations and experimental conditions are available on the Gene Expression Omnibus website under accession numbers GSE50642     v6.7b functional tool (G93A and wild-type groups) were microdissected as described previously . RNA was extracted from the microdissected motor neurons using the PicoPure RNA isolation kit . Linear amplification of RNA was performed following Eberwine's procedure  were rapidly removed and frozen in ice cold isopentane at −45°C and stored at −80°C until use. The labeling procedure was performed according to adaptation of a previous description  and their sciatic nerves were dissected under aseptic conditions. Nerves were then placed in 60 mm dishes containing Leibovitz-15 medium , divested of their epineurial sheaths and chopped into 1 mm pieces. The fragments were then transferred to new 60 mm dishes containing D-10 culture medium [composed by DMEM  supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin ] and were maintained there in 5% CO2 at 37°C for 5 days. The sciatic nerve fragments were then transferred to 30 mm dishes containing 2.5 ml Hanks' Balanced Salt solution , 0.05% trypsin , and 1 mg collagenase . The fragments were kept in that solution for 2 h in 5% CO2 at 37°C. Tissue fragments were washed with D-10 and dissociated by trituration through a 200 μl-pipette and a 19-gauge sterile needle. The suspension was centrifuged at 1500 rpm for 5 min at 4°C and the cells were resuspended in D-10 medium. This step was repeated and cells were passed through a 70 μm-cell strainer .Schwann cells were isolated by means of flow cytometry sorting from the sciatic nerve explants of 60 days old SOD16 cells were resuspended in 500 μl of buffer. Flow cytometry dot plot Schwann cell profiles are shown in Figure The cells were centrifuged at 1,500 rpm for 5 min at 4°C and the pellets were resuspended in PBS containing 10% fetal bovine serum and 0.01% sodium azide, . Sciatic nerve-derived cell suspension was incubated with a fluorescein isothiocyanate (FITC)-conjugated mouse p75NGF receptor antibody  diluted in the buffer solution 1/200) for 1 h at room temperature as mentioned above. The p75NGF receptor labeling was employed in the cell sorting experiments because it is a well-characterized surface marker for Schwann cells  according to the manufacturer's protocol. The quantity (NanoDrop 1000 Spectrophotometer) and quality  of RNA were analyzed as described above. Also, the Schwann cell samples were submitted to PCRs in order to access contamination from other cell types. Protocol and results regarding specificity of separated Schwann cell samples are presented in the supplementary material Figures .G93A and wild-type mice as described above. Nerve pieces were transferred weekly to new 60 mm dishes filled with 1 ml of D-10 for 5 weeks. Dishes were replaced every other day with a fresh medium  according to the manufacturer's protocol. The quantity (NanoDrop 1000 Spectrophotometer) and quality  of RNA were analyzed as described above. Cultured Schwann cell RNA samples were submitted to PCRs in order to access fibroblast contamination; the protocol and results are shown in the supplementary material Figure .Kif1b in the spinal cord (40 days old mice) and sciatic nerve (60 days old mice) and it was the only gene that its product has been described in the context of ALS . Sciatic nerve cDNA was synthesized from 100 ng of total RNA by using a Maxima First Strand cDNA Synthesis Kit  according to manufacturers.Kif1b—Forward 5′-3′: CTGCTAGCCCTTTAAGACTCG; Reverse 5′-3′: AAACTCCTAGACAAACGCTCC; Gapdh—Forward 5′-3′: GAGTAAGAAACCCTGGACCAC; Reverse 5′-3′: TCTGGGATGGAAATTGTGAGG) in a 20 μl final volume reaction.qPCR reactions were carried out in duplicate by means of the PikoReal-Time PCR System  employing 40 ng cDNA for spinal cord and 15 ng cDNA for sciatic nerve, the DyNAmo ColorFlash SYBR Green qPCR kit  and finally 400 nM of each primer . Gapdh was chosen as a housekeeping gene to normalize the qPCR values because the microarray analysis showed no alteration in the gene expression across samples.The cycling for SYBR reactions was composed by an initial denaturation at 95°C for 10 min. Templates were amplified by 40 cycles of 95°C for 15 s and of 60°C for 30 s. A dissociation curve was then generated to ensure amplification of a single product and absence of primer dimers. A standard curve was generated for each primer pair in order to determine the efficiency of the PCR reaction over a range of template concentrations from 0.032 to 20 ng/μ l, using cDNA synthesized from reference mouse RNA. The efficiency for each set of primers was 100 ± 5%. Gene expressions, which were normalized by The statistical method employed in the microarray analysis is described above , which are shown in Table G93A mice , cytoskeleton part (53 genes), actin cytoskeleton (16 genes), neurofilament cytoskeleton (three genes), and cytoskeleton (76 genes). Those genes are overlapped with the 76 deregulated genes of cytoskeleton category (25 down and 51 upregulated).The DAVID analysis of differentially expressed genes of 40 days old SOD1ce Table . The CCOce Table  in the sG93A mice. Furthermore, the CCO indicated four GO terms related with cytoskeleton, specifically the actin cytoskeleton (43 genes), cytoskeleton part (101 genes), microtubule cytoskeleton (64 genes), and cytoskeleton (146 genes). The 146 genes of the cytoskeleton GO term (74 down and 72 upregulated) are overlapped with all other GO terms  were deregulated in both spinal cord and sciatic nerve of SOD1G93A mice at the presymptomatic ages 40 and 60 days, respectively, and 10 genes  were deregulated in the above regions of 80 and 60 days mice, respectively, as shown by the Venn diagram  were obtained by means of single cell laser microdissection and the sciatic nerve Schwann cells were achieved by means of laser microdissection, flow cytometry cell sorting and cell culture. The levels of cDNA specific cell type marker that demonstrated the enrichment for each cell type obtained by respective technique are shown in the Supplementary Material , S3.Kif1b expression showed an upregulation (1.21 fold) of the gene in spinal cord of presymptomatic 40 days old SOD1G93A mice  was seen in laser microdissected motor neurons from 40 days old SOD1G93A mice  by means of single cell laser microdissection (6.35 fold), cell sorting (3.53 fold), and cell culture (2.70 fold)  Figure , regulatGene regulation of cytoskeleton-related molecules employing microarray technology has been described in the spinal cord and/or microdissected survival neurons from post mortem material of ALS patients  gene was in agreement to our analysis. Also, from 11 deregulated genes of the cytoskeleton and motor activity categories described in microdissected toluidine blue-labeled neurons from the spinal cord of asymptomatic SOD1G93A mice  and Kif1b (upregulation) deregulated genes seen in the spinal cord of 40 days old SOD1G93A mice were in line with descriptions of reduction KIF3Aβ in motor cortices of ALS human and animal model  in the spinal cord of 40 days old presymptomatic ALS mice already indicates a very early presymptomatic event related to cytoskeleton with a possible implication to physiopathological mechanisms of the disease onset. From those genes, only Actg1, Adora, Akt1, App, Dctn1, Kif1a, Sirt2, and Stmn1) or related molecules have been studied in the context of ALS.From the 76 deregulated genes (25 down and 51 upregulated) in the spinal cord of 80 days old presymptomatic ALS mice, only eight genes  of ALS mouse model, thus denoting dynactin impairment as a mechanism in the presymptomatic phase of the disease. Mutation in Dctn1 gene has been associated to motor neuron degeneration in ALS  are both enriched on endoplasmic reticulum and Golgi membranes and are capable to interact with cytoskeleton elements in order to maintain the organelle morphology  in the sciatic nerve of 60 days presymptomatic old SOD1Aif1, Ccnb1, and Mapt in the presymptomatic ALS mice is likely to participate in the early events in the ALS peripheral nerve pathology. Aif1 encodes the allograft inflammatory factor-1 (AIF-1) and AIF-1 positive microglia/macrophages are among the earliest cells to respond to nerve injury , an actin-capping protein for the slow-growing end of filamentous actin  and qPCR analysis  and Schwann cells (downregulation). The results were coincident to those obtained in qPCR and microarray analyses of whole spinal cord and sciatic nerve of presymptomatic ALS mice.It is of substantial importance that the methodology allowed the observation of a differential regulation of Kif1b upregulation in immunolabeled motor neurons was in line to a previous work that employed toluidine blue-enriched putative motor neurons of presymptomatic ALS mice  and sciatic nerve (downregulation) was coincident to that found in the enriched motor neurons and Schwann cells and emerged as an important event in the pathogenic mechanism of ALS.In conclusion, the present work demonstrated cytoskeleton gene regulation as an important occurrence in motor neurons and Schwann cells in the presymptomatic stages of ALS and may be of importance in the dying back mechanisms of neuronal death in the neurodegenerative disease. The differential regulation of Jessica R. Maximino, Gabriela P. de Oliveira, and Chrystian J. Alves performed the experiments. Jessica R. Maximino and Gerson Chadi designed the study and analyzed the results. Gerson Chadi wrote the manuscript. All authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Arabidopsis PIKLE and rice CHR729 are identified to play critical roles in the regulation of series of genes involved in developmental or stress responding process. In this review we focus on how plant CHD proteins regulate gene expression and the role of these proteins in controlling plant development and stress response.Chromodomain-Helicase-DNA (CHD)-binding proteins have been characterized in various species as important transcription regulators by their chromatin remodeling activity. However, in plant the function of these proteins has hardly been analyzed before except that  Figure 1). Double chromodomains of human CHD1 are associated with methylated histone H3 lysine 4 (H3K4me) which is a hallmark of active chromatin -binding proteins, which are Snf2 family ATP-dependent chromatin remodeling factors, play important roles in the regulation of gene expression. Sequence analysis indicates that CHD proteins contain double chromodomains and Helicase-like region comprising SNF2_N and Helicase_C domains, which are critical for chromatin remodeling activity of the proteins. Based on the structure and function conservation CHD proteins could be divided into three subfamilies: Chd1 subfamily , Mi-2 subfamily , and CHD7 subfamily . In addihromatin . Yeast Cgulation . In Mi-2gulation . The effproteins .Arabidopsis PKL, which is a CHD3 protein. In recent years two papers reported the function of CHR729, a rice CHD3 protein  and its homologs in rice evolved from CHD3 chromatin remodelers were not considered to be CHD protein as they do not have conserved Helicase_C domain in the helicase-like region (Arabidopsis (CHR5) and rice  and rice . All of these proteins contain single PHD domain and two chromodomains except Arabidopsis CHR7 which lacks PHD domain at N-terminal part of the protein . Phylogenetic analysis using helicase-like region showed that CHR7 was the close homolog of PKL . However, later results show that dCHD3 which is homologous to dMi-2 but lacks one PHD domain is associated with actively transcribed sites as well as dMi-2 . This indicates that CHD3 protein may also positively participate in the process of transcription of series of genes.It has long been found that dMi-2 is involved in the repression of hox genes. The NuRD (Nucleosome Remodeling Deacetylase) complex which contains CHD3 protein exhibits histone deacetylase activity further improving the repressive function of the protein . In rice, CHR729, which is the homolog of Arabidopsis CHR4, was found to bind to H3K27me3 via its PHD domain in vitro. Loss-of-function of CHR729 results in genome-wide decrease of H3K27me3 level .In plant, there are also controversial arguments on the function of PKL. It was initially characterized to repress embryonic identity related genes and later was found to negatively regulate several genes expression. The repression mediated by PKL was found to involve trimethylation of histone H3 lysine 27 (H3K27me3), a repressive epigenetic mark. PKL target genes were enriched for H3K27me3 and mutation of PKL leads to the loss of H3K27me3 at these loci , 2012. Re3 level . This inFigure 2). Since many transcription factors were down-regulated in chr729 plants it would be interesting to test if these genes are direct targets of CHR729 to clarify if the protein also participate in the process of transcription  genes which are activated by PKL and CHR7 synergistically . They alcription . As ArabArabidopsis, PKL was found to play important roles in plant development as reviewed recently (pkl showed characteristics of embryonic tissue (LEAFY COTYLEDON 1 (LEC1), LEAFY COTYLEDON 2 (LEC2), FUSCA3 (FUS3) and PHERES1 were derepressed in the mutants  and ARF9 by SOLITARY-ROOT (SLR)/IAA14 requires PKL, also named SSL2 in the study  technique affects plant growth, demonstrating that this gene is also important in controlling plant development  and ABI5 in response to ABA implicating the protein may play a role in osmoic stress (Direct evidence that plant CHD3 proteins are involved in stress response has not been present yet. However, in c stress . PKL andc stress . In ricec stress .Arabidopsis PKL and rice CHR729 have revealed that the proteins contribute to epigenetic regulation of gene expression which involves polycomb mediated H3K27me3 albeit the mechanism remains to be explored. PKL was found to control expression of several developmental genes. Despite direct targets are not clear mutants of CHR729 showed multiple developmental defects suggesting that both genes have effects on plant development. In addition, Arabidopsis CHD3 proteins are also involved in stress response. In light of the idea that H3K27me3 is an epigenetic mark associated with regulation of developmental and stress responsive genes it would be intriguing to disclose how CHD3 proteins and H3K27me3 are related in the matter of repressing gene expression (Arabidopsis respectively. Since distinct molecular function of CHD1 protein has been found in human, fly and yeast it is necessary to analyzed whether the protein in plant functions in the same way or adopt a novel mechanism to activate gene expression. Genetic analysis is also required to show whether plant CHD1 protein play important roles in development or stress response as CHD3 protein.Studies on pression . Plant CThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The reports on polymorphism in the KLK2 gene in relation to prostate cancer are contradictory among American and Korean population. Reports are not available regarding polymorphism in KLK2 gene in relation to prostate cancer in Indian population. The relationship between mutants (T) for wild-type (C) allele substitution of the KLK2, circulating human kallikrein-2 (hK2) levels and prostate cancer risk in Indian population has been examined.Prostate cancer is the third most common cancer among men in the world. The human kallikrein-2 gene (KLK2 gene) contains a mutant (T) for wild-type (C) allele substitution, resulting in a Trp to Arg change on the 250The study was limited to 15 subjects that include seven patients with prostate cancer and six controls who were persons with no cancer. Peripheral blood samples were collected from subject and control. DNA was extracted from these samples using standard protocol. DNA was dissolved in 1X TE buffer and used for PCR reactions for the amplification of 322bp KLK2 gene. Polymerase chain reaction- Restriction fragment length polymorphism (PCR-RFLP) procedure was employed using MspI to detect this polymorphism. The enzyme recognizes the 4bp sequence 5ʹCCGG3ʹ at codon 226 of KLK2 gene. PCR-RFLP products ware separated in 2% agarose gel and viewed under Gel-doc system.The median age of the eight subjects diagnosed for prostate cancer were 60, out of which 6 are found to be homozygous for CC allele of KLK2 gene, 1 was heterozygous having CT allele and 1 was homozygous for TT allele. From 7 subjects diagnosed negative for prostate cancer 2 were having the CT genotype and 5 were found to have the TT genotype.The study indicates a possible correlation among the C/T polymorphism of the KLK2 gene and circulating levels of hK2 and, in combination, may be predictive for prostate cancer. Though significant association has been found with small subjects, uses of allele marker for prostate cancer need further extensive study with large number of subject.